A . Overexpression of MavC but not inactive mutant MavCC74A in ∆ mavC ∆ mvcA mutant background causes IκBα accumulation in infected . U937 infected with indicated strains at an MOI of 5 were probed for IκBα . Tubulin was probed as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Overexpression of MavC but not the inactive mutant MavCC74A in the ∆mavC∆mvcA mutant background causes IκBα accumulation in infected cells. U937 cells infected with the indicated bacterial strains at an MOI of 5 were probed for IκBα. Tubulin was probed as a loading control.


B . Prolonged UBE2N modification by MavC restricts vacuole maturation . HEK293 transfected to express FcγII receptor 4xFlag - MavC or C74A mutant were challenged at an MOI of 1 with opsonized L . pneumophila that expresses GFP for 12 h . vacuole was scored categorized by number of it contained . Data ( mean + / - SE ) shown were from three independent experiments , each with at least 300 vacuoles counted . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Prolonged UBE2N modification by MavC restricts bacterial vacuole maturation. HEK293 cells transfected to express the FcγII receptor and 4xFlag-MavC or the C74A mutant were challenged at an MOI of 1 with opsonized L. pneumophila that expresses GFP for 12 h. The vacuole was scored and categorized by the number of bacteria it contained. Data (mean +/- SE) shown were from three independent experiments, each with at least 300 vacuoles counted. *, p<0.05; **, p<0.01; n.s., not significant.


C . Overexpression of MavC but not MavCC74A affects intracellular growth of ∆ mavC ∆ mvcA mutant of L . pneumophila . Raw264 . 7 were infected with indicated strains intracellular were determined at indicated time points . Each was examined in triplicate values shown were mean + / - SE . Similar results were obtained in two independent experiments . * , p < 0 . 1 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ( t - by comparing p ( MavC ) with p ( MavCC74A ) ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Overexpression of MavC but not MavCC74A affects intracellular growth of the ∆mavC∆mvcA mutant of L. pneumophila. Raw264.7 cells were infected with the indicated bacterial strains and intracellular bacteria were determined at the indicated time points. Each strain was examined in triplicate and the values shown were mean +/- SE. Similar results were obtained in two independent experiments. *, p<0.1; **, p<0.01; ***, p<0.001(t-test by comparing p(MavC) with p(MavCC74A)).


D . MvcA is required for reversal of UBE2N modification during L . pneumophila infection . U937 infected with wild type or ∆ mvcA mutant were withdrawn at indicated time points . Samples resolved by SDS - PAGE were probed for UBE2N , translocated MavC MvcA . Tubulin was probed as a loading control . Note that in samples infected with ∆ mvcA , level of UBE2N - Ub decreased at slower rates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O

D. MvcA is required for reversal of UBE2N modification during L. pneumophila infection. U937 cells infected with wild type or the ∆mvcA mutant were withdrawn at the indicated time points. Samples resolved by SDS-PAGE were probed for UBE2N, translocated MavC and MvcA. Tubulin was probed as a loading control. Note that in samples infected with the ∆mvcA strain, the level of UBE2N-Ub decreased at slower rates.


E . MvcA becomes detectable in intracellular 5 h after uptake . U937 macrophages infected with wild type L . pneumophila at an MOI of 10 were lysed at indicated time recovered were probed by immunoblotting for MavC MvcA , respectively . isocitrate dehydrogenase ( ICDH ) was probed sequentially as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. MvcA becomes detectable in intracellular bacteria 5 h after uptake. U937 macrophages infected with wild type L. pneumophila at an MOI of 10 were lysed at the indicated time and recovered bacteria were probed by immunoblotting for MavC and MvcA, respectively. The bacterial isocitrate dehydrogenase (ICDH) was probed sequentially as a loading control.


F . MvcA restores translocation of p65 inhibited by MavC during L . pneumophila infection . Raw264 . 7 were infected with indicated L . pneumophila strains for 2 h at an MOI of 1 . Fixed were immunostained with antibodies specific for p65 bacterium , respectively . Host nuclei were labeled with Hoechst . Representative of in each sample category were shown ( upper panels ) ; localization of p65 was determined by scoring at least 300 infected ( lower panel ) . Results shown were from three independent experiments done in triplicate values shown were mean + / - SE ; t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. MvcA restores the nuclear translocation of p65 inhibited by MavC during L. pneumophila infection. Raw264.7 cells were infected with the indicated L. pneumophila strains for 2 h at an MOI of 1. Fixed cells were immunostained with antibodies specific for p65 and the bacterium, respectively. Host nuclei were labeled with Hoechst. Representative images of cells in each sample category were shown (upper panels); nuclear localization of p65 was determined by scoring at least 300 infected cells (lower panel). Results shown were from three independent experiments done in triplicate and the values shown were mean +/- SE; t-test.


G . Overexpression of MvcA but not its inactive mutant MvcAC83A promotes IκBα degradation during L . pneumophila infection . U937 were infected with indicated strains at an MOI of 5 . Lysates of infected for indicated time were probed for IκBα . Tubulin was probed as a loading control . IκBα for infected with strains ∆ mvA ( pMvcA ) ∆ mvA ( pMvcAC83A ) at indicated time points were evaluated by measuring band from three independent experiments ( lower panel ) , values shown were mean + / - SE ; t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Overexpression of MvcA but not its inactive mutant MvcAC83A promotes IκBα degradation during L. pneumophila infection. U937 cells were infected with the indicated bacterial strains at an MOI of 5. Lysates of cells infected for the indicated time were probed for IκBα. Tubulin was probed as a loading control. IκBα levels for cells infected with the strains ∆mvA(pMvcA) and ∆mvA(pMvcAC83A) at the indicated time points were evaluated by measuring the band intensity from three independent experiments (lower panel), the values shown were mean +/- SE; t-test.


Live imaging of histone - GFP ( green ) ParB - mCherry ( magenta ) expressed in larval salivary nuclei using 1151 - Gal4 . H3 . 3 - GFP are increased at E ( spl ) - C in presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ ( A ) . ParB - mCherry binds to its cognate int inserted within E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( ) = 5 µm . Quantifications of relative of histone - GFP ParB - mCherry across E ( spl ) - C in Notch - OFF ( upper ) Notch - ON ( lower ) conditions . Mean + / - ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 nglands = 5 , 5 , 5 , 5 , 5 , 4 where each represents a biological replicate ( from top to bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. H3.3-GFP levels are increased at the E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Live imaging of histone - GFP ( green ) ParB - mCherry ( magenta ) expressed in larval salivary nuclei using 1151 - Gal4 . E ( spl ) - C in presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ but there is little change in H3 - GFP between Notch - OFF Notch - ON nuclei ParB - mCherry binds to its cognate int inserted within E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( ) = 5 µm . Quantifications of relative of histone - GFP ParB - mCherry across E ( spl ) - C in Notch - OFF ( upper ) Notch - ON ( lower ) conditions . Mean + / - ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 nglands = 5 , 5 , 5 , 5 , 5 , 4 where each represents a biological replicate ( from top to bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ but there is little change in H3-GFP between Notch-OFF and Notch-ON nuclei ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Live imaging of histone - GFP ( green ) ParB - mCherry ( magenta ) expressed in larval salivary nuclei using 1151 - Gal4 . E ( spl ) - C in presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ ( A ) . with H3 . 3core - GFP ParB - mCherry binds to its cognate int inserted within E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( ) = 5 µm . Quantifications of relative of histone - GFP ParB - mCherry across E ( spl ) - C in Notch - OFF ( upper ) Notch - ON ( lower ) conditions . Mean + / - ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 nglands = 5 , 5 , 5 , 5 , 5 , 4 where each represents a biological replicate ( from top to bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). with H3.3core-GFP ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Effects from depleting remodelers histone chaperones , as indicated ( wide range shown in A , on recruitment of Su ( H ) - GFP in Notch - ON nuclei ( expressing NΔECD ) of larval salivary . Different OSA RNAi stocks used in C are denoted by ( 1 ) ( 2 ) . In conditions except Moira RNAi ( A ) nuclei exhibit a bright accumulation of Su ( H ) - GFP at a single locus when imaged live . Scale bars ( yellow ) = 5 µm . of Notch - ON nuclei retaining a single clear band of Su ( H ) - GFP when indicated RNAi is co - expressed with NΔECD . For each genotype , 5 nuclei from each of 10 were scored ( 50 nuclei total ) . * * * * A significant fraction of nuclei lost band when components of BRM were perturbed ; p < 0 . 0001 , two - Fisher ' s exact calculated using raw ( non - ) scoring data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effects from depleting chromatin remodelers and histone chaperones, as indicated (wide range shown in A, on recruitment of Su(H)-GFP in Notch-ON nuclei (expressing NΔECD) of larval salivary glands. Different OSA RNAi stocks used in C are denoted by (1) and (2). In all conditions except Moira RNAi (A) nuclei exhibit a bright accumulation of Su(H)-GFP at a single locus when imaged live. Scale bars (yellow) = 5 µm. Percentage of Notch-ON nuclei retaining a single clear band of Su(H)-GFP when the indicated RNAi is co-expressed with NΔECD. For each genotype, 5 nuclei from each of 10 glands were scored (50 nuclei total). **** A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed; p<0.0001, two-tailed Fisher's exact test calculated using the raw (non-percentage) scoring data.


Effects from depleting remodelers histone chaperones , BRM components shown in C ) , on recruitment of Su ( H ) - GFP in Notch - ON nuclei ( expressing NΔECD ) of larval salivary . w RNAi is a control BrmK804R is expression of dominant - negative Brm . Different OSA RNAi stocks used in C are denoted by ( 1 ) ( 2 ) . , Snr1 RNAi BrmK804R expression ( C ) , nuclei exhibit a bright accumulation of Su ( H ) - GFP at a single locus when imaged live . Scale bars ( yellow ) = 5 µm . of Notch - ON nuclei retaining a single clear band of Su ( H ) - GFP when indicated RNAi is co - expressed with NΔECD . For each genotype , 5 nuclei from each of 10 were scored ( 50 nuclei total ) . * * * * A significant fraction of nuclei lost band when components of BRM were perturbed ; p < 0 . 0001 , two - Fisher ' s exact calculated using raw ( non - ) scoring data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effects from depleting chromatin remodelers and histone chaperones , BRM complex components shown in C), on recruitment of Su(H)-GFP in Notch-ON nuclei (expressing NΔECD) of larval salivary glands. w RNAi is a control and BrmK804R is expression of dominant-negative Brm. Different OSA RNAi stocks used in C are denoted by (1) and (2). , Snr1 RNAi and BrmK804R expression (C), nuclei exhibit a bright accumulation of Su(H)-GFP at a single locus when imaged live. Scale bars (yellow) = 5 µm. Percentage of Notch-ON nuclei retaining a single clear band of Su(H)-GFP when the indicated RNAi is co-expressed with NΔECD. For each genotype, 5 nuclei from each of 10 glands were scored (50 nuclei total). **** A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed; p<0.0001, two-tailed Fisher's exact test calculated using the raw (non-percentage) scoring data.


accessibility in Notch - OFF ( B ) Notch - ON ( NΔECD expression , C ) salivary nuclei measured by ATAC - qPCR ; fold enrichment at indicated regions compared to a " closed ctrl " region . Expression of dominant - negative Brm , BrmK804R , led to reduced accessibility of E ( spl ) mβ - HLH E ( spl ) m3 - HLH enhancer regions in Notch - OFF conditions , to a more widespread reduction in accessibility in Notch - ON conditions . " Eip78C enh " corresponds to ecdysone receptor - binding region of Eip78C enhancer , which is highly accessible but not Notch - responsive ; " Rab11 tr " " Mst87F tr " represent highly lowly - expressed control genes respectively . Mean + / - ; n = 3 ; * p < 0 . 05 with two - Welch ' s t - comparing LacZ BrmK804R samples .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromatin accessibility in Notch-OFF (B) and Notch-ON (NΔECD expression, C) salivary gland nuclei measured by ATAC-qPCR; fold enrichment at the indicated regions compared to a "closed ctrl" region. Expression of dominant-negative Brm, BrmK804R, led to reduced accessibility of E(spl)mβ-HLH and E(spl)m3-HLH enhancer regions in Notch-OFF conditions, and to a more widespread reduction in accessibility in Notch-ON conditions. "Eip78C enh" corresponds to the ecdysone receptor-binding region of the Eip78C enhancer, which is highly accessible but not Notch-responsive; "Rab11 tr" and "Mst87F tr" represent highly and lowly-expressed control genes respectively. Mean +/- SEM; n = 3; * p<0.05 with two-tailed Welch's t-test comparing LacZ and BrmK804R samples.


accessibility in salivary nuclei depleted for Su ( H ) by RNAi , measured by ATAC - qPCR ; fold enrichment at indicated regions compared to a " closed ctrl " region . Accessibility is increased across most of E ( spl ) - C compared to controls expressing LacZ . Control primer regions are as in B C . Mean + / - ; n = 3 ; * p < 0 . 05 with two - Welch ' s t - compared to LacZ controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromatin accessibility in salivary gland nuclei depleted for Su(H) by RNAi, measured by ATAC-qPCR; fold enrichment at the indicated regions compared to a "closed ctrl" region. Accessibility is increased across most of the E(spl)-C compared to controls expressing LacZ. Control primer regions are as in B and C. Mean +/- SEM; n = 3; * p<0.05 with two-tailed Welch's t-test compared to LacZ controls.


Effect of brm Snr1 RNAi on brm Snr1 cDNA respectively , measured by reverse transcription - qPCR in Kc167 ; cDNA compared to GFP RNAi . knockdowns are highly effective , with only 1 - 2 % of brm Snr1 cDNA remaining detectable . Mean + / - ; n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of brm and Snr1 RNAi on brm and Snr1 cDNA levels respectively, measured by reverse transcription-qPCR in Kc167 cells; percentage cDNA compared to GFP RNAi. The knockdowns are highly effective, with only 1-2% of brm and Snr1 cDNA remaining detectable. Mean +/- SEM; n = 3.


Knockdown of components of BRM reduces Su ( H ) recruitment both in Notch - OFF ( B ) Notch - ON ( C ) conditions . Fold enrichment of Su ( H ) occupancy at indicated positions detected by ChIP , relative to input , in Kc167 treated with brm , Snr1 or GFP RNAi as a control . Notch - ON conditions ( C ) were induced by 30 minutes of EGTA treatment . Mean + / - , n = 3 ( B ) ; Mean , n = 2 ( C ) ; * p < 0 . 05 with one - student ' s t - compared to GFP RNAi control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Knockdown of components of the BRM complex reduces Su(H) recruitment both in Notch-OFF (B) and Notch-ON (C) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with brm, Snr1 or GFP RNAi as a control. Notch-ON conditions (C) were induced by 30 minutes of EGTA treatment. Mean +/- SEM, n = 3 (B); Mean, n = 2 (C); * p<0.05 with one-tailed student's t-test compared to GFP RNAi control.


Effect of brm RNAi on E ( spl ) mβ - HLH ( mβ ) E ( spl ) m3 - HLH ( m3 ) induction by Notch activation ( EGTA treatment ) measured by reverse transcription - qPCR ; shown as fold difference to lacZ RNAi control . Mean + / - ; n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of brm RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) induction by Notch activation (EGTA treatment) measured by reverse transcription-qPCR; shown as fold difference to lacZ RNAi control. Mean +/- SEM; n = 3.


Effect of Brm dominant - negative on expression of E ( spl ) mβ - HLH ( mβ ) E ( spl ) m3 - HLH ( m3 ) measured by reverse transcription - qPCR . Expression was analyzed in stable lines containing pMT - inducible BrmWT or BrmK804R in absence ( left , uninduced ) or presence of copper sulfate ( right , Cu2 + induced ) . response of E ( spl ) mβ - HLH E ( spl ) m3 - HLH to Notch activation ( " N - On " = EGTA treatment vs . " N - Off " = PBS control ) was reduced in BrmK804R - expressing compared to BrmWT - expressing , only when induced with copper ( right graph ) . Mean , n = 2 ( left ) ; Mean + / - , n = 3 ( right ) ; * p < 0 . 05 with one - student ' s t - comparing BrmWT BrmK804R .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of Brm dominant-negative on expression of E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) measured by reverse transcription-qPCR. Expression was analyzed in stable cell lines containing pMT-inducible BrmWT or BrmK804R in the absence (left, uninduced) or presence of copper sulfate (right, Cu2+ induced). The response of E(spl)mβ-HLH and E(spl)m3-HLH to Notch activation ("N-On" = EGTA treatment vs. "N-Off" = PBS control) was reduced in the BrmK804R-expressing cells compared to BrmWT-expressing cells, only when induced with copper (right graph). Mean, n = 2 (left); Mean +/- SEM, n = 3 (right); * p<0.05 with one-tailed student's t-test comparing BrmWT and BrmK804R.


Knockdown of actin - related subunit , BAP55 , reduces Su ( H ) recruitment in both Notch - OFF ( PBS treatment ) Notch - ON ( EGTA treatment ) conditions . Fold enrichment of Su ( H ) occupancy at indicated positions detected by ChIP , relative to input , in Kc167 treated with Bap55 or lacZ RNAi as a control . Mean + / - ; n = 3 ; * p < 0 . 05 with one - student ' s t - compared to lacZ RNAi control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Knockdown of actin-related subunit, BAP55, reduces Su(H) recruitment in both Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with Bap55 or lacZ RNAi as a control. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.


Effect of Bap55 RNAi on E ( spl ) mβ - HLH ( mβ ) E ( spl ) m3 - HLH ( m3 ) expression measured by reverse transcription - qPCR in Notch - OFF ( PBS treatment ) Notch - ON ( EGTA treatment ) conditions . Expression level of RpII215 is shown as a control gene . Mean + / - ; n = 3 ; * p < 0 . 05 with one - student ' s t - compared to lacZ RNAi control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of Bap55 RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) expression levels measured by reverse transcription-qPCR in Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Expression level of RpII215 is shown as a control gene. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.


Nucleosome turnover measured by CATCH - IT - qPCR ; fold enrichment over input samples compared to Sec15 tr control region . Su ( H ) - bound enhancers show increased nucleosome turnover in response to Notch signaling . Notch signaling is activated in Kc167 by 6 hours of copper induction of pMT - NICD with copper excluded in control . Positions of E ( spl ) - C primers are shown in Fig 3A ; remaining primers are control non - Notch - responsive regions . Mean , n = 2 .

Nucleosome turnover measured by CATCH-IT-qPCR; fold enrichment over input samples compared to Sec15 tr control region. Su(H)-bound enhancers show increased nucleosome turnover in response to Notch signaling. Notch signaling is activated in Kc167 cells by 6 hours of copper induction of pMT-NICD with copper excluded in the control. Positions of E(spl)-C primers are shown in Fig 3A; the remaining primers are control non-Notch-responsive regions. Mean, n = 2.


brm RNAi reduces nucleosome turnover at Notch - responsive regions . CATCH - IT - qPCR results as in A after brm or lacZ RNAi as a control . Mean + / - ; n = 5 ; * p < 0 . 05 with one - student ' s t - comparing brm lacZ RNAi .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

brm RNAi reduces nucleosome turnover at Notch-responsive regions. CATCH-IT-qPCR results as in A after brm or lacZ RNAi as a control. Mean +/- SEM; n = 5; * p<0.05 with one-tailed student's t-test comparing brm and lacZ RNAi.


Brm is required for Notch - responsive nucleosome turnover . CATCH - IT - qPCR results after brm or lacZ RNAi as in B pMT - NICD expression as in A . Mean + / - ; n = 3 ; * p < 0 . 05 with two - student ' s t - compared to control ( lacZ RNAi Notch - ON bars are compared to lacZ RNAi Notch - OFF bars brm RNAi bars are compared to their respective lacZ RNAi control bars ) .

Brm is required for Notch-responsive nucleosome turnover. CATCH-IT-qPCR results after brm or lacZ RNAi as in B and pMT-NICD expression as in A. Mean +/- SEM; n=3; * p<0.05 with two-tailed student's t-test compared to control (lacZ RNAi Notch-ON bars are compared to lacZ RNAi Notch-OFF bars and brm RNAi bars are compared to their respective lacZ RNAi control bars).


brm RNAi reduces incorporation of histone H3 . 3 . V5 ChIP - qPCR in Kc167 after lacZ or brm RNAi treatment in with H3 - V5 or H3 . 3 - V5 expression induced from pMT promoter by 3 hours of copper treatment , shown as fold enrichment over input samples . Mean + / - ; n = 3 . * p < 0 . 05 with two - Welch ' s t - compared to lacZ RNAi control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

brm RNAi reduces incorporation of histone H3.3. V5 ChIP-qPCR in Kc167 cells after lacZ or brm RNAi treatment in cells with H3-V5 or H3.3-V5 expression induced from the pMT promoter by 3 hours of copper treatment, shown as fold enrichment over input samples. Mean +/- SEM; n = 3. * p<0.05 with two-tailed Welch's t-test compared to lacZ RNAi control.


( B ) Epg5 wild - type copy of Epg5 are absent in Epg5 − / − mice . There is an intervening lane between Epg5 + / − Epg5 − / − lanes in Western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Epg5 protein and the wild-type copy of Epg5 are absent in Epg5−/− mice. There is an intervening lane between Epg5+/− and Epg5−/− lanes in the Western blot.


( C ) curves of Epg5 + / − Epg5 − / − . Means ± of 12 mice are shown .

O O O O O O O O O O O O O O O O O O O O O O

(C) Body weight curves of Epg5+/− and Epg5−/− males. Means ± SEM of 12 mice are shown.


( D ) Time spent on a rotarod . horizontal lines indicate mean times .

(D) Time spent on a rotarod. The horizontal lines indicate mean times.


( E ) Epg5 − / − show gradually worsening limb - clasping reflexes .

(E) Epg5−/− males show gradually worsening limb-clasping reflexes.


( F ) hindquarters of Epg5 − / − mice are completely paralyzed at 10 mo of .

(F) The hindquarters of Epg5−/− mice are completely paralyzed at 10 mo of age.


( G ) Spinal deformation in an Epg5 − / − at 10 mo of .

(G) Spinal deformation in an Epg5−/− male at 10 mo of age.


( H ) Epg5 − / − gradually develop limb - clasping phenotype .

(H) Epg5−/− females gradually develop the limb-clasping phenotype.


( A ) Nissl of fifth layer of cortex in 10 - mo - old Epg5 + / − Epg5 − / − mice . ( B ) number of pyramidal in fifth layer per millimeters squared .

(A) Nissl staining of the fifth layer of the cortex in 10-mo-old Epg5+/− and Epg5−/− mice. (B) The number of pyramidal cells in the fifth layer per millimeters squared.


( C D ) H & amp ; amp ; E of hippocampus shows reduced pyramidal numbers in Epg5 − / − mice . Means ± of three mice are shown in B D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C and D) H&amp;E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5−/− mice. Means ± SEM of three mice are shown in B and D.


( E ) normally developed cerebellum in Epg5 − / − mice . ( F ) thickness of molecular layer in cerebellum in mutant control mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) The normally developed cerebellum in Epg5−/− mice. (F) The thickness of the molecular layer in the cerebellum in mutant and control mice.


( G H ) Purkinje , stained by calbindin , are not reduced in number in Epg5 − / − mice . Means ± of five mice are shown in F H .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G and H) Purkinje cells, stained by calbindin, are not reduced in number in Epg5−/− mice. Means ± SEM of five mice are shown in F and H.


( I ) Arrows indicate Pyknotic neurons in anterior horn of Epg5 − / − mice .

(I) Arrows indicate Pyknotic neurons in the anterior horn of Epg5−/−mice.


( J ) number of motor neurons in spinal cord ( SC ) in Epg5 + / − Epg5 − / − mice .

O O O O O O O O O O O O O O O O O O O O O O O O

(J) The number of motor neurons in the spinal cord (SC) in Epg5+/− and Epg5−/−mice.


( K ) numbers of Nissl - stained interneurons in spinal cord are similar in control Epg5 − / − mice . Means ± of three mice are shown in J K .

(K) The numbers of Nissl-stained interneurons in the spinal cord are similar in control and Epg5−/− mice. Means ± SEM of three mice are shown in J and K.


( L ) Eosinophilic spheroids ( arrows ) accumulate in thoracic spinal cord of Epg5 − / − mice .

(L) Eosinophilic spheroids (arrows) accumulate in the thoracic spinal cord of Epg5−/− mice.


( M ) Eosinophilic spheroids are swollen degenerating axons ( arrow ) .

(M) Eosinophilic spheroids are swollen degenerating axons (arrow).


( N ) EM pictures showing degenerated axons ( arrows ) in spinal cord of Epg5 − / − mice .

O O O O O O O O O O O O O O O O O O O O O

(N) EM pictures showing degenerated axons (arrows) in the spinal cord of Epg5−/− mice.


( O P ) GFAP in Epg5 + / − Epg5 − / − mice . DCST , dorsal corticospinal tract .

(O and P) GFAP staining in Epg5+/− and Epg5−/−mice. DCST, dorsal corticospinal tract.


( A ) of LC3 p62 in brain spinal cord extracts from Epg5 + / − Epg5 − / − mice . SC , spinal cord .

(A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/− mice. SC, spinal cord.


( B ) Polyubiquitinated in detergent - soluble ( S ) - insoluble ( I ) fractions from brain spinal cord homogenates of Epg5 + / − Epg5 − / − mice .

(B) Polyubiquitinated proteins in detergent-soluble (S) and -insoluble (I) fractions from brain and spinal cord homogenates of Epg5+/− and Epg5−/− mice.


( C ) p62 ubiquitin - positive are absent from Epg5 + / − mice but dramatically accumulate are colocalized ( arrows ) in Epg5 − / − mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) p62 aggregates and ubiquitin-positive aggregates are absent from Epg5+/− mice but dramatically accumulate and are colocalized (arrows) in Epg5−/− mice.


( D - F ) p62 are detected in neurons ( arrows ; D ) oligodendrocytes ( arrows ; E ) but absent from astrocytes ( F ) in Epg5 − / − mice .

(D-F) p62 aggregates are detected in neurons (arrows; D) and oligodendrocytes (arrows; E) but absent from astrocytes (F) in Epg5−/− mice.


( G ) TDP - 43 accumulate in motor neurons in anterior horn of spinal cord in Epg5 − / − mice . Arrows show TDP - 43 . DSCT , dorsal corticospinal tract . ( H ) Percentages of TDP - 43 aggregate - positive motor neurons in fifth layer of cortex anterior horn of spinal cord in Epg5 + / − Epg5 − / − mice . Means ± of three mice are shown . LC , lateral column . Bars , 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Cytoplasmic TDP-43 aggregates accumulate in motor neurons in the anterior horn of spinal cord in Epg5−/− mice. Arrows show cytoplasmic TDP-43 aggregates. DSCT, dorsal corticospinal tract. (H) Percentages of cytoplasmic TDP-43 aggregate-positive motor neurons in the fifth layer of cortex and anterior horn of the spinal cord in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown. LC, lateral column. Bars, 10 µm.


( A B ) Electromyography of gastrocnemius muscle from a 3 - mo - old Epg5 − / − mouse showing fibrillations ( A ) positive sharp waves ( B ) . These defects were absent in control mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A and B) Electromyography of the gastrocnemius muscle from a 3-mo-old Epg5−/− mouse showing fibrillations (A) and positive sharp waves (B). These defects were absent in control mice.


( C ) At 10 mo , Epg5 − / − mice showed high - amplitude long - duration action potentials when conducting MUAP . blue lines show start end of an action potential . Dur , duration ; Amp , amplitude .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) At 10 mo, Epg5−/−mice showed high-amplitude and long-duration action potentials when conducting MUAP tests. The blue lines show the start and end of an action potential. Dur, duration; Amp, amplitude.


( D - F ) H & amp ; amp ; E of gastrocnemius showed features of muscle degeneration in Epg5 − / − mice . arrow in E indicates centrally nucleated .

(D-F) H&amp;E staining of gastrocnemius muscles showed features of muscle degeneration in Epg5−/−mice. The arrow in E indicates centrally nucleated fibers.


( G ) transcription of atrophy - related genes were up - regulated in gastrocnemius of Epg5 − / − mice .

(G) The transcription levels of the atrophy-related genes were up-regulated in gastrocnemius muscles of Epg5−/−mice.


(A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/−mice. SC, spinal cord.


( J ) of LC3 - II p62 in muscle extracts from Epg5 + / − Epg5 − / − mice .

(J) Levels of LC3-II and p62 in muscle extracts from Epg5+/− and Epg5−/− mice.


( K ) of p62 aggregate - positive myofibers in Epg5 + / − Epg5 − / − mice . Means ± of three mice are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Percentage of p62 aggregate-positive myofibers in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown.


( L ) Accumulation colocalization of p62 ubiquitin in gastrocnemius of Epg5 − / − mice but not controls . Bars : ( D - F L ) 10 µm ; ( H I ) 1 µm .

(L) Accumulation and colocalization of p62 and ubiquitin aggregates in the gastrocnemius muscles of Epg5−/− mice but not controls. Bars: (D-F and L) 10 µm; (H and I) 1 µm.


( A ) Number of LC3 in Epg5 + / − ( control ) Epg5 − / − MEFs .

(A) Number of LC3 puncta in Epg5+/− (control) and Epg5−/− MEFs.


( B ) Western blot showing of LC3 - I LC3 - II in control Epg5 − / − MEFs upon indicated treatment ( Starv . , starvation ; Rapa . , rapamycin ; Bafilo . , bafilomycin A1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O

(B) Western blot showing levels of LC3-I and LC3-II in control and Epg5−/− MEFs upon indicated treatment (Starv., starvation; Rapa., rapamycin; Bafilo., bafilomycin A1).


( C ) Proteinase K ( ProK ) protection in different MEFs . PNS , postnuclear supernatant ; P , pellet fractions .

O O O O O O O O O O O O O O O O O O O O O O O

(C) Proteinase K (ProK) protection assay in different MEFs. PNS, postnuclear supernatant; P, pellet fractions.


( D ) Colocalization of LC3 LAMP - 1 in control Epg5 − / − MEFs . 50 were examined per time point in A D .

(D) Colocalization ratio of LC3 and LAMP-1 in control and Epg5−/− MEFs. 50 cells were examined per time point in A and D.


( E ) Colocalization of LC3 lysosomes in control Epg5 − / − MEFs 4 h after starvation .

(E) Colocalization of LC3 puncta and lysosomes in control and Epg5−/− MEFs 4 h after starvation.


( F ) Under nutrient repletion conditions , almost no autophagic elements are detected in Epg5 + / − MEFs . RER , rough ER ; M , mitochondrion ; N , nucleus . arrowhead indicates a vacuole with a late residual - like appearance ( aAV - III ) . ( G ) Under nutrient repletion conditions , Epg5 − / − MEFs accumulate a large number of autophagic vacuoles . Red arrow , a likely autophagosome ; red arrowheads , vacuoles of aAV - I type ; arrowheads , aAV - II

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Under nutrient repletion conditions, almost no autophagic elements are detected in Epg5+/− MEFs. RER, rough ER; M, mitochondrion; N, nucleus. The arrowhead indicates a vacuole with a late residual body-like appearance (aAV-III). (G) Under nutrient repletion conditions, Epg5−/− MEFs accumulate a large number of autophagic vacuoles. Red arrow, a likely autophagosome; red arrowheads, complex vacuoles of the aAV-I type; white arrowheads, aAV-II vacuoles


( H I ) High magnification of autophagic elements in unstarved Epg5 − / − MEFs . ( H ) red arrow indicates a autophagic vacuole containing multilayered structures ( arrowheads ) a lipid - like region ( L ) . ( I ) red arrow indicates an autophagosome containing cytoplasm ribosomes . red arrowhead points to an autophagosome containing a mitochondrion material with membranous structures . arrow shows an aAV - III vacuole .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H and I) High magnification of autophagic elements in unstarved Epg5−/− MEFs. (H) The red arrow indicates a complex autophagic vacuole containing multilayered membrane structures (white arrowheads) and a lipid-like region (L). (I) The red arrow indicates an autophagosome containing cytoplasm and ribosomes. The red arrowhead points to an autophagosome containing a mitochondrion and cytoplasmic material with membranous structures. The white arrow shows an aAV-III vacuole.


( J ) Compared with control , Rab5 - labeled early endosomes are significantly enlarged in Epg5 siRNA - treated .

(J) Compared with control cells, Rab5-labeled early endosomes are significantly enlarged in Epg5 siRNA-treated cells.


( K L ) EGFR degradation in control Epg5 knockdown .

(K and L) EGFR degradation in control and Epg5 knockdown cells.


( M N ) Distribution of Alexa Fluor 488 - conjugated transferrin at different time points in control siRNA - Epg5 siRNA - treated . Higher magnification views are shown in insets . Error bars indicate SEMs .

(M and N) Distribution of Alexa Fluor 488-conjugated transferrin at different time points in control siRNA- and Epg5 siRNA-treated cells. Higher magnification views are shown in the insets. Error bars indicate SEMs.


( A ) Co - immunoprecipitation ( Co - IP ) of Beclin1 with WT or mutant vBcl - 2 . 293T were transiently transfected with indicated constructs , followed by immunoprecipitation of HA - tagged vBcl - 2 immunoblotting of V5 - tagged Beclin1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Co-immunoprecipitation (Co-IP) of Beclin1 with WT or mutant vBcl-2. 293T cells were transiently transfected with the indicated constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of V5-tagged Beclin1.


( B ) Co - IP of WT or mutant vBcl - 2 with endogenous Beclin1 . 293T were transfected with indicated vBcl - 2 constructs , followed by immunoprecipitation of HA - tagged vBcl - 2 immunoblotting of endogenous Beclin1 . 1 % whole - lysates ( WCLs ) was used as input .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Co-IP of WT or mutant vBcl-2 with endogenous Beclin1. 293T cells were transfected with the indicated vBcl-2 constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of endogenous Beclin1. 1% whole-cell lysates (WCLs) was used as the input.


( C ) vBcl - 2 interaction with Bak . 293T were transfected with WT mutant forms of vBcl - 2 as indicated . At 48 h posttransfection , WCLs were mixed either with GST - BakΔTM fusion ( left panel ) or with GST alone ( right panel ) for an in vitro GST pull - down ( GST PD ) assays . GST fusion used for pulldown are indicated ( bottom panel ) . 1 % WCL was used as input . Data are representative of at least three experiments yielding similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) vBcl-2 interaction with Bak protein. 293T cells were transfected with WT and mutant forms of vBcl-2 as indicated. At 48 h posttransfection, WCLs were mixed either with GST-BakΔTM fusion protein (left panel) or with GST alone (right panel) for an in vitro GST pull-down (GST PD) assays. GST fusion proteins used for the pulldown assay are indicated (bottom panel). 1% WCL was used as the input. Data are representative of at least three experiments yielding similar results.


( A ) NIH3T3 stably expressing WT mutant forms of vBcl - 2 were transfected with GFP - LC3 . At 18 h posttransfection , were incubated under normal or starvation conditions for 4 h . Autophagy was quantified as mean ± of combined results from three independent experiments . * * , P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) NIH3T3 cells stably expressing the WT and mutant forms of vBcl-2 were transfected with GFP-LC3. At 18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as mean±SEM of the combined results from three independent experiments. **, P<0.0001.


( B ) NIH3T3 expressing WT or mutant forms of vBcl - 2 as indicated were transfected with GFP - LC3 treated with 2 µM rapamycin for 6 h . GFP - LC3 was detected using an inverted microscope ( top ) . Autophagy was quantified as mean ± of combined results from three independent experiments . Scale bar , 5 µm ; * * , P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) NIH3T3 cells expressing the WT or mutant forms of vBcl-2 as indicated were transfected with GFP-LC3 and treated with 2 µM rapamycin for 6 h. GFP-LC3 was detected using an inverted fluorescence microscope (top). Autophagy was quantified as mean±SEM of the combined results from three independent experiments. Scale bar, 5 µm; **, P<0.01.


( C ) NIH3T3 stably expressing WT or mutant forms of vBcl - 2 , as indicated , were treated with 2 µM rapamycin . LC3 - I LC3 - II were then determined by immunoblotting with an antibody against LC3 ( top ) . Densitometric of LC3 - II / LC3 - I ratios under normal rapamycin treatment conditions is shown at bottom . Similar results were obtained from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(C) NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2, as indicated, were treated with 2 µM rapamycin. LC3-I and LC3-II levels were then determined by immunoblotting with an antibody against LC3 (top). Densitometric quantification of the LC3-II/LC3-I ratios under normal and rapamycin treatment conditions is shown at the bottom. Similar results were obtained from three independent experiments.


( top ) Representative confocal of GFP - LC3 HA - tagged vBcl - 2 ( WT mutants ) in NIH3T3 infected with indicated ( MOI = 5 ) . Nuclei were counterstained with 4 ′ , 6 ′ - diamidino - 2 - phenylindole ( DAPI ) . Scale bar , 5 µm . ( bottom ) of of virally infected with GFP - LC3 punctate . Results shown represent mean ± of combined results from three independent experiments ( 200 per experimental condition ) . * , P < 0 . 01 versus HA - WT ; * * , P < 0 . 001 versus HA - WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(top) Representative confocal images of GFP-LC3 and HA-tagged vBcl-2 (WT and mutants) in NIH3T3 cells infected with the indicated viruses (MOI = 5). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Scale bar, 5 µm. (bottom) Quantification of the percentage of virally infected cells with GFP-LC3 punctate staining. Results shown represent mean±SEM of combined results from three independent experiments (200 cells per experimental condition). *, P<0.01 versus HA-WT; **, P<0.001 versus HA-WT.


NIH3T3 stably expressing WT or mutant forms of vBcl - 2 were treated with TNFα cycloheximide ( CHX ) for 12 h , then assayed for viability by trypan blue exclusion ( A ) ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A),


NIH3T3 stably expressing WT or mutant forms of vBcl - 2 were treated with TNFα cycloheximide ( CHX ) for 12 h , then assayed for viability by trypan blue exclusion ( A ) , or for apoptosis by TUNEL ( B )

NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B)


NIH3T3 stably expressing WT or mutant forms of vBcl - 2 were treated with TNFα cycloheximide ( CHX ) for 12 h , then assayed for viability by trypan blue exclusion ( A ) , or for apoptosis by TUNEL ( B ) or for caspase 3 activation using flow cytometry ( C ) . Apoptotic in ( B ) were counted under high power magnification ( 60 × magnification ) . Mock , untreated condition . Data represents mean ± of combined results from three independent experiments . Scale bars , 100 µm ( A ) , 5 µm ( B ) . * * , P < 0 . 0001 versus vector .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B) or for the caspase 3 activation using flow cytometry (C). Apoptotic cells in (B) were counted under high power magnification (60×magnification). Mock, untreated condition. Data represents mean±SEM of combined results from three independent experiments. Scale bars, 100 µm (A), 5 µm (B). **, P<0.0001 versus vector cells.


( A ) Single - step ( right ) multiple - step ( left ) growth curves of WT recombinant γHV68 in BHK21 .

O O O O O O O O O O O O O O O O O O O O O O O O

(A) Single-step (right) and multiple-step (left) growth curves of the WT and recombinant γHV68 viruses in BHK21 cells.


( B ) Acute replication of WT mutant vBcl - 2 γHV68 in lungs of BALB / c mice at 5 dpi ( up ) 7 dpi ( down ) after intranasal infection determined by viral titers in lungs of infected mice ( left ) or by real - time PCR of viral genomic ( right ) . Mean ± of five mice per group / experiment . Data of 7 dpi is pooled from two separate experiments . vBcl - 2 Δα1 ΔBH2 mutants did not yield significantly different results when compared to WT in infectious titers [ for Δα1 , P = 0 . 82 ( day 5 ) ; P = 0 . 08 ( day 7 ) ; for ΔBH2 , P = 0 . 49 ( day 5 ) ; P = 0 . 54 ( day 7 ) ; unpaired t - ] in viral genome loads in lungs [ for Δα1 , P = 0 . 25 ( day 5 ) ; P = 0 . 28 ( day 7 ) ; for ΔBH2 , P = 0 . 21 ( day 5 ) ; P = 0 . 37 ( day 7 ) ; unpaired t - ] . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Acute replication of the WT and mutant vBcl-2 γHV68 viruses in the lungs of BALB/c mice at 5 dpi (up) and 7 dpi (down) after intranasal infection determined by viral titers in the lungs of the infected mice (left) or by real-time PCR of the viral genomic DNA (right). Mean±SEM of five mice per group/experiment. Data of 7 dpi is pooled from two separate experiments. The vBcl-2 Δα1 and ΔBH2 mutants did not yield significantly different results when compared to the WT in infectious virus titers [for Δα1, P = 0.82 (day 5); P = 0.08 (day 7); for ΔBH2, P = 0.49 (day 5); P = 0.54 (day 7); unpaired t-tests] and in viral genome loads in the lungs [for Δα1, P = 0.25 (day 5); P = 0.28 (day 7); for ΔBH2, P = 0.21 (day 5); P = 0.37 (day 7); unpaired t-tests]. n.s., not significant.


Splenic infectious centers measured at 12 dpi ( A , left ) , 14 dpi ( B , left ) , 21 dpi ( C , left ) , 28 dpi ( D , up ) , 35 dpi ( E , up ) , or 42 dpi ( F , up ) viral genome load measured by real - time PCR at 12 dpi ( A , right ) , 14 dpi ( B , right ) , 21 dpi ( C , right ) , 28 dpi ( D , down ) , 35 dpi ( E , down ) , or 42 dpi ( F , down ) , in BALB / cmice intranasally infected with WT or recombinant γHV68 mutants , as indicated ( Mean ± of five mice per group / time point / experiment ) . Data of 14 dpi 28 dpi is pooled from two three separate experiments , respectively . Preformed infectious was negligible in spleen samples . No significant difference was detected at 12 dpi ( A ) , 14 dpi ( B ) , 21 dpi ( C ) with Δα1 ΔBH2 mutant when compared to WT in infectious center titers [ for Δα1 , P = 0 . 58 ( day 12 ) ; P = 0 . 75 ( day 14 ) ; P = 0 . 18 ( day 21 ) ; for ΔBH2 , P = 0 . 64 ( day 12 ) ; P = 0 . 73 ( day 14 ) ; P = 0 . 81 ( day 21 ) ; unpaired t - ] in viral genome loads [ for Δα1 , P = 0 . 85 ( day 12 ) ; P = 0 . 76 ( day 14 ) ; P = 0 . 96 ( day 21 ) ; for ΔBH2 , P = 0 . 56 ( day 12 ) ; P = 0 . 73 ( day 14 ) ; P = 0 . 25 ( day 21 ) ; unpaired t - ] . At 28 dpi ( D ) , 35 dpi ( E ) , 42 dpi ( F ) , decreased infectious center titers ( top ) of vBcl - 2 mutant , as compared to WT γHV68 , were statistically significant [ vBcl - 2Δα1 versus vBcl - 2AAA ( 42 dpi ) , P = 0 . 46 ; vBcl - 2Δα1 versus WT γHV68 ( 42 dpi ) , P = 0 . 08 ; unpaired t - ] . At 28 dpi ( D ) , 35 dpi ( E ) , 42 dpi ( F ) , decreased viral loads ( bottom ) of vBcl - 2Δα1 , vBcl - 2AAA , vBcl - 2 null mutants , as compared to WT γHV68 vBcl - 2ΔBH2 mutant , were statistically significant as follows ( unpaired t - ) : at day 28 , vBcl - 2Δα1 versus WT γHV68 , P < 0 . 01 ; vBcl - 2AAA versus WT γHV68 , P < 0 . 01 ; vBcl - 2 - null versus WT γHV68 , P < 0 . 01 ; vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2 - null versus vBcl - 2ΔBH2 , P < 0 . 001 ; at day 35 , vBcl - 2Δα1 versus WT γHV68 , P < 0 . 05 ; vBcl - 2AAA versus WT γHV68 , P < 0 . 05 ; vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 01 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 05 ; at day 42 , vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2ΔBH2 versus WT γHV68 , P = 0 . 33 . I . C . , infectious center . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Splenic infectious centers measured at 12 dpi (A, left), 14 dpi (B, left), 21 dpi (C, left), 28 dpi (D, up), 35 dpi (E, up), or 42 dpi (F, up) and viral genome load measured by real-time PCR at 12 dpi (A, right), 14 dpi (B, right), 21 dpi (C, right), 28 dpi (D, down), 35 dpi (E, down), or 42 dpi (F, down), in the BALB/cmice intranasally infected with the WT or recombinant γHV68 mutants, as indicated (Mean±SEM of five mice per group/time point/experiment). Data of 14 dpi and 28 dpi is pooled from two and three separate experiments, respectively. Preformed infectious virus was negligible in all spleen samples. No significant difference was detected at 12 dpi (A), 14 dpi (B), and 21 dpi (C) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [for Δα1, P = 0.58 (day 12); P = 0.75 (day 14); P = 0.18 (day 21); for ΔBH2, P = 0.64 (day 12); P = 0.73 (day 14); P = 0.81 (day 21); unpaired t-tests] and in viral genome loads [for Δα1, P = 0.85 (day 12); P = 0.76 (day 14); P = 0.96 (day 21); for ΔBH2, P = 0.56 (day 12); P = 0.73 (day 14); P = 0.25 (day 21); unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased infectious center titers (top) of vBcl-2 mutant viruses, as compared to WT γHV68, were statistically significant [vBcl-2Δα1 versus vBcl-2AAA (42 dpi), P = 0.46; vBcl-2Δα1 versus WT γHV68 (42 dpi), P = 0.08; unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased viral DNA loads (bottom) of the vBcl-2Δα1, vBcl-2AAA, and vBcl-2 null mutants, as compared to WT γHV68 and the vBcl-2ΔBH2 mutant, were statistically significant as follows (unpaired t-tests): at day 28, vBcl-2Δα1 versus WT γHV68, P<0.01; vBcl-2AAA versus WT γHV68, P<0.01; vBcl-2-null versus WT γHV68, P<0.01; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2-null versus vBcl-2ΔBH2, P<0.001; at day 35, vBcl-2Δα1 versus WT γHV68, P<0.05; vBcl-2AAA versus WT γHV68, P<0.05; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.01; vBcl-2AAA versus vBcl-2ΔBH2, P<0.05; at day 42, vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2ΔBH2 versus WT γHV68, P = 0.33. I.C., infectious center. *, P<0.05; **, P<0.01; ***, P<0.001.


( A ) Volcano plot displaying associations between mutational status of genes activity of kinases . x - axis contains mutation coefficients ( effect sizes ) y - axis adjusted P - values . vertical in x - axis is at x = 0 separates positive ( right ) from negative ( left ) associations . horizontal lines in y - axis represent points of statistical significance in plot ( adjusted P - values < 0 . 05 < 0 . 01 ) . associations are represented in form of a mutated gene - kinase . colour gradient represents string network edge interval of pair ( grey if pair is not in string network ) . ( B ) Same as ( A ) for TFs . inner plot shows effects of TF mutations on their own activities . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Volcano plot displaying the associations between the mutational status of genes and the activity of kinases. The x-axis contains the mutation coefficients (effect sizes) and the y-axis the adjusted P-values. The vertical line in the x-axis is at x=0 and separates the positive (right) from the negative (left) associations. The horizontal lines in the y-axis represent the points of statistical significance in the plot (adjusted P-values < 0.05 and < 0.01).The associations are represented in the form of a mutated gene - kinase. The colour gradient represents the string network edge weight interval of the pair (grey if the pair is not in the string network). (B) Same as (A) for the TFs. The inner plot shows the effects of TF mutations on their own activities. (


( C ) ( D ) Examples of genetic associations highlighted in volcano plots . x - axis represents associations y - axis activities . colours stratify samples by their mutational status in respective genes . outliers ( defined as data points beyond Q1 - 1 . 5 * IQR Q3 + 1 . 5 * IQR , where Q1 Q3 are first third quartiles IQR is interquartile range ) were removed from distributions for representation purposes . number of activity quantifications ( including outliers ) are shown beneath each boxplot . P - values from Wilcoxon rank sum comparing both distributions are shown . data points ( including outliers ) were used to calculate P - values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) (D) Examples of the genetic associations highlighted in the volcano plots. The x-axis represents the associations and the y-axis the protein activities. The colours stratify the samples by their mutational status in the respective genes. The outliers (defined as the data points beyond Q1-1.5*IQR and Q3+1.5*IQR, where Q1 and Q3 are the first and third quartiles and IQR is the interquartile range) were removed from the distributions for representation purposes. The number of protein activity quantifications (including outliers) are shown beneath each boxplot. The P-values from Wilcoxon rank sum tests comparing both distributions are shown. All data points (including outliers) were used to calculate the P-values.


( C ) Kinase activity gradient along samples for a selection of kinases shown in ( B ) .

(C) Kinase activity gradient along the samples for a selection of the kinases shown in (B).


( F ) Linear regression between of samples where kinase is regulated in perturbed conditions in tumour samples . trend Pearson ' s r , with respective P - value , are shown . In red green are kinases preferably regulated in tumours in conditions , respectively . In blue are kinases regulated in both . 95 % confidence interval around regression is represented by shaded .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Linear regression between the percentage of samples where the kinase is regulated in the perturbed conditions and in the tumour samples. The trend line and the Pearson's r, with the respective P-value, are shown. In red and green are the kinases preferably regulated in the tumours and in the conditions, respectively. In blue are the kinases regulated in both. The 95% confidence interval around the regression line is represented by the shaded area.


( C - F ) Kaplan - Meier survival plots comparing survival probabilities ( y - axes ) as a function of time in days ( x - axes ) for cancer types shown in ( A ) ( B ) . ( C ) MYC / brain cancer ( inactive = 71 , neutral = 39 , active = 67 ) . ( D ) MYC / liver cancer ( inactive = 58 , neutral = 59 , active = 40 ) . ( E ) MAP3K8 / renal cancer ( neutral = 95 , active = 7 ) . ( F ) PRKCA / renal cancer ( inactive = 3 , neutral = 92 , active = 7 ) . log - rank P - values are shown in plots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-F) Kaplan-Meier survival plots comparing the survival probabilities (y-axes) as a function of time in days (x-axes) for proteins and cancer types shown in (A) and (B). (C) MYC/brain cancer (inactive = 71, neutral = 39, active = 67). (D) MYC/liver cancer (inactive = 58, neutral = 59, active = 40). (E) MAP3K8/renal cancer (neutral = 95, active = 7). (F) PRKCA/renal cancer (inactive = 3, neutral = 92, active = 7). The log-rank P-values are shown in the plots.


( c ) Shown are responses of glucose sensor promoter ( PgluA7 ) , a negative control lacking CRP operator ( PgluA7 * ) , a constitutive promoter ( BBa _ J23101 ) to presence ( + ) absence ( - ) of

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Shown are the responses of the glucose sensor promoter (PgluA7), a negative control lacking the CRP operator (PgluA7*), and a constitutive promoter (BBa_J23101) to the presence (+) and absence (-) of glucose


( d ) Shown are responses of oxygen sensor promoter ( PfnrF8 ) , a negative control lacking FNR operator ( PfnrF8 * ) , a constitutive promoter ( BBa _ J23101 ) to presence ( + ) absence ( - ) of

(d) Shown are the responses of the oxygen sensor promoter (PfnrF8), a negative control lacking the FNR operator (PfnrF8*), and a constitutive promoter (BBa_J23101) to the presence (+) and absence (-) of oxygen


Shown are schematics responses for glucose sensor response functions ( center ) are shown for each sensor ( black circles ) compared to promoter variants where operators are removed ( PgluA7 * , PfnrF8 * ) ( open circles ) text ) . Representative cytometry florescence distributions for Figures

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Shown are the schematics and responses for the glucose sensor The response functions (center) are shown for each sensor (black circles) compared to promoter variants where the operators are removed (PgluA7*, PfnrF8*) (open circles) text). Representative cytometry florescence distributions for Figures 1f


Shown are schematics responses for th oxygen sensors Horizontal error bars in PfnrF8 response reflect one standard deviation of three DO measurements Representative cytometry florescence distributions for Figure

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Shown are the schematics and responses for th oxygen sensors Horizontal error bars in the PfnrF8 response reflect one standard deviation of three DO measurements Representative cytometry florescence distributions for Figure 1g


Shown are schematics responses for th acetate sensors For acetate sensor , response of sensor is shown in unmodified E . coli MG1655 with glnL intact ( open diamonds ) . dynamics of induction are shown ( right graph ) where are induced at time indicated by dashed ( see text ) . Representative cytometry florescence distributions for Figure 1h are shown

O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Shown are the schematics and responses for th acetate sensors For the acetate sensor, the response of the sensor is shown in unmodified E. coli MG1655 with glnL intact (open diamonds). The dynamics of induction are shown (right graph) where cells are induced at the time indicated by the dashed line (see text). Representative cytometry florescence distributions for Figure 1h are shown i


responses of glucose sensors during growth in shake flasks are

The responses of the glucose sensors during growth in shake flasks are shown


responses of th e , oxygen sensors during growth in shake flasks are

O O O O O B-CONTROLLED_VAR O O O O O O O

The responses of th e, oxygen sensors during growth in shake flasks are shown


The responses of th acetate sensors during growth in shake flasks are shown


( e ) responses of a 3 - input , 2 - output circuit are shown . ( f ) Shown are circuit ( left ) genetic diagram ( right ) of circuit corresponding to part e . In genetic diagram , dashed * indicates a second copy of PgluA7 promoter that drives rfp expression is repressed by PhlF via an immediately downstream PhlF operator . ( g ) response of circuit in parts e f to different combinations of stimuli under same conditions as Figure 1 ( Methods ) . ( + ) ( - ) indicate whether output promoter of each sensor is active under those conditions . Bars where circuit is predicted to be on are shown in grey when predicted to be

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) The responses of a 3-input, 2-output circuit are shown. (f) Shown are the circuit (left) and genetic diagram (right) of the circuit corresponding to part e. In the genetic diagram, the dashed line and * indicates a second copy of the PgluA7 promoter that drives rfp expression and is repressed by PhlF via an immediately downstream PhlF operator. (g) The response of the circuit in parts e and f to different combinations of stimuli under the same conditions as Figure 1 (Methods). The (+) and (-) indicate whether the output promoter of each sensor is active under those conditions. Bars where the circuit is predicted to be on are shown in grey and white when predicted to be off


( h ) response of a 3 - input 1 - output circuit is shown . ( i ) Shown are circuit ( left ) genetic diagram ( right ) of circuit corresponding to part h . ( j ) response of circuit in parts h i to different combinations of stimuli under same conditions as Figure 1 ( Methods ) . Representative cytometry florescence distributions for Figure 2a / b / c Figure 2e / g / h / j are shown in Appendix Figures S7 S10 , respectively . Error bars represent one standard deviation of three independent experiments done on different

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) The response of a 3-input 1-output circuit is shown. (i) Shown are the circuit (left) and genetic diagram (right) of the circuit corresponding to part h. (j) The response of the circuit in parts h and i to different combinations of stimuli under the same conditions as Figure 1 (Methods). Representative cytometry florescence distributions for Figure 2a/b/c and Figure 2e/g/h/j are shown in Appendix Figures S7 and S10, respectively. Error bars represent one standard deviation of three independent experiments done on different days


( b ) Reduction of of RFP by different mechanisms of repression after 18 hours of growth ( Methods ) . inducers are either 1 mM IPTG ( sRNA ) or 25 μM DAPG ( sgRNA , mf - LON ) . sgRNA mf - LON are co - transcribed on a single transcript , processed by ribozymes ( Appendix Figure S13 ) . ( c ) dynamics of repression by each of mechanisms is shown . Empty circles are uninduced black circles are induced ( 1 mM IPTG or 25 µM DAPG ) at 2 - hour time point ( dashed ) ( Methods )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Reduction of fluorescence of RFP by the different mechanisms of repression after 18 hours of growth (Methods). The inducers are either 1 mM IPTG (sRNA) or 25 μM DAPG (sgRNA, mf-LON). sgRNA and mf-LON are co-transcribed on a single transcript, processed by ribozymes (Appendix Figure S13). (c) The dynamics of repression by each of the mechanisms is shown. Empty circles are uninduced and black circles are induced (1 mM IPTG or 25 µM DAPG) at the 2-hour time point (dashed line) (Methods)


( f ) impact on acetate production for a double mutant where poxB is complemented on a BAC ( Methods ) . Note that complementation causes a ~ 3 - fold reduction in acetate compared to WT in part d . N - ter E170 refer to location of SuMMV tag . ( g ) Reduction of acetate production by expression of SuMMV from an DAPG - inducible promoter ( - no inducer , + 25 µM ) . PoxB variant with degron inserted at position E170 is either carried on a BAC or at its native location in

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) The impact on acetate production for a double mutant where poxB is complemented on a BAC (Methods). Note that complementation causes a ~3-fold reduction in acetate compared to WT in part d. N-ter and E170 refer to the location of the SuMMV tag. (g) Reduction of acetate production by the expression of SuMMV from an DAPG-inducible promoter (- no inducer, + 25 µM). The PoxB variant with the degron inserted at position E170 is either carried on a BAC or at its native location in the genome


( b ) transcription of poxB over time ( E . coli MG1655 ∆ glnL ) , as calculated from - seq data ( Methods ) . colored bars indicate times at which glucose / acetate sensors will be on on metabolite measurements . It should be noted that these are right - shifted compared to Figure 2 due to slower growth of tested

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) The transcription of poxB over time (E. coli MG1655∆glnL), as calculated from RNA-seq data (Methods). The colored bars indicate the times at which the glucose/acetate sensors will be on based on metabolite measurements. It should be noted that these are right-shifted compared to Figure 2 due to slower growth of the tested strains


( c ) production of acetate is shown over time for E . coli MG1655 ∆ glnL ∆ pta poxB : : E170 ( black ) as compared to same containing complete circuit ( green ) . A version of circuit in which sgRNA is targeted to rfp ( not present in system ) is tested in MG1655 ∆ glnL ∆ pta containing an untagged poxB as a control ( red ) . Empty circles connected by dashed represent MG1655 ∆ glnL ∆ pta ∆

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) The production of acetate is shown over time for E. coli MG1655∆glnL∆pta poxB::E170 (black) as compared to the same strain containing the complete circuit (green). A version of the circuit in which the sgRNA is targeted to rfp (not present in the system) and is tested in MG1655∆glnL∆pta containing an untagged poxB as a control (red). Empty circles connected by the dashed line represent MG1655∆glnL∆pta∆poxB


( e ) transcription of pta over time ( E . coli MG1655 ∆ glnL ) , as calculated from - seq data ( Methods ) . colored bars indicate times at which glucose / oxygen sensors will be on on metabolite

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O

(e) The transcription of pta over time (E. coli MG1655∆glnL), as calculated from RNA-seq data (Methods). The colored bars indicate the times at which the glucose/oxygen sensors will be on based on metabolite measurements


( f ) production of acetate is shown over time for E . coli MG1655 ∆ glnL ∆ poxB pta : : pdt3 as compared to a containing complete circuit ( green ) tested in same . A version of circuit in which sgRNA is targeted to rfp ( not present in system ) is tested in MG1655 ∆ glnL ∆ poxB containing an untagged pta as a control ( red ) Empty circles connected by dashed represent MG1655 ∆ glnL ∆ pta ∆

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) The production of acetate is shown over time for E. coli MG1655∆glnL∆poxB pta::pdt3 as compared to a strain containing the complete circuit (green) tested in same strain. A version of the circuit in which the sgRNA is targeted to rfp (not present in the system) and is tested in MG1655∆glnL∆poxB containing an untagged pta as a control (red) Empty circles connected by the dashed line represent MG1655∆glnL∆pta∆poxB


BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with

Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SM


BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with TAK1i for 6 or 4 h respectively .

O O O O O O O O O O O O O O O O O O O O O O O O

Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively.


Time - lapse confocal ( hour : min ) of BMDMs stimulated with recombinant murine TNF ( 100 ng / ml ) SM ( 250 nM ) stained with propidium iodide ( red ) for 6 h . Black arrowheads indicate ballooning while arrowheads indicate apoptotic bodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time-lapse confocal images (hour:min) of BMDMs stimulated with recombinant murine TNF (100 ng/ml) and SM (250 nM) stained with propidium iodide (red) for 6 h. Black arrowheads indicate membrane ballooning while white arrowheads indicate apoptotic bodies.


immortalized BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with

immortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SM


immortalized BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with TAK1i for 6 or 4 h respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O

immortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively.


BMDMs were stimulated with TNF ( 100 ng / ml ) in combination with TAK1i ( 125 nM ) for indicated time points . LDH

BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. LDH release


BMDMs were stimulated with TNF ( 100 ng / ml ) in combination with TAK1i ( 125 nM ) for indicated time points . mixed supernatant extracts were analysed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. mixed supernatant and cell extracts were analysed.


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h in presence or absence of KCl ( 50 mM ) . Where indicated , were pre - incubated with MCC950 ( 10 μM ) 20 - 30 min prior to TNF / TAK1i stimulation .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h in the presence or absence of KCl (50 mM). Where indicated, cells were pre-incubated with MCC950 (10 μM) 20-30 min prior to TNF/TAK1i stimulation.


BMDMs were costimulated with TNF ( 100 ng / ml ) SM ( 250 nM ; 6 h ) mixed supernatant extracts were analysed by

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

BMDMs were costimulated with TNF (100 ng/ml) and SM (250 nM; 6 h) and mixed supernatant and cell extracts were analysed by immunoblot


BMDMs were costimulated with TNF ( 100 ng / ml ) SM LDH release in culture supernatant were quantified at indicated time points .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

BMDMs were costimulated with TNF (100 ng/ml) and SM LDH release in the cell culture supernatant were quantified at the indicated time points.


BMDMs were costimulated with TNF ( 100 ng / ml )

BMDMs were costimulated with TNF (100 ng/ml) and SM


HEK 293T were transfected with doxycycline - inducible DmrB - caspase - 8 indicated GSDMD constructs . were stimulated with doxycycline ( 10 μg / ml ) for 18 h to induce DmrB - caspase - 8 expression exposed to B / B homodimerizer ( 12 . 5 nM ) for another 2 h to activate caspase - 8 . Mixed supernatant extracts were analysed by immunoblot .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

HEK 293T cells were transfected with doxycycline-inducible DmrB-caspase-8 and the indicated GSDMD constructs. Cells were stimulated with doxycycline (10 μg/ml) for 18 h to induce DmrB-caspase-8 expression and exposed to B/B homodimerizer (12.5 nM) for another 2 h to activate caspase-8. Mixed supernatant and extracts were analysed by immunoblot.


Immortalized Gsdmd - / - BDMDs expressing GSDMDWT GSDMDD88A were costimulated with TNF ( 100 ng / ml ) SM for 6 h LDH release was quantified .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were costimulated with TNF (100 ng/ml) and SM for 6 h and LDH release was quantified.


Immortalized Gsdmd - / - BDMDs expressing GSDMDWT GSDMDD88A were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) stimulated with LCL161 ( 1 μM ) for 24 h LDH release was quantified .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with LCL161 (1 μM) for 24 h and LDH release was quantified.


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i for 4 h ( D ) mixed supernatant extracts were analysed by

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and (D) mixed supernatant and extracts were analysed by immunoblot


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i for 4 h LDH release was quantified at indicated time points .

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and LDH release was quantified at the indicated time points.


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h LDH release was quantified Where indicated , were treated with inhibitors Nec - 1s ( 50 μM )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and LDH release was quantified Where indicated, cells were treated with the inhibitors Nec-1s (50 μM)


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h LDH release was

O O O O O O O O O O O O O O O O O O O O O O

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and LDH release was quantified


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h mixed supernatant extracts were analysed by immunoblot . Where indicated , were treated with inhibitors MCC950 ( 10 μM ) , GSK ' 872 ( 1 μM ) , 20 - 30 min prior to stimulation . KCl ( 50 mM ) was added together with TNF TAK1i . To induce necroptosis , BMDMs were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) Q - VD - OPh ( 10 μM ) was added at last 20 - 30 min of priming stimulated with SM ( 500 nM ) for 4 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors MCC950 (10 μM), GSK'872 (1 μM), 20-30 min prior to cell stimulation. KCl (50 mM) was added together with TNF and TAK1i. To induce necroptosis, BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and Q-VD-OPh (10 μM) was added at the last 20-30 min of priming and stimulated with SM (500 nM) for 4 h.


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h mixed supernatant extracts were analysed by immunoblot . Where indicated , were treated with inhibitors GSK ' 872 ( 1 μM ) , 20 - 30 min prior to stimulation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors GSK'872 (1 μM), 20-30 min prior to cell stimulation.


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h mixed supernatant extracts were analysed by immunoblot . Where indicated , were treated with inhibitors probenecid ( 1 mM ) 20 - 30 min prior to stimulation . To activate NLRP3 inflammasome , BMDM were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 4 h stimulated with nigericin ( 10 μM ) for 1 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors probenecid (1 mM) 20-30 min prior to cell stimulation. To activate the NLRP3 inflammasome, BMDM were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 4 h and stimulated with nigericin (10 μM) for 1 h.


BMDMs were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) stimulated with SM ( 0 . 5 μM ) for a further 4 h . Probenecid ( 1 mM ) was added 20 - 30 min prior to stimulation mixed supernatant extracts were analysed by immunoblot .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with SM (0.5 μM) for a further 4 h. Probenecid (1 mM) was added 20-30 min prior to cell stimulation and mixed supernatant and extracts were analysed by immunoblot.


BMDMs were stimulated with TNF ( 100 ng / ml ) SM ( 0 . 5 μM ) for 6 h ( G ) mixed supernatant extracts were analysed by

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and (G) mixed supernatant and extracts were analysed by immunoblot


BMDMs were stimulated with TNF ( 100 ng / ml ) SM ( 0 . 5 μM ) for 6 h LDH release was quantified .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and LDH release was quantified.


BMDMs were stimulated with an increasing dose of S63845 in presence of ABT - 737 ( 0 . 5 μM ) LDH release was quantified at 6 h .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT-737 (0.5 μM) and LDH release was quantified at 6 h.


BMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) S63845 ( 0 . 5 μM ) LDH release was

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O

BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and LDH release was quantified


BMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) S63845 ( 0 . 5 μM ) mixed supernatant extracts were analysed by immunoblot at 6

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) mixed supernatant and extracts were analysed by immunoblot at 6 h


BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and mixed supernatant and extracts were analysed by immunoblot at 6 h


BMDMs were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 3 h further stimulated with ABT - 737 ( 1 μM ) S63845 ( 1 μM ) for 24 h mixed supernatant extracts were analysed by

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and mixed supernatant and extracts were analysed by immunoblot


BMDMs were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 3 h further stimulated with ABT - 737 ( 1 μM ) S63845 ( 1 μM ) for 24 h IL - 1β in - free supernatant were quantified by

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and IL-1β in cell-free supernatant were quantified by ELISA


A Ccno mRNA is expressed in embryonic node at E8 as shown by in situ hybridization . Occasionally , additional expression is observed at posterior tip of ( asterisk ) . Double labelling by for LacZ expression from CcnoTA allele ( shown in B ) in situ hybridization for Dand5 , marking crown at circumference of node . Ccno expression is restricted to ciliated , ventral pit of node as detailed in transverse section at indicated plane .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization. Occasionally, additional expression is observed at the posterior tip of the embryo (asterisk). Double labelling by staining for LacZ expression from the CcnoTA allele (shown in B) and in situ hybridization for Dand5, marking crown cells at the circumference of the node. Ccno expression is restricted to ciliated, ventral pit cells of the node as detailed in the transverse section at the indicated plane.


C Whole X ‐ Gal of E16 CcnoTA / + wild ‐ type control indicated section planes of ( D - F ' ) .

O O O O O O O O O O O O O O O O O O O O O O O O O

C Whole embryo X‐Gal staining of E16 CcnoTA/+ and wild‐type control embryos and indicated section planes of (D-F').


D - F ' Histological sections of LacZ ‐ stained CcnoTA / + at E16 reveal Ccno expression in epithelium of ( D ) plexus choroideus ependyme , ( E ) snout epithelium , ( F , F ' ) trachea bronchi . pc , plexus choroideus ; se , snout epithelium ; tr , trachea ; br , bronchus .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-F' Histological sections of LacZ‐stained CcnoTA/+ embryo at E16 reveal Ccno expression in the epithelium of (D) plexus choroideus and ependyme, (E) snout epithelium, and (F, F') trachea and bronchi. pc, plexus choroideus; se, snout epithelium; tr, trachea; and br, bronchus.


G Co ‐ expression of Ccno Foxj1 in multiciliated of trachea shown by double for X ‐ Gal immunohistochemistry using a FOXJ1 ‐ specific antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O

G Co‐expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X‐Gal and immunohistochemistry using a FOXJ1‐specific antibody.


A Ccno ‐ deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 ( arrow ) .

A Ccno‐deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 (arrow).


B , C Cranial MRI ( B ) shows drastically enlarged ventricular cavity ( marked by V ) diminished cerebral cortex tissue , ( C ) also seen when brains are cut in coronal orientation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C Cranial MRI analysis (B) shows the drastically enlarged ventricular cavity (marked by V) and diminished cerebral cortex tissue, (C) also seen when brains are cut in coronal orientation.


D , D ' Alcian ‐ blue of paranasal cavities reveals mucus congestion along nasal epithelium ( arrows ) .

D, D' Alcian‐blue staining of paranasal cavities reveals mucus congestion along the nasal epithelium (arrows).


F , F ' Scanning electron microscopy ( ) of P21 adult trachea shows reduced numbers of cilia of Ccno ‐ deficient MCCs . Remaining cilia are found in central regions of surface , margins frequently lack ciliary decoration ( arrows ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, F' Scanning electron microscopy (SEM) of P21 adult trachea shows reduced numbers of cilia of Ccno‐deficient MCCs. Remaining cilia are found in the central regions of the cell surface, and cell margins frequently lack the ciliary decoration (arrows).


G Transmission electron microscopy ( ) of wild ‐ type CcnoRA / RA MCCs from adult trachea . Ccno ‐ deficient MCCs show reduced numbers of basal bodies cilia that correctly docked to apical surface ( arrows ) . Ectopic electron ‐ dense material is found within cytoplasm of Ccno ‐ deficient MCCs ( arrowheads ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Transmission electron microscopy (TEM) of wild‐type and CcnoRA/RA MCCs from adult trachea. Ccno‐deficient MCCs show reduced numbers of basal bodies and cilia that correctly docked to the apical cell surface (arrows). Ectopic electron‐dense material is found within the cytoplasm of Ccno‐deficient MCCs (arrowheads).


A - H X ‐ Gal ‐ of ( A - C ) control ( D - H ) CcnoTA / + embryonic lungs at indicated stages showing onset of Ccno expression from E13 in proximal trachea in main bronchi . Expression is extending to more distal regions , from E16 is also found in bronchioli . I X ‐ Gal ‐ indicating LacZ expression from CcnoTA / + allele in mTEC cultures after 5 days of differentiation ‐ onset by switching to air - liquid interface ( ALI ) conditions ( ALI d5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-H X‐Gal‐staining of (A-C) control and (D-H) CcnoTA/+ embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi. Expression is extending to more distal regions, and from E16 staining is also found in bronchioli.I X‐Gal‐staining indicating LacZ expression from the CcnoTA/+ allele in mTEC cultures after 5 days of differentiation‐onset by switching to air-liquid interface (ALI) conditions (ALI d5).


J Transcript of Ccno indicated genes with known functions for generation of multiple cilia during mTEC differentiation . mRNA from three independent experiments were measured by qRT - PCR of expression set as 1 on day 0 of ALI cultures . Scales for Ccno Deup1 are indicated on left , for Cep152 , Cep63 Ift57 on right side .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation. mRNA levels from three independent experiments were measured by qRT-PCR and levels of expression set as 1 on day 0 of ALI cultures. Scales for Ccno and Deup1 are indicated on the left, and for Cep152, Cep63 and Ift57 on the right side.


K Relative mRNA expression for indicated genes were measured by qRT - PCR in wild ‐ type Ccno ‐ deficient mTEC cultures at indicated days after differentiation ‐ onset . Relative values were calculated as in ( J ) relative to day 0 of ALI cultures .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K Relative mRNA expression levels for indicated genes were measured by qRT-PCR in wild‐type and Ccno‐deficient mTEC cultures at indicated days after differentiation‐onset. Relative values were calculated as in (J) relative to day 0 of ALI cultures.


A - B ' of tracheae from ( A , A ' ) wild ‐ type ( B , B ' ) CcnoRA / RA at E17 . In comparison to wild ‐ type MCCs , Ccno ‐ deficient MCCs almost completely lack formation of multiple cilia only single , or doublet ( arrow ) cilia are observed at E17 .

A-B' SEM of tracheae from (A, A') wild‐type and (B, B') CcnoRA/RA embryos at E17. In comparison to wild‐type MCCs, Ccno‐deficient MCCs almost completely lack formation of multiple cilia and only single, or doublet (arrow) cilia are observed at E17.


C - E lack of multiple cilia at E17 stages is reflected by almost complete absence of acetylated α ‐ tubulin as marker for axonemes in ( D ) Ccno ‐ deficient MCCs of E17 embryonic trachea as quantified in ( E ) . FOV , field of view .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-E The lack of multiple cilia at E17 stages is reflected by the almost complete absence of acetylated α‐tubulin staining as marker for axonemes in (D) Ccno‐deficient MCCs of E17 embryonic trachea as quantified in (E). FOV, field of view.


F - I of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in ( F , F ' , H ) Ccno ‐ proficient ( G , G ' , I ) Ccno ‐ deficient MCCs . Asterisks in ( F , G ) indicate microvilli that extend from apical surface . ( F ' , G ' ) Deuterosomes in Ccno ‐ deficient MCCs are significantly enlarged ( identical bars in F ' G ' ) show irregular , which is different to annular shape of wild ‐ type deuterosomes as seen in ( F ' , H ) . Procentrioles ( arrowheads in F ' I ) that are found at deuterosomes of ( I ) Ccno ‐ deficient MCCs were recurrently found being shorter appeared less structured in comparison to ( F ' , H ) wild ‐ type .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-I TEM of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in (F, F', H) Ccno‐proficient and (G, G', I) Ccno‐deficient MCCs. Asterisks in (F, G) indicate microvilli that extend from the apical cell surface. (F', G') Deuterosomes in Ccno‐deficient MCCs are significantly enlarged (identical size bars in F' and G') and show irregular morphology, which is different to the annular shape of wild‐type deuterosomes as seen in (F', H). Procentrioles (arrowheads in F' and I) that are found at deuterosomes of (I) Ccno‐deficient MCCs were recurrently found being shorter and appeared less structured in comparison to (F', H) wild‐type cells.