( a ) Experimental testing of predicted SR ( DU ) rescuers of DNMT1 via drug combination experiments in 18 NSCLC lines insensitive to Decitabine . matrix displays drug interactions between Decitabine , a DNMT1 inhibitor , inhibitors of its predicted rescuer genes ( X - axis ) across 18 NSCLC lines ( Y - axis ) that are insensitive to Decitabine . Row labels present rescuer genes their inhibitors . Colors in matrix show whether interactions found are significantly synergistic ( red ) , antagonistic ( green ) or non - significant ( in gray ) . Values in matrix show average synergism ( < 1 synergism > 1 antagonism Thirteen predicted DU - SR rescuers ( red lines ) , two predicted DD SR rescuers ( green lines ) of DNMT1 one random control ( JAK3i ) were tested .

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(a) Experimental testing of the predicted SR (DU) rescuers of DNMT1 via drug combination experiments in 18 NSCLC cell lines insensitive to Decitabine. The matrix displays drug interactions between Decitabine, a DNMT1 inhibitor, and inhibitors of its predicted rescuer genes (X-axis) across 18 NSCLC cell lines (Y-axis) that are insensitive to Decitabine. Row labels present rescuer genes and their inhibitors. Colors in the matrix show whether the interactions found are significantly synergistic (red), antagonistic (green) or non-significant (in gray). Values in the matrix show average synergism (<1 synergism and >1 antagonism Thirteen predicted DU-SR rescuers (red lines), two predicted DD SR rescuers (green lines) of DNMT1 and one random control (JAK3i) were tested.


transcriptomic alterations of rescuer genes post PD1 / PDL1 blockade in biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after anti - PD1 ( b ) Each panel displays expression fold change of each predicted rescuer gene ( rows ) for different samples ( columns ) P - value of over - paired Wilcoxon of expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models .

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The transcriptomic alterations of rescuer genes post PD1/PDL1 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-PD1 (b) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.


transcriptomic alterations of rescuer genes post CTLA4 blockade in biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after anti - CTLA4 ( c ) Each panel displays expression fold change of each predicted rescuer gene ( rows ) for different samples ( columns ) P - value of over - paired Wilcoxon of expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models .

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The transcriptomic alterations of rescuer genes post CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-CTLA4 (c) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.


transcriptomic alterations of rescuer genes post PD1 / PDL1 CTLA4 blockade in biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after PD1 + CTLA4 combination therapies ( d ) . Each panel displays expression fold change of each predicted rescuer gene ( rows ) for different samples ( columns ) P - value of over - paired Wilcoxon of expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models .

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The transcriptomic alterations of rescuer genes post PD1/PDL1 and CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after PD1 + CTLA4 combination therapies (d). Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.


a , Phase partitioning of Apg8 . Wild - type Δapg4Δapg8 producing Apg8FG Δapg7 were subjected to phase partitioning ( see Methods ) .

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a, Phase partitioning of Apg8. Wild-type and Δapg4Δapg8 cells producing Apg8FG and Δapg7 cells were subjected to phase partitioning (see Methods).


b , MALDI mass spectrum of His6 - Apg8 . Purified His6 - Apg8 ( predominantly Apg8FG - X a small amount of Apg8FG ) was extracted with 0 . 4 % β - octylglycoside from a polyacrylamide gel desalted with ZipTipC4 ( Millipore ) before mass measurement . principal MH + ion signal at m / z 15 , 003 is shifted in mass by about + 635 from MH + value corresponding to His6 - Apg8FG .

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b, MALDI mass spectrum of His6-Apg8. Purified His6-Apg8 (predominantly Apg8FG-X and a small amount of Apg8FG) was extracted with 0.4% β-octylglycoside from a polyacrylamide gel and desalted with ZipTipC4(Millipore) before mass measurement. The principal MH+ ion signal at m/z 15,003 is shifted in mass by about +635 from the MH+ value corresponding to His6-Apg8FG.


c , MS / MS spectra of C - terminal peptides of Apg8FG - X ( top ) Apg8FG ( bottom ) . C - terminal fragments were generated from Apg8FG - X Apg8FG ( see Methods ) . Each fragment that was observed only as non - 18O - labelled signals was subjected to MS / MS . MS / MS spectra , deconvoluted with MaxEnt3 , were interpreted by SeqMS software26 . Arrows show sequences from N C terminus on y ″ m bl ions , respectively ( where m l denote positions counted from C N terminus ) that were produced by cleavage of peptide bonds during MS / MS . shown in three - letter code denote immonium ions . nomenclature of these ions is as in previous work28 .

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c, MS/MS spectra of the C-terminal peptides of Apg8FG-X (top) and Apg8FG (bottom). C-terminal fragments were generated from Apg8FG-X and Apg8FG (see Methods). Each fragment that was observed only as non-18O-labelled signals was subjected to MS/MS. The MS/MS spectra, deconvoluted with MaxEnt3, were interpreted by SeqMS software26. Arrows show the sequences from the N and C terminus based on y″m and bl ions, respectively (where m and l denote positions counted from the C and N terminus) that were produced by cleavage of peptide bonds during MS/MS. Amino acids shown in three-letter code denote immonium ions. The nomenclature of these ions is as in previous work28.


d , Thioester formation between Apg8 Apg7 . extracts of Δapg7Δapg8 expressing Apg7 - Myc / or HA - Apg8 from 2µ plasmids were immunoprecipitated with anti - HA antibody , immunoblotted ( IB ) with anti - Myc anti - HA antibody . DTT - , no DTT ; DTT + , 100 mM DTT ; asterisk , Apg8 - Apg7 conjugate .

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d, Thioester formation between Apg8 and Apg7. Cell extracts of Δapg7Δapg8 cells expressing Apg7-Myc and/or HA-Apg8 from 2µ plasmids were immunoprecipitated with anti-HA antibody, and immunoblotted (IB) with anti-Myc and anti-HA antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg7 conjugate.


b , Interaction between Apg8 Apg7 . Δapg7Δapg8 were transformed with APG7 - Myc / or APG8 on 2µ plasmids . lysates were immunoprecipitated with anti - Myc antibody , immunoblotted with anti - Myc or anti - Apg8 antibody . Apg8 co - immunoprecipitated with Apg7 - Myc .

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b, Interaction between Apg8 and Apg7. Δapg7Δapg8 cells were transformed with APG7-Myc and/or APG8 on 2µ plasmids. Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc or anti-Apg8 antibody. Apg8 co-immunoprecipitated with Apg7-Myc.


c , Effect of Apg8 C - terminal processing on Apg8 - Apg7 interaction . Δapg4 co - expressing Apg7 - Myc HA - Apg8 ( FGR , FG F ) from 2µ plasmids . FGR , wild - type Apg8 ; FG , Apg8 lacking C - terminal arginine ; F , Apg8 lacking C - terminal glycine arginine . lysates were immunoprecipitated with anti - HA antibody , immunoblotted with anti - HA anti - Myc antibody .

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c, Effect of Apg8 C-terminal processing on the Apg8-Apg7 interaction. Δapg4 cells co-expressing Apg7-Myc and HA-Apg8 (FGR, FG and F) from 2µ plasmids. FGR, wild-type Apg8; FG, Apg8 lacking C-terminal arginine; F, Apg8 lacking C-terminal glycine and arginine. Cell lysates were immunoprecipitated with anti-HA antibody, and immunoblotted with anti-HA and anti-Myc antibody.


b , Interaction between Apg8 Apg3 . Lysates of Δapg3 expressing APG3 / or Myc - APG8 from 2µ plasmids were immunoprecipitated with anti - Apg3 antibody , immunoblotted with anti - Apg3 anti - Myc antibody .

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b, Interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing APG3 and/or Myc-APG8 from 2µ plasmids were immunoprecipitated with anti-Apg3 antibody, and immunoblotted with anti-Apg3 and anti-Myc antibody.


c , Effect of mutated Cys 234 in Apg3 on interaction between Apg8 Apg3 . Lysates of Δapg3 expressing Myc - APG8 either wild - type ( wt ) APG3 or mutant APG3 ( C234S C234A ) from 2µ plasmids were immunoprecipitated with anti - Myc antibody , immunoblotted with anti - Myc anti - Apg3 antibodies .

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c, Effect of mutated Cys 234 in Apg3 on the interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing Myc-APG8 and either wild-type (wt) APG3 or mutant APG3 (C234S and C234A) from 2µ plasmids were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc and anti-Apg3 antibodies.


d , Thioester formation between Apg8 Apg3 . Δapg3 , Δapg7 Δapg10 were transformed with both Myc - APG8 Apg3 , or only Apg3 , on 2µ plasmids . extracts were immunoprecipitated with anti - Myc antibody , subjected to immunoblot ( IB ) using anti - Myc anti - Apg3 antibody . DTT - , no DTT ; DTT + , 100 mM DTT ; asterisk , Apg8 - Apg3 conjugate ; diamond , possible Myc - Apg8 dimer .

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d, Thioester formation between Apg8 and Apg3. Δapg3, Δapg7 and Δapg10 cells were transformed with both Myc-APG8 and Apg3, or only Apg3, on 2µ plasmids. Cell extracts were immunoprecipitated with anti-Myc antibody, and subjected to immunoblot (IB) using anti-Myc and anti-Apg3 antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg3 conjugate; diamond, possible Myc-Apg8 dimer.


a , Apg7 Apg3 are necessary for attachment of Apg8 to PE . Δapg lysates were subjected to SDS - PAGE containing 6 M urea , immunoblotted with anti - Apg8 antibody .

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a, Apg7 and Apg3 are necessary for attachment of Apg8 to PE. Δapg cell lysates were subjected to SDS-PAGE containing 6 M urea, and immunoblotted with anti-Apg8 antibody.


b , Autophagic activity in apg3 mutants . Wild - type , Δapg3 apg3C243A were grown in rich medium ( open bar ) subjected to nitrogen starvation for 4 h ( filled bar ) . autophagic activity of each was assessed by measuring alkaline phosphatase activity . apg3C243A mutant showed defective autophagy .

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b, Autophagic activity in apg3 mutants. Wild-type, Δapg3 and apg3C243A cells were grown in rich medium (open bar) and subjected to nitrogen starvation for 4 h (filled bar). The autophagic activity of each cell was assessed by measuring the alkaline phosphatase activity. The apg3C243A mutant showed defective autophagy.


( A , B ) BMDCs were infected with Pru ( at a MOI of 4 / BMDC ) or treated with tunicamycin ( TN ) for 16h expression of UPR target genes were analyzed by RT - qPCR . results are expressed as ( Log2 ) Fold change ( A ) or relative mRNA level ( B ) compared to non - infected ( NI ) conditions . IRE1α / XBP1s pathway : Ern1 ( IRE1α ) , Xbp1s , Dnajb9 ( ERdj4 ) , Sec24D , Bloc1s1 Sec61a1 ; PERK / ATF4 pathway : Atf4 , Ddit3 ( CHOP ) Ppp1r15a ( GADD34 ) ; ATF6 pathway : Atf6 Herpud1 ; classical ER chaperons : Hspa5 ( BIP ) Hsp90b1 ( GRP94 ) . Results are normalized to housekeeping gene Gapdh . Unpaired Student T - , ns : p > 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; mean ± S . E . M ( n = 2 - 5 independent experiments depending of studied gene , 3 mice / independent experiment ) .

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(A, B) BMDCs were infected with Pru parasites (at a MOI of 4 parasites/BMDC) or treated with tunicamycin (TN) for 16h and the expression of UPR target genes were analyzed by RT-qPCR. The results are expressed as (Log2) Fold change (A) or relative mRNA level (B) compared to non-infected (NI) conditions. IRE1α/XBP1s pathway: Ern1 (IRE1α), Xbp1s, Dnajb9 (ERdj4), Sec24D, Bloc1s1 and Sec61a1; PERK/ATF4 pathway: Atf4, Ddit3 (CHOP) and Ppp1r15a (GADD34); ATF6 pathway: Atf6 and Herpud1; classical ER chaperons: Hspa5 (BIP) and Hsp90b1 (GRP94). Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=2-5 independent experiments depending of the studied gene, 3 mice / independent experiment).


( C , D ) Immunoblot of IRE1α , PERK , Phospho - PERK ( detected by presence of a second upper band after migration on a 6 % acrylamide gel ) , Phospho - eIF2α , CHOP XBP1s expression in non - infected BMDCs , BMDCs treated with TN Pru infected BMDCs ( C ) in non - infected Pru infected Flt3 / Notch - DCs ( D ) for indicated times . GAPDH tubulin were used as loading controls . same blots are shown in Figure EV3B - C .

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(C, D) Immunoblot analysis of IRE1α, PERK, Phospho-PERK (detected by the presence of a second upper band after migration on a 6% acrylamide gel), Phospho-eIF2α, CHOP and XBP1s protein expression in non-infected BMDCs, BMDCs treated with TN and Pru infected BMDCs (C) and in non-infected and Pru infected Flt3/Notch-DCs (D) for the indicated times. GAPDH and tubulin were used as loading controls. The same blots are shown in Figure EV3B-C.


( D ) mRNA relative expression level of Xbp1s , Dnajb9 ( ERdj4 ) , Ddit3 ( CHOP ) Ppp1r15a ( GADD34 ) from WT MyD88KO BMDCs infected with Pru for 16h compared to non - infected ( NI ) conditions . Results are normalized to housekeeping gene Gapdh . Two - way ANOVA Tukey ' s multiple comparisons , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 , * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 4 - 5 mice pooled from 2 independent experiments ) .

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(D) mRNA relative expression level of Xbp1s, Dnajb9 (ERdj4), Ddit3 (CHOP) and Ppp1r15a (GADD34) from WT and MyD88KO BMDCs infected with Pru parasites for 16h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001, ****: p<0.0001; mean ± S.E.M (n=4-5 mice pooled from 2 independent experiments).


( A ) BMDCs ( CD11c + ) were incubated with Pru - Tomato for 16h . Infected ( red gating ) non - infected bystander ( green gating ) were sorted - purified further analyzed . ( B ) expression of UPR target genes were analyzed by RT - qPCR in infected non - infected bystander compared to non - infected BMDCs ( NI ) . results are expressed as relative mRNA level compared to non - infected conditions . Results are normalized to housekeeping gene Gapdh . Unpaired Student T - , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; mean ± S . E . M ( n = 6 mice pooled from 2 independent experiments ) .

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(A) BMDCs (CD11c+) were incubated with Pru-Tomato parasites for 16h. Infected cells (red gating) and non-infected bystander cells (green gating) were sorted-purified and further analyzed. (B) The expression of UPR target genes were analyzed by RT-qPCR in infected and non-infected bystander cells compared to non-infected BMDCs (NI). The results are expressed as relative mRNA level compared to non-infected conditions. Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=6 mice pooled from 2 independent experiments).


( E ) mRNA relative expression level of Il - 6 , Il - 12 Il - 23 from XBP1fl / flIRE1fl / fl , XBP1ΔDC IRE1truncDC XBP1ΔDC BMDCs infected with Pru for 16 h compared to non - infected ( NI ) conditions . Results are normalized to housekeeping gene Gapdh . Two - way ANOVA Tukey ' s multiple comparisons , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 3 - 5 mice pooled from 2 independent experiments ) .

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(E) mRNA relative expression level of Il-6, Il-12 and Il-23 from XBP1fl/flIRE1fl/fl, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs infected with Pru parasites for 16 h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).


( I ) Graph depicts expression ( indicated as Mean ) of H - 2Kb by Pru Tomato SAG1 ‐ OVA infected XBP1fl / fl IRE1fl / fl treated or not with 4μ8C , XBP1ΔDC IRE1truncDC XBP1ΔDC BMDCs . Bar graphs depict mean ± S . E . M ( n = 3 mice , one representative experiment from 2 independent experiments ) . Tukey ' s ANOVA one - way , ns : p > 0 . 05 .

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(I) Graph depicts the expression (indicated as the Mean Fluorescence Intensity) of H-2Kb by Pru Tomato SAG1‐OVA infected XBP1fl/fl IRE1fl/fl treated or not with 4μ8C, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs. Bar graphs depict mean ± S.E.M (n=3 mice, one representative experiment from 2 independent experiments). Tukey's ANOVA one-way test, ns: p>0.05.


( A , B ) Expression of VenusFP by splenic cDC1 ( CD26 + CD11c + MHCII + XCR1 + CD172a - , upper panels ) cDC2 ( CD26 + CD11c + MHCII + XCR1 - CD172a + , lower panel ) from WT ( A ) ERAI ( B ) mice non - infected ( NI ) or infected with Pru ( 6 days post - infection ( dpi ) ) reporting on IRE1α pathway activation .

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(A, B) Expression of VenusFP by splenic cDC1 (CD26+ CD11c+ MHCII+ XCR1+ CD172a-, upper panels) and cDC2 (CD26+ CD11c+ MHCII+ XCR1- CD172a+, lower panel) from WT (A) and ERAI (B) mice non-infected (NI) or infected with Pru (6 days post-infection (dpi)) reporting on IRE1α pathway activation.


( E , F ) RT - qPCR of indicated UPR genes among total prepared from splenic cDC1 ( E ) cDC2 ( F ) sorted by flow cytometry 6 dpi from wild - type mice , non - infected ( NI ) or infected with Pru . Results were normalized to housekeeping gene Gapdh are expressed as relative mRNA level compared to NI condition . Bar graphs depict mean ± S . E . M . Unpaired Student T - , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; mean ± S . E . M ( n = 3 - 5 mice pooled from 2 independent experiments ) .

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(E, F) RT-qPCR analysis of the indicated UPR genes among total RNA prepared from splenic cDC1 (E) and cDC2 (F) sorted by flow cytometry 6 dpi from wild-type mice, non-infected (NI) or infected with Pru parasites. Results were normalized to the housekeeping gene Gapdh and are expressed as relative mRNA level compared to the NI condition. Bar graphs depict mean ± S.E.M. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).


( A ) Survival curves of XBP1fl / flIRE1fl / fl XBP1ΔDC IRE1truncDC mice infected intraperitoneally with 500 Pru . Log - rank ( Mantel - Cox ) , p = 0 , 0004 ; n = 10 mice per genotype .

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(A) Survival curves of XBP1fl/flIRE1fl/fl and XBP1ΔDC IRE1truncDC mice infected intraperitoneally with 500 Pru parasites. Log-rank (Mantel-Cox) test, p=0,0004; n=10 mice per genotype.


( D , E ) of number of total ( left histogram ) activated ( right histogram ) ( IFNγ - positive ) CD4 + T ( D ) CD8 + T ( E ) in spleen of control mice ( XBP1fl / flIRE1fl / fl ) XBP1ΔDC IRE1truncDC infected for 6 days . Two - way ANOVA Tukey ' s multiple compare , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 2 independent experiments combined ) .

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(D, E) Quantification of the number of total (left histogram) and activated (right histogram) (IFNγ-positive) CD4+ T cells (D) and CD8+ T cells (E) in the spleen of control mice (XBP1fl/flIRE1fl/fl) and XBP1ΔDC IRE1truncDC infected for 6 days. Two-way ANOVA Tukey's multiple compare, ns: p> 0.05; *: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; mean ± S.E.M (n=2 independent experiments combined).


b , c Phospho - sites identified after TiO2 enrichment b phospho - tyrosine immunoprecipitation ( pY - IP ) c . Left panel : pie chart presenting proportion of identified phosphorylated serines ( pS ) , threonines ( pT ) tyrosines ( pY ) in two data sets . Right panel : cumulative number of unique identified phospho - sites across biological replicates ( 3 4 independent experiments for TiO2 pY - IP , respectively ) . Phosphorylated serines , threonines tyrosines are presented individually next to total number of identified sites ( pSTY ) . phospho - sites that were not previously reported in any species in PhosphoSitePlus database ( www . phosphosite . org ) are reported in isolation in graph on right .

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

b, c Phospho-sites identified after TiO2 enrichment b and phospho-tyrosine immunoprecipitation (pY-IP) c. Left panel: pie chart presenting the proportion of identified phosphorylated serines (pS), threonines (pT) and tyrosines (pY) in the two data sets. Right panel: cumulative number of unique identified phospho-sites across the biological replicates (3 and 4 independent experiments for the TiO2 and pY-IP, respectively). Phosphorylated serines, threonines and tyrosines are presented individually next to the total number of identified sites (pSTY). The phospho-sites that were not previously reported in any species in the PhosphoSitePlus database (www.phosphosite.org) are reported in isolation in the graph on the right.


a , b Output of statistical analyses of TiO2 - fractionated a pY - IP - fractionated b data sets . Left panel : number of phospho - sites significantly regulated upon TCR stimulation presented in bar plots for each time point , next to total number of regulated sites across entire time course ( total ) . number of phosphorylated threonines ( pT ) serines ( pS ) regulated in TiO2 data set are indicated in yellow green , respectively . Right panel : representative volcano plot presenting statistical significance distribution against log2 - transformed fold change between 300 seconds unstimulated control . For each condition , phospho - sites were considered significantly up - ( red ) or down - ( blue ) regulated when displaying a corrected P - value ≤ 0 . 05 ( Anova ) an absolute fold change ≥ 1 . 75 ( see Materials Methods for more detailed information ) .

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

a, b Output of the statistical analyses of the TiO2-fractionated a and the pY-IP-fractionated b data sets. Left panel: number of phospho-sites significantly regulated upon TCR stimulation presented in bar plots for each time point, next to the total number of regulated sites across the entire time course (total). The number of phosphorylated threonines (pT) and serines (pS) regulated in the TiO2 data set are indicated in yellow and green, respectively. Right panel: representative volcano plot presenting the statistical significance distribution against the log2-transformed fold change between 300 seconds and the unstimulated control. For each condition, phospho-sites were considered significantly up- (red) or down- (blue) regulated when displaying a corrected P-value ≤ 0.05 (Anova test) and an absolute fold change ≥ 1.75 (see Materials and Methods for more detailed information).


a showing kinase activities on enrichment of their specific substrates for each cluster . Black dots indicate clusters with significantly enriched kinase activity ( hypergeometric P - value ≤ 0 . 05 , fold - change ≥ 2 , number of annotated sites ≥ 2 ; Grey squares : fold - change < 1 ) .

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a Heatmap showing kinase activities based on enrichment of their specific substrates for each cluster. Black dots indicate clusters with significantly enriched kinase activity (hypergeometric test P-value ≤ 0.05, fold-change ≥ 2, number of annotated sites ≥ 2; Grey squares: fold-change < 1).


d Immunoblot of equal amounts of from total lysates of CD4 + T treated with DMSO , U0126 ( 10 μM ) or AKTi VIII ( 10 μM ) left unstimulated ( - ) or stimulated for 15 , 120 , or 300 sec with anti - CD3 anti - CD4 antibodies , probed with indicated phospho - specific antibodies . Anti - ERK1 / 2 immunoblot served as a loading control .

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 O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot analysis of equal amounts of proteins from total lysates of CD4+ T cells treated with DMSO, U0126 (10 μM) or AKTi VIII (10 μM) and left unstimulated (-) or stimulated for 15, 120, or 300 sec with anti-CD3 and anti-CD4 antibodies, probed with the indicated phospho-specific antibodies. Anti-ERK1/2 immunoblot served as a loading control.


e Equal amounts of from total lysates of CD4 + T left unstimulated ( - ) or stimulated for 120 , 300 or 600 sec with anti - CD3 anti - CD4 antibodies were analyzed by immunoblot with antibodies probing indicated phospho - sites / .

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e Equal amounts of proteins from total lysates of CD4+ T cells left unstimulated (-) or stimulated for 120, 300 or 600 sec with anti-CD3 and anti-CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho-sites/proteins.


b Cas9 - EGFP OT - I CD8 + T were transfected with control sgRNA ( sgEGFP ) or with two different sgRNA targeting Itsn2 ( sgITSN2 - 1 or sgITSN2 - 2 ) . Transfected were stimulated for 48 h with N4 peptide MHC tetramers ( 0 . 1 nM ) in presence or absence of soluble anti - CD28 antibody or with IL - 7 as control . surface expression of CD69 CD5 were analyzed by flow cytometry ( Z - score normalization of Geometric Means of MFI per experiment ) in four independent experiments . Comparison between sgEGFP sgITSN2 conditions were performed using a paired t - . Boxplot elements : center corresponds to median , box limits correspond to first third quartiles , whiskers indicate variability from Q1 - 1 . 5 . IQR to Q3 + 1 . 5 IQR . Outliers are shown as black dots .

O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers (0.1 nM) in presence or absence of soluble anti-CD28 antibody or with IL-7 as control. Cell surface expression of CD69 and CD5 were analyzed by flow cytometry (Z-score normalization of the Geometric Means of fluorescence MFI per experiment) in four independent experiments. Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t-test. Boxplot elements: center line corresponds to median, box limits correspond to the first and third quartiles, whiskers indicate variability from Q1-1.5. IQR to Q3+1.5 IQR. Outliers are shown as black dots.


b Cas9 - EGFP OT - I CD8 + T were transfected with control sgRNA ( sgEGFP ) or with two different sgRNA targeting Itsn2 ( sgITSN2 - 1 or sgITSN2 - 2 ) . Transfected were stimulated for 48 h with N4 peptide

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b Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHC


c Proliferation of OT - I Cas9 - EGFP CD8 + T transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers ( 0 . 01 - 0 . 1 nM ) in presence or absence of soluble anti - CD28 antibody or with PMA ionomycin ( PMA / Iono ) or with IL - 7 . Data are presented as mean ± standard deviation ( SD ) from four proliferative measures of two independent sgEGFP sgITSN2 transfections . Data are representative of three independent experiments .

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c Proliferation of OT-I Cas9-EGFP CD8+ T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers (0.01-0.1 nM) in the presence or absence of soluble anti-CD28 antibody or with PMA and ionomycin (PMA/Iono) or with IL-7. Data are presented as mean ± standard deviation (SD) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections. Data are representative of three independent experiments.


d IFN - γ production of treated as in c was assessed by intracellular after 24 h of stimulation .

d IFN-γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation.


e Proliferation of similar as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in presence of soluble anti - CD28 antibody .

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

e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti-CD28 antibody.


a Immunoblot of equal amounts of from total lysates of Cas9 - EGFP OT - I CD8 + T transfected with sgEGFP , sgITSN2 - 1 or sgITSN2 - 2 left unstimulated ( - ) or stimulated for 2 or 5 min with N4 tetramers ( 20 nM ) . Upper panel : probed with antibody to phosphorylated tyrosine ( Anti - p - Tyr ) or anti - LAT ( loading control ) . Lower panel : probed with antibodies to anti - CBL - pY774 , anti - ZAP70 - pY319 / 352 , anti - AKT - pT308 , anti - ERK1 / 2 - pY204 / T202 or anti - ERK1 / 2 ( 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR 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

a Immunoblot analysis of equal amounts of proteins from total lysates of Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP, sgITSN2-1 or sgITSN2-2 and left unstimulated (-) or stimulated for 2 or 5 min with N4 tetramers (20 nM). Upper panel: membrane probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-LAT (loading control). Lower panel: membrane probed with antibodies to anti-CBL-pY774, anti-ZAP70-pY319/352, anti-AKT-pT308, anti-ERK1/2-pY204/T202 or anti-ERK1/2 (loading control).


b Heat map showing normalized ( z - score ) of indicated surface markers phosphorylations ( left margin ) from Cas9 - EGFP OT - I CD8 + T transfected with sgEGFP or sgITSN2 - 1 left unstimulated ( - ) or stimulated for 1 , 3 or 10 min with N4 tetramers ( 1 or 10 nM ) analyzed by mass cytometry . Z - scores were calculated from hyperbolic arcsine ( arcsinh ) transformed intensities .

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b Heat map showing normalized levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 left unstimulated (-) or stimulated for 1, 3 or 10 min with N4 tetramers (1 or 10 nM) and analyzed by mass cytometry. Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities.


c Similar as in a stimulated with 1 nM of N4 peptide for indicated time points ( left panel ) or stimulated with increased doses of N4 peptides for 4 h ( right panel ) , were analyzed by FACS for surface TCR expression . Data are presented as mean ± comparisons were performed using a paired t - ( * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ) . A representative of three independent experiments is shown .

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 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

c Similar cells as in a stimulated with 1 nM of N4 peptide for the indicated time points (left panel) or stimulated with increased doses of N4 peptides for 4 h (right panel), were analyzed by FACS for surface TCR expression. Data are presented as mean ± SEM and comparisons were performed using a paired t-test (*, P ≤ 0.05, **, P ≤ 0.01; ***, P ≤ 0.001). A representative analysis of three independent experiments is shown.


d showing scaled expression ( z - score ) of indicated surface markers phosphorylations ( left margin ) from Cas9 - EGFP OT - I CD8 + T transfected with sgEGFP or sgITSN2 - 1 stimulated for 6 h with increasing doses of N4 peptides or PMA / Ionomycin ( PI ) analyzed by single - mass cytometry ( left ) . Z - scores were calculated from hyperbolic arcsine ( arcsinh ) transformed intensities . Signal difference ( mean arcsinh difference ) between sgEGFP sgITSN2 - 1 transfected is depicted on right panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heatmap showing scaled expression levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 and stimulated for 6 h with increasing doses of N4 peptides or PMA/Ionomycin (PI) analyzed by single-cell mass cytometry (left). Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities. Signal difference (mean arcsinh difference) between sgEGFP and sgITSN2-1 transfected cells is depicted on the right panel.


( A ) Differential interference contrast micrograph of live T . agilis . arrow points to anterior , where rostrum is located ; arrowheads point to some of flagella .

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(A) Differential interference contrast micrograph of live T. agilis cell. The arrow points to the cell anterior, where the rostrum is located; arrowheads point to some of the flagella.


( B ) Transmission electron of T . agilis centriole embedded in resin - longitudinal view ; hub ( arrowhead ) is visible in cartwheel - bearing region ( pink ) , but not in distal region ( ) .

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(B) Transmission electron micrographs of T. agilis centriole embedded in resin - longitudinal view; the hub (arrowhead) is visible in the cartwheel-bearing region (pink line), but not in the distal region (white line).


( C ) Transmission electron of T . agilis centriole embedded in resin in transverse view from distal end ( left ) corresponding image circularized symmetrized with CentrioleJ plugin ( middle ) , with schematic indicating principal architectural elements ( right ) .

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(C) Transmission electron micrographs of T. agilis centriole embedded in resin in transverse view from distal end (left) and corresponding image circularized and symmetrized with the CentrioleJ plugin (middle), with schematic indicating principal architectural elements (right).


( A ) Transverse 2D slices through T . spp . central cartwheel STA at indicated height from proximal ( 0 nm ) to distal ( 8 . 7 nm ) . dashed box indicates corresponding region shown in ( C ) . ( B ) Transverse view of T . spp . central cartwheel STA 3D map ; 9 spoke densities emanate from hub CID is present within hub . diameter of hub is 23 . 0 ± 0 nm ( N = 3 ; here thereafter in figure legends , N corresponds to 2D measurements from STA ) . An electron dense structure is present inside CID ( grey ) , which is also visible to some extent before symmetrizing . Note that coloring of elements in this other figure panels is merely illustrative not meant to reflect underlying molecular boundaries . ( C ) 2D longitudinal view of T . spp . central cartwheel STA delineated by a dashed box in ( A ) . Arrowheads denote position of scans along vertical axis at level of CID ( purple ) hub ( blue ) , with corresponding pixel intensities in arbitrary units ( AU ) . Plot profiles are shifted horizontally relative to each other for better visibility . Some maxima are highlighted with dashed lines ; average between two CID elements is 8 . 5 ± 0 . 2 nm ( N = 4 ) . Note that hub densities are elongated in vertical direction , resulting in broad peak profiles where two maxima can be discerned ( dashed blue lines ) . ( D ) Longitudinal view of T . spp . central cartwheel STA . average between emanating spokes is 8 . 0 ± 1 . 5 nm ( N = 2 ) ; these are measurements on STA are within error of CID periodicities reported ( C ) ; same applies for other measurements hereafter . Discontinuous densities in center of CID ( grey ) are visible , as well as densities that vertically bridge successive hub elements ( arrows ) . Boxed is shown magnified in ( E ) . Proximal is down in this other figure panels showing STA longitudinal views . ( E ) CID axis is located distal relative to axis of spoke densities , corresponding to an average shift of 0 . 9 ± 0 . 7 nm ( N = 3 ) .

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(A) Transverse 2D slices through T. spp. central cartwheel STA at indicated height from proximal (0 nm) to distal (8.7 nm). The dashed box indicates corresponding region shown in (C). (B) Transverse view of T. spp. central cartwheel STA 3D map; 9 spoke densities emanate from the hub and the CID is present within the hub. The diameter of the hub is 23.0 ± 0 nm (N=3; here and thereafter in the figure legends, N corresponds to 2D measurements from STA). An electron dense structure is present inside the CID (grey), which is also visible to some extent before symmetrizing. Note that the coloring of elements in this and other figure panels is merely illustrative and not meant to reflect underlying molecular boundaries. (C) 2D longitudinal view of T. spp. central cartwheel STA delineated by a dashed box in (A). Arrowheads denote position of line scans along the vertical axis at the level of the CID (purple) and hub (blue), with corresponding pixel intensities in arbitrary units (AU). Plot profiles are shifted horizontally relative to each other for better visibility. Some maxima are highlighted with dashed lines; the average distance between two CID elements is 8.5 ± 0.2 nm (N=4). Note that hub densities are elongated in the vertical direction, resulting in broad peak profiles where two maxima can be discerned (dashed blue lines). (D) Longitudinal view of T. spp. central cartwheel STA. The average distance between emanating spokes is 8.0 ± 1.5 nm (N=2); these are measurements on STA and are within the error of the CID periodicities reported (C); the same applies for other measurements hereafter. Discontinuous densities in the center of the CID (grey) are visible, as well as densities that vertically bridge successive hub elements (arrows). Boxed area is shown magnified in (E). Proximal is down in this and all other figure panels showing STA longitudinal views. (E) The CID axis is located distal relative to the axis of spoke densities, corresponding to an average shift of 0.9 ± 0.7 nm (N=3) .


( F ) Transverse 2D slice through T . agilis central cartwheel STA at indicated height from proximal ( 0 nm ) to distal ( 12 nm ) . dashed box indicates corresponding region shown in ( H ) . ( G ) Transverse view of T . agilis central cartwheel STA 3D map ; 9 spoke densities emanate from hub CID is present within hub , which has a diameter of 22 . 7 ± 0 . 2 nm ( N = 3 ) . ( H ) 2D longitudinal view of T . agilis central cartwheel STA delineated by a dashed box in ( F ) . Arrowheads denote position of scans along vertical axis at level of CID ( pink ) hub ( pink ) , with corresponding pixel intensities in arbitrary units ( AU ) . Plot profiles are shifted horizontally relative to each other for better visibility . Some maxima are highlighted with dashed lines ; average between two CID elements is 8 . 3 ± 0 . 5 nm ( N = 9 ) . Note that hub densities are elongated in vertical direction , resulting in broad peak profiles where two maxima can be discerned ( dashed pink lines ) . ( I ) Longitudinal view of T . agilis central cartwheel STA . average between emanating spokes is 8 . 2 ± 0 . 5 nm ( N = 9 ) . Discontinuous densities in center of CID ( grey ) are visible at this lower contour level compared to ( G ) . Note densities vertically bridging successive hub elements ( arrows ) . Boxed is shown magnified in ( J ) . ( J ) CID axis is located distal relative to axis of spoke densities , corresponding to an average shift of 0 . 8 ± 0 . 3 nm ( N = 9 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transverse 2D slice through T. agilis central cartwheel STA at indicated height from proximal (0 nm) to distal (12 nm). The dashed box indicates corresponding region shown in (H). (G) Transverse view of T. agilis central cartwheel STA 3D map; 9 spoke densities emanate from the hub and the CID is present within the hub, which has a diameter of 22.7 ± 0.2 nm (N=3). (H) 2D longitudinal view of T. agilis central cartwheel STA delineated by a dashed box in (F). Arrowheads denote position of line scans along the vertical axis at the level of the CID (dark pink) and hub (light pink), with corresponding pixel intensities in arbitrary units (AU). Plot profiles are shifted horizontally relative to each other for better visibility. Some maxima are highlighted with dashed lines; the average distance between two CID elements is 8.3 ± 0.5 nm (N=9). Note that hub densities are elongated in the vertical direction, resulting in broad peak profiles where two maxima can be discerned (dashed light pink lines). (I) Longitudinal view of T. agilis central cartwheel STA. The average distance between emanating spokes is 8.2 ± 0.5 nm (N=9). Discontinuous densities in the center of the CID (grey) are visible at this lower contour level compared to (G). Note densities vertically bridging successive hub elements (arrows). Boxed area is shown magnified in (J). (J) The CID axis is located distal relative to axis of spoke densities, corresponding to an average shift of 0.8 ± 0.3 nm (N=9).


( A ) ( Top ) 2D slice through STA transverse view of T . spp . microtubule triplet , with insets showing position of pinhead ( dashed green box ) A - C linker ( dashed red box ) . ( Bottom ) Longitudinal 2D slice of STA centered on pinhead ( left ) or A - C linker ( right ) . ( B ) Transverse view of T . spp . microtubule triplet STA . Microtubule protofilament numbers are indicated , as are pinhead A - C linker ( only C - link is visible ; A - link lies on edge of volume is thus less well resolved in this STA - for better view see STA centered on A - C linker in ( D ) ) . Prominent microtubule inner densities within A - microtubule are highlighted ( empty arrowhead next to A9 , chevron next to A5 ) , as are additional external densities at A - B B - C inner junctions ( black arrowheads ) . Double arrowheads point to viewing direction in indicated panels . ( C ) Longitudinal view of T . spp . STA centered on pinhead from viewing point indicated in ( B ) . pinfeet ( PinF1 PinF2 ) pinbody ( PinB ) are indicated , as are microtubule protofilaments A3 A4 . average between pinfeet elements is 8 . 6 ± 0 . 4 nm 7 . 9 ± 0 . 4 nm ( N = 3 each ) . Corresponding transverse views are shown below , illustrating connection of PinF2 with protofilament A3 . ( D ) Longitudinal view of T . spp . STA centered on A - C linker from viewing point indicated in ( B ) . Microtubule protofilaments A8 / 9 C9 / C10 of two adjacent triplets are indicated , as are connected A - C - links . average between A - C - links is 8 . 4 ± 0 . 4 nm 8 . 4 ± 0 . 3 nm ( both N = 6 ) . Corresponding transverse views are shown below , chevrons point to connection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (Top) 2D slice through STA transverse view of T. spp. microtubule triplet, with insets showing position of pinhead (dashed green box) and A-C linker (dashed red box). (Bottom) Longitudinal 2D slice of STA centered on the pinhead (left) or A-C linker (right). (B) Transverse view of T. spp. microtubule triplet STA. Microtubule protofilament numbers are indicated, as are the pinhead and A-C linker (only the C-link is visible; the A-link lies on the edge of the volume and is thus less well resolved in this STA -for better view see STA centered on A-C linker in (D)). Prominent microtubule inner densities within the A-microtubule are highlighted (empty arrowhead next to A9, chevron next to A5), as are additional external densities at the A-B and B-C inner junctions (black arrowheads). Double arrowheads point to viewing direction in indicated panels. (C) Longitudinal view of T. spp. STA centered on the pinhead from the viewing point indicated in (B). The pinfeet (PinF1 and PinF2) and pinbody (PinB) are indicated, as are microtubule protofilaments A3 and A4. The average distance between pinfeet elements is 8.6 ± 0.4 nm and 7.9 ± 0.4 nm (N=3 each). Corresponding transverse views are shown below, illustrating the connection of PinF2 with protofilament A3. (D) Longitudinal view of T. spp. STA centered on the A-C linker from the viewing point indicated in (B). Microtubule protofilaments A8/9 and C9/C10 of two adjacent triplets are indicated, as are the connected A- and C-links. The average distance between A- and C-links is 8.4 ± 0.4 nm and 8.4 ± 0.3 nm (both N=6). Corresponding transverse views are shown below, chevrons point to connection.


( E ) ( Top ) 2D slice through STA transverse view of T . agilis microtubule triplet , with insets showing position of pinhead ( dashed green box ) A - C linker ( dashed red box ) . ( Bottom ) Longitudinal 2D slice of STA centered on pinhead ( left ) or A - C linker ( right ) . ( F ) Transverse view of T . agilis microtubule triplet STA . Microtubule protofilament numbers are indicated , as are pinhead A - C linker ( only C - link is visible ; A - link lies on edge of volume is thus less well resolved in this STA - for better view see STA centered on A - C linker in ( H ) ) . Prominent microtubule inner densities ( MIPs ) within A - microtubule are highlighted ( empty arrowhead next to A9 ) , as are additional external densities at A - B B - C inner junctions ( black arrowheads ) . Double arrowheads point to viewing direction in indicated panels . ( G ) Longitudinal view of T . agilis STA centered on pinhead from viewing point indicated in ( F ) . pinfeet ( PinF1 PinF2 ) pinbody ( PinB ) are indicated , as are microtubule protofilaments A3 / A4 . average between pinfeet elements is 8 . 4 ± 0 nm in each case ( both N = 3 ) . Corresponding transverse views are shown below , illustrating connection of PinF2 with protofilament A3 . ( H ) Longitudinal view of T . agilis STA centered on A - C linker from viewing point indicated in ( F ) . Microtubule protofilaments A8 / 9 C9 / C10 of two adjacent triplets are indicated , as are connected A - C - links . average between A - C - links is 8 . 4 ± 0 . 3 nm 8 . 4 ± 0 . 9 nm ( N = 6 N = 5 , respectively ) . Corresponding transverse views are shown below , chevrons point to connection ; connection of A - link with A9 is only partially visible in transverse view at this height , as indicated by dashed chevron .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (Top) 2D slice through STA transverse view of T. agilis microtubule triplet, with insets showing position of pinhead (dashed green box) and A-C linker (dashed red box). (Bottom) Longitudinal 2D slice of STA centered on the pinhead (left) or A-C linker (right). (F) Transverse view of T. agilis microtubule triplet STA. Microtubule protofilament numbers are indicated, as are the pinhead and A-C linker (only the C-link is visible; the A-link lies on the edge of the volume and is thus less well resolved in this STA -for better view see STA centered on A-C linker in (H)). Prominent microtubule inner densities (MIPs) within the A-microtubule are highlighted (empty arrowhead next to A9), as are additional external densities at the A-B and B-C inner junctions (black arrowheads). Double arrowheads point to viewing direction in indicated panels. (G) Longitudinal view of T. agilis STA centered on the pinhead from the viewing point indicated in (F). The pinfeet (PinF1 and PinF2) and pinbody (PinB) are indicated, as are microtubule protofilaments A3/A4. The average distance between pinfeet elements is 8.4 ± 0 nm in each case (both N=3). Corresponding transverse views are shown below, illustrating the connection of PinF2 with protofilament A3. (H) Longitudinal view of T. agilis STA centered on the A-C linker from the viewing point indicated in (F). Microtubule protofilaments A8/9 and C9/C10 of two adjacent triplets are indicated, as are the connected A- and C-links. The average distance between A- and C-links is 8.4 ± 0.3 nm and 8.4 ± 0.9 nm (N=6 and N=5, respectively). Corresponding transverse views are shown below, chevrons point to connection; the connection of the A-link with A9 is only partially visible in the transverse view at this height, as indicated by the dashed chevron.


( A ) Transverse 2D slices through STA comprising 64 % 36 % of sub - volumes of T . mirabilis central cartwheel at indicated height from proximal ( 0 nm ) to distal ( 8 . 0 nm ) . Note filamentous cartwheel inner densities ( fCID ) in center of hub .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transverse 2D slices through STA comprising 64 % and 36 % of sub-volumes of T. mirabilis central cartwheel at indicated height from proximal (0 nm) to distal (8.0 nm). Note filamentous cartwheel inner densities (fCID) in the center of the hub.


( D , E ) 2D longitudinal view of T . mirabilis central region of cartwheel STA 64 % ( D ) 36 % ( E ) classes in region delineated by dashed boxes in ( A ) . Arrowheads denote position of scans along vertical axis at hub level , with corresponding normalized pixel intensities ( in arbitrary units ) . Some maxima are highlighted by dashed lines . average between hub units in 64 % class is 8 . 4 ± 0 . 6 nm ( N = 8 ; D ) ; in 36 % class , between hub densities alternates between 3 . 2 ± 0 . 3 nm 5 . 2 ± 0 . 3 nm ( both N = 9 ; E ) . Dashed in ( E ) indicates offset between two superimposed hub units at level of spoke densities , which occurs every other hub unit pair .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) 2D longitudinal view of T. mirabilis central region of the cartwheel STA 64% (D) and 36% (E) classes in the region delineated by dashed boxes in (A). Arrowheads denote position of line scans along vertical axis at the hub level, with corresponding normalized pixel intensities (in arbitrary units). Some maxima are highlighted by dashed lines. The average distance between hub units in the 64 % class is 8.4 ± 0.6 nm (N=8; D); in the 36 % class, the distance between hub densities alternates between 3.2 ± 0.3 nm and 5.2 ± 0.3 nm and (both N=9; E). Dashed white line in (E) indicates the offset between two superimposed hub units at the level of spoke densities, which occurs every other hub unit pair.


( H ) ( Top ) 2D slice through STA transverse view of microtubule triplet in T . mirabilis , with insets showing pinhead ( dashed green box ) A - C linker ( dashed red box ) . ( Bottom ) Longitudinal 2D slice of STA centered on pinhead ( left ) or A - C

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (Top) 2D slice through STA transverse view of microtubule triplet in T. mirabilis, with insets showing pinhead (dashed green box) and A-C linker (dashed red box). (Bottom) Longitudinal 2D slice of STA centered on the pinhead (left) or A-C linker


( E ) Purified CrSAS - 6 [ NL ] self - organized into stacks analyzed by cryo - ET ; one longitudinal one transverse view are highlighted by a dashed rectangle a dashed circle , respectively . ( F , G ) 2D views through STA of in vitro self - assembled CrSAS - 6 [ NL ] . Transverse sections at indicated heights through one assembly unit ( F ) , as well as longitudinal view ( G ) of delineated by a dashed box in ( F ) ; arrowhead denotes position of scan along vertical axis at level of hub , with corresponding pixel intensities in arbitrary units ( AU ) . average between two hub elements is 4 . 5 ± 0 . 3 nm ( N = 7 ; dashed lines ) . ( H ) Longitudinal view of in vitro self - assembled CrSAS - 6 [ NL ] STA 3D map . Note densities bridging successive hubs vertically ( arrows ) . ( I ) Two CrSAS - 6 [ HR ] single rings fitted into 3D map of in vitro self - assembled CrSAS - 6 [ NL ] . Dashed box indicates longitudinal section through hub element ( left ) , double chevron indicates viewing point of longitudinal external view ( right ) . Note that 3D map can accommodate rings stacked 4 . 4 nm apart ( measured on fitted ring models ) , that vertical densities linking hubs are partially occupied .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Purified CrSAS-6[NL] protein self-organized into stacks and analyzed by cryo-ET; one longitudinal and one transverse view are highlighted by a dashed rectangle and a dashed circle, respectively. (F, G) 2D views through STA of in vitro self-assembled CrSAS-6[NL] proteins. Transverse sections at the indicated heights through one assembly unit (F), as well as longitudinal view (G) of the area delineated by a dashed box in (F); white arrowhead denotes position of line scan along the vertical axis at the level of the hub, with corresponding pixel intensities in arbitrary units (AU). The average distance between two hub elements is 4.5 ± 0.3 nm (N=7; dashed lines). (H) Longitudinal view of in vitro self-assembled CrSAS-6[NL] proteins STA 3D map. Note densities bridging successive hubs vertically (arrows). (I) Two CrSAS-6[HR] single rings fitted into the 3D map of in vitro self-assembled CrSAS-6[NL] proteins. Dashed box indicates longitudinal section through hub element (left), double chevron indicates viewing point of longitudinal external view (right). Note that the 3D map can accommodate rings stacked 4.4 nm apart (measured on fitted ring models), and that vertical densities linking hubs are partially occupied.


Green : Normalized population expression level averaged over OxyR , SoxS - regulated promoters ; red : average over LexA - regulated promoters as measured with a promoter - GFP plasmid library in a plate reader . Oxidative stress promoters clearly precede SOS response promoters in response to NIT stress , when measured at population level Error bars show standard deviation over detected oxidative stress SOS promoters , respectively It is not clear if this temporal order correctly reflects temporal order in single . B . Schematic showing response of two different genes ( green red ) in three different ( solid , dashed , dotted ) . temporal order observed at population level correctly reflects temporal order in each . C . As B . Here temporal order at population level is not same in every single : Although in most , green gene responds before red one , one expresses green gene before red one ( dashed lines ) .

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

Green line: Normalized population expression level averaged over all OxyR, and SoxS-regulated promoters; red line: average over all LexA-regulated promoters as measured with a promoter-GFP plasmid library in a plate reader. Oxidative stress promoters clearly precede SOS response promoters in response to NIT stress, when measured at the population level Error bars show standard deviation over all detected oxidative stress and SOS promoters, respectively It is not clear if this temporal order correctly reflects the temporal order in single cells. B. Schematic showing response of two different genes (green and red) in three different cells (solid, dashed, dotted line). The temporal order observed at the population level correctly reflects the temporal order in each individual cell. C. As B. Here the temporal order at the population level is not the same in every single cell: Although in most cells, the green gene responds before the red one, one cell expresses the green gene before the red one (dashed lines).


Single - gene expression time traces from dnaK promoter in response to TET stress , normalized to median full response . Response times were determined as time point at which a threshold expression level was reached This threshold was low enough so that most exceed it high enough to avoid false positives due to low signal - to - noise at time point zero . Brown : median of . Time traces are from one microcolony . B . Histogram of response times for dnaK promoter with mean µt = 1 . 87 h standard deviation σt = 0 . 37 h , fit of an Erlang distribution with shape parameter n = 37 Response times are from two microcolonies .

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

Single-cell gene expression time traces from the dnaK promoter in response to TET stress, normalized to the median full response. Response times were determined as the time point at which a threshold expression level was reached This threshold was low enough so that most cells exceed it and high enough to avoid false positives due to the low signal-to-noise ratio at time point zero. Brown line: median of all cells. Time traces are from one microcolony. B. Histogram of the response times for the dnaK promoter with mean µt = 1.87 h and standard deviation σt = 0.37 h, and fit of an Erlang distribution with shape parameter n = 37 Response times are from two microcolonies.


C . Standard deviation σt versus mean response time µt for 23 different promoters in three antibiotic stress conditions ( TMP , TET , NIT ) . standard deviation of response time σt grows with mean response time µt does not fall below a ' precision limit ' ( dashed ) that increases linearly with a slope ~ 0 . 165 . dotted indicates upper bound to timing variability where σt = µt , see text . promoter dnaK under TET has low timing variability , whereas promoters recA , fpr , osmC , wrbA under TMP stress have high timing variability . response time mean standard deviation are from subsampling of descendants of single that were present at time of stress addition Subsampling for each promoter was done from at least 2 microcolonies descendants of at least 17 present at time of stress addition .

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 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 B-CONTROLLED_VAR 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 O O O

C. Standard deviation σt versus mean response time µt for 23 different promoters in three antibiotic stress conditions (TMP, TET, and NIT). The standard deviation of the response time σt grows with the mean response time µt and does not fall below a 'precision limit' (dashed line) that increases linearly with a slope ~0.165. The dotted line indicates the upper bound to timing variability where σt = µt, see text. The promoter dnaK under TET has low timing variability, whereas the promoters recA, fpr, osmC, and wrbA under TMP stress have high timing variability. The response time mean and standard deviation are from subsampling of descendants of single cells that were present at the time of stress addition Subsampling for each promoter was done from at least 2 microcolonies and the descendants of at least 17 individual cells present at the time of stress addition.


E . Standard deviation σt versus mean response time µt for LlacO - 1 promoter induced with different IPTG concentrations .

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

E. Standard deviation σt versus mean response time µt for the LlacO-1 promoter induced with different IPTG concentrations.


D . Response times of LlacO - 1 promoter ( Lutz & Bujard , 1997 ) , induced with 1 mM IPTG at time zero , measured with YFP CFP constructs in same ( r = 0 . 81 ± 0 . 04 , p = 6 · 10 - 12 ) .

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

D. Response times of the LlacO-1 promoter (Lutz & Bujard, 1997), induced with 1 mM IPTG at time zero, measured with YFP and CFP constructs in the same cell (r = 0.81 ± 0.04, p = 6 · 10-12).


A . Normalized expression from promoters ybjC recA in response to NIT stress , measured at population level using GFP reporters 4 μg / mL NIT was added at time zero . oxidative stress promoter ybjC clearly precedes SOS response promoter recA , when measured at population level Error bars show standard deviation of three replicate experiments measured on different days . B . Normalized ybjC recA expression over time in response to addition of 4 μg / mL NIT at time zero in single from one microcolony . Dashed : threshold used to determine response times Lower panels , upper row : Dual - color of oxidative stress reporter ybjC ( green ) SOS reporter recA ( red ) in single at 4 different time points after NIT addition in one microcolony . Note that ybjC signal ( green ) is stronger at t = 2h , whereas recA signal ( red ) is only apparent at t = 4h 6h . Lower row : Constitutively expressed mCherry used for segmentation . C . Response times for ybjC recA from with dual reporters ( Figure 3 ) , combined from two microcolonies . response times for recA are higher than for ybjC response times for both reporters are strongly correlated ( r = 0 . 74 ± 0 . 04 , p = 3 . 8 · 10 - 6 ) . error of correlation coefficient is from subsampling of descendants of single that were present at time of NIT

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

A. Normalized expression from the promoters ybjC and recA in response to NIT stress, measured at the population level using GFP reporters 4 μg/mL NIT was added at time zero. The oxidative stress promoter ybjC clearly precedes the SOS response promoter recA, when measured at the population level Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized ybjC and recA expression over time in response to the addition of 4 μg/mL NIT at time zero in single cells from one microcolony. Dashed line: threshold used to determine response times Lower panels, upper row: Dual-color images of the oxidative stress reporter ybjC (green) and the SOS reporter recA (red) in single cells at 4 different time points after NIT addition in one microcolony. Note that the ybjC signal (green) is stronger at t = 2h, whereas the recA signal (red) is only apparent at t = 4h and 6h. Lower row: Constitutively expressed mCherry used for segmentation. C. Response times for ybjC and recA from individual cells with dual reporters (Figure 3), combined from two microcolonies. All response times for recA are higher than for ybjC and response times for both reporters are strongly correlated (r = 0.74 ± 0.04, p = 3.8 · 10-6). The error of the correlation coefficient is from subsampling of descendants of single cells that were present at the time of NIT addition


A . Expression from gadB recA promoters , measured at population level using GFP reporters over time ; 0 . 5 μg / mL TMP was added at time zero . acid stress response ( gadB ) clearly precedes SOS response ( recA ) . Error bars show standard deviation of three replicate experiments measured on different days . B . Normalized expression of gadB recA expression over time in responding to addition of 0 . 5 μg / mL TMP at time zero shown for one microcolony . Dashed : threshold used to determine response times ( Methods ) . Lower panels , upper row : Dual - color of gadB ( blue ) recA ( red ) in single at 4 different time points . Most are predominantly either red or blue , i . e . acid stress SOS response are typically not both strongly expressed in same . Lower row : Constitutively expressed mCherry used for segmentation . C . Scatter plot of response times for gadB recA in single ; there is no clear temporal order no significant correlation ( r = 0 . 08 ± 0 . 08 , p = 0 . 68 ) ; data is combined from three microcolonies . Error is from subsampling of descendants of single present at time point of TMP

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

A. Expression from the gadB and recA promoters, measured at the population level using GFP reporters over time; 0.5 μg/mL TMP was added at time zero. The acid stress response (gadB) clearly precedes the SOS response (recA). Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized expression of gadB and recA expression over time in individual cells responding to the addition of 0.5 μg/mL TMP at time zero shown for one microcolony. Dashed line: threshold used to determine response times (Methods). Lower panels, upper row: Dual-color images of gadB (blue) and recA (red) in single cells at 4 different time points. Most cells are predominantly either red or blue, i.e. acid stress and SOS response are typically not both strongly expressed in the same cells. Lower row: Constitutively expressed mCherry used for segmentation. C. Scatter plot of response times for gadB and recA in single cells; there is no clear temporal order and no significant correlation (r = 0.08 ± 0.08, p = 0.68); data is combined from three microcolonies. Error is from subsampling of descendants of single cells present at the time point of TMP addition


D . Expression of gadB recA in inosine - supplemented medium , averaged over 13 single in a microcolony . Insets : Dual color image of gadB recA segmentation image at t = 6h .

O O O O O O O O O O O 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. Expression of gadB and recA in inosine-supplemented medium, averaged over 13 single cells in a microcolony. Insets: Dual color image of gadB and recA and segmentation image at t = 6h.


Figure 1 : Endothelial Ang - 2 upregulation correlates with WHO grading in human gliomas . ELISA displaying human Ang - 2 ( A ) Ang - 1 ( B ) level in serum of healthy ( Ang - 2 n = 4 ; Ang - 1 n = 21 ) or with low grade glioma ( WHOII ) ( n = 5 ) , anaplastic astrocytoma ( WHOIII ) ( n = 7 ) or glioblastoma ( WHOIV ) ( n = 39 ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1: Endothelial Ang-2 upregulation correlates with WHO grading in human gliomas. ELISA displaying human Ang-2 (A) and Ang-1 (B) level in serum of healthy patients (Ang-2 n=4; Ang-1 n=21) or patients with low grade glioma (WHOII) (n=5), anaplastic astrocytoma (WHOIII) (n=7) or glioblastoma (WHOIV) (n=39) are shown.


Figure 1 : Endothelial Ang - 2 upregulation correlates with WHO grading in human gliomas . ELISA displaying human Ang - 2 ( A ) Ang - 1 ( B ) level in serum of healthy ( Ang - 2 n = 4 ; Ang - 1 n = 21 ) or with low grade glioma ( WHOII ) ( n = 5 ) , anaplastic astrocytoma ( WHOIII ) ( n = 7 ) or glioblastoma ( WHOIV ) ( n = 39 ) are shown . TCGA database was accessed to obtain gene expression level for Ang - 1 , Ang - 2 , Tie2 Tie1 in GBM in comparison to normal brain ( C ) .

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

Figure 1: Endothelial Ang-2 upregulation correlates with WHO grading in human gliomas. ELISA displaying human Ang-2 (A) and Ang-1 (B) level in serum of healthy patients (Ang-2 n=4; Ang-1 n=21) or patients with low grade glioma (WHOII) (n=5), anaplastic astrocytoma (WHOIII) (n=7) or glioblastoma (WHOIV) (n=39) are shown. The TCGA database was accessed to obtain gene expression level for Ang-1, Ang-2, Tie2 and Tie1 in GBM in comparison to normal brain (C).


Ang - 2 expression quantitative of Ang - 2 in healthy human brain ( n = 3 ) , low grade diffuse glioma ( n = 14 ) , anaplastic astrocytoma ( n = 12 ) or glioblastoma ( n = 11 ) is shown ( 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 O O O O O O O O O O O O O O

Ang-2 expression and quantitative analysis of Ang-2 in healthy human brain (n=3), low grade diffuse glioma (n=14), anaplastic astrocytoma (n=12) or glioblastoma (n=11) is shown (D).


Co - of Ang - 2 vWF ( endothelial ) , αSMA ( mural ) IBA1 ( microglia ) in different glioblastoma specimen . Normal brain tissue was used to assess Ang - 2 specificity ( E ) .

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Co-staining of Ang-2 and vWF (endothelial cells), αSMA (mural cells) and IBA1 (microglia) in different glioblastoma specimen. Normal brain tissue was used to assess Ang-2 staining specificity (E).


Ang - 2 predicts survival of glioblastoma ( F ) . If not indicated differently , Kruskall - Wallis ( Dunn ´ s post ) was applied , * * p < 0 . 05 , * * * p < 0 . 005 ; data are mean ±

Ang-2 predicts survival of glioblastoma patients (F). If not indicated differently, Kruskall-Wallis (Dunn´s post test) was applied, ** p < 0.05, *** p < 0.005; data are mean ± SEM


Figure 2 : Endothelial Ang - 2 expression reduced pericyte coverage increased macrophage infiltration in experimental glioblastoma . GL261 were intracerebrally transplanted in wild type or Ang - 2 gain - of - function mice ( Ang - 2 DT ) . Brain sections were stained with antibodies against CD31 desmin ( A ) , microvessel densities ( MVD ) pericyte coverage were determined ( WT n = 14 ; Ang - 2 DT n = 6 ) ( B ) .

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Figure 2: Endothelial Ang-2 expression reduced pericyte coverage and increased macrophage infiltration in experimental glioblastoma. GL261 cells were intracerebrally transplanted in wild type or Ang-2 gain-of-function mice (Ang-2 DT). Brain tumor sections were stained with antibodies against CD31 and desmin (A), and microvessel densities (MVD) and pericyte coverage were determined (WT n=14; Ang-2 DT n=6) (B).


FACS of infiltrating macrophages in Ang - 2 DT normal brain ( w / o ) compared to WT is shown ( C , dot plot ; D , ) ( WT n = 5 ; Ang - 2 DT n = 3 ) .

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FACS analysis of infiltrating macrophages in Ang-2 DT normal brain (w/o tumor) compared to WT is shown (C, dot plot; D, quantification) (WT n=5; Ang-2 DT n=3).


) . Immunohistochemistry for monocytes / macrophages ( F4 / 80 ) neutrophils ( Ly6G ; arrowheads ) in brain tumors derived from WT or Ang - 2 overexpressing mice ( E ) , Quantitative of C ( F ) ( WT n = 14 ; Ang - 2 DT n = 10 )

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

). Immunohistochemistry staining for monocytes/macrophages (F4/80) and neutrophils (Ly6G; arrowheads) in brain tumors derived from WT or Ang-2 overexpressing mice (E), Quantitative analysis of C (F) (WT n=14; Ang-2 DT n=10)


Kaplan - Meier survival curve of GL261 - bearing WT Ang - 2 DT mice ( WT n = 17 ; Ang - 2 DT n = 14 ) ( E ) . Statistical analyses were performed using an unpaired Student ´ s t - ( B , F ) , one - way Anova ( Tukey post ) ( D ) , Log - rank Wilcoxon ( G ) * p < 0 . 05 , * * p < 0 . 01 ; data are mean ±

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Kaplan-Meier survival curve of GL261 tumor-bearing WT and Ang-2 DT mice (WT n=17; Ang-2 DT n=14) (E). Statistical analyses were performed using an unpaired Student´s t-test (B, F), one-way Anova (Tukey post test) (D), Log-rank and Wilcoxon (G) * p < 0.05, ** p < 0.01; data are mean ± SEM


Figure 3 : Infiltrating leukocyte subsets in human glioma . Paraffin sections of healthy human brain ( n = 3 ) , low grade glioma ( n = 12 ) , anaplastic astrocytoma ( n = 7 ) glioblastoma ( n = 9 ) were stained for IBA1 ( macrophages / microglia ) , CD15 ( granulocytes ) CD3 ( T - ) ( A ) . of leukocyte subsets in human glioma specimens ( B ) . For statistical Kruskall - Wallis ( Dunn ´ s post ) was applied . * p < 0 . 05 , * * p < 0 . 01 ; data are mean ±

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

Figure 3: Infiltrating leukocyte subsets in human glioma. Paraffin sections of healthy human brain (n=3), low grade glioma (n=12), anaplastic astrocytoma (n=7) and glioblastoma (n=9) were stained for IBA1 (macrophages/microglia), CD15 (granulocytes) and CD3 (T-cells) (A). Quantification of leukocyte subsets in human glioma specimens (B). For statistical analysis Kruskall-Wallis (Dunn´s post test) was applied.* p < 0.05, ** p < 0.01; data are mean ± SEM


Figure 4 : Dual anti - Ang - 2 anti - VEGF therapy acts synergistic on vascular normalization macrophage infiltration in experimental GBM . Immunofluorescence of CD31 desmin in GL261 glioblastoma after single dual treatment with anti - Ang - 2 ( AMG386 ) anti - VEGF ( aflibercept ) ( A ) . Corresponding quantitative of microvessel densities pericytes numbers ( B ; control n = 24 ; AMG386 n = 10 ; aflibercept n = 12 ; AMG386 + aflibercept n = 13 ) .

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

Figure 4: Dual anti-Ang-2 and anti-VEGF therapy acts synergistic on vascular normalization and macrophage infiltration in experimental GBM. Immunofluorescence staining of CD31 and desmin in GL261 glioblastoma after single and dual treatment with anti-Ang-2 (AMG386) and anti-VEGF (aflibercept) (A). Corresponding quantitative analysis of microvessel densities and pericytes numbers (B; control n=24; AMG386 n=10; aflibercept n=12; AMG386 + aflibercept n=13).


. for mouse IgG ( red ) as a surrogate parameter for vascular leakage CD31 ( green ) is shown in GL261 glioblastoma sections following different anti - angiogenic treatments ( C ) with in ( D ; n = 3 ) .

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. Staining for mouse IgG (red) as a surrogate parameter for vascular leakage and CD31 (green) is shown in GL261 glioblastoma sections following different anti-angiogenic treatments (C) with quantification in (D; n=3).


of necrotic areas in GL261 glioblastoma sections after anti - angiogenic treatment is shown as of whole ( E ; control , aflibercept , AMG386 + aflibercept n = 4 ; AMG386 n = 3 ) .

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

Quantification of necrotic areas in GL261 glioblastoma sections after anti-angiogenic treatment is shown as percent of whole tumor area (E; control, aflibercept, AMG386 + aflibercept n=4; AMG386 n=3).


of Glut1 immunoreactivity in GL261 glioblastoma sections ( F ; n = 3 ) .

Analysis of Glut1 immunoreactivity in GL261 glioblastoma sections (F; n=3).


Double - immunofluorescence stainings of macrophages ( F4 / 80 ) vessels ( vWF ) in mouse GBM after treatment with AMG386 , aflibercept or combination of both are shown in ( G )

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

Double-immunofluorescence stainings of macrophages (F4/80) and tumor vessels (vWF) in mouse GBM after treatment with AMG386, aflibercept or the combination of both are shown in (G)


Quantitatve of infiltrating leukocytes ( F4 / 80 + , CD3 + , Ly - 6G + ) following anti - angiogenic treatment is displayed in ( H ; control n = 28 ; AMG386 n = 12 ; aflibercept n = 11 ; AMG386 + aflibercept n = 12 ) .

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

Quantitatve analysis of tumor infiltrating leukocytes (F4/80+, CD3+, Ly-6G+) following anti-angiogenic treatment is displayed in (H; control n=28; AMG386 n=12; aflibercept n=11; AMG386 + aflibercept n=12).


Kaplan - Meier survival of GL261 tumors grown in C57BL / 6 mice following anti - angiogenic treatment ( I ; control n = 38 ; AMG386 n = 13 ; aflibercept n = 12 ; AMG386 + aflibercept n = 13 ) .

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

Kaplan-Meier survival analysis of GL261 tumors grown in C57BL/6 mice following anti-angiogenic treatment (I; control n=38; AMG386 n=13; aflibercept n=12; AMG386 + aflibercept n=13).


Double - immunofluorescence stainings with anti - F4 / 80 anti - CD206 in brain sections of mice treated with anti - Ang - 2 ( AMG386 ) , anti - VEGF ( aflibercept ) or combination of both are shown in ( J ) . Corresponding quantitative analyses of infiltrating F4 / 80 + CD206 + , of CD206 + vs . F4 / 80 + upon anti - angiogenic therapy are displayed in ( K ; control n = 21 ; AMG386 n = 13 ; aflibercept n = 9 ; AMG386 + aflibercept n = 4 ) .

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

Double-immunofluorescence stainings with anti-F4/80 and anti-CD206 in brain tumor sections of mice treated with anti-Ang-2 (AMG386), anti-VEGF (aflibercept) or the combination of both are shown in (J). Corresponding quantitative analyses of tumor infiltrating F4/80+ cells and CD206+ cells, and the ratio of CD206+ vs. F4/80+ upon anti-angiogenic therapy are displayed in (K; control n=21; AMG386 n=13; aflibercept n=9; AMG386 + aflibercept n=4).


Flow cytometry of infiltrating macrophages following enzymatic dissociation of mouse GL261 brain tumors plus / minus anti - angiogenic therapy . of CD206 + ( L ) MHC class IIHI ( M ) among CD45 + CD11b + GR1 - F4 / 80 + are displayed ( L , M ; control n = 4 ; AMG386 n = 4 ; aflibercept n = 5 ; AMG386 + aflibercept n = 5 ) . Statistical analyses were performed using one - way ANOVA Tukey ' s multiple comparison except for Log - rank Wilcoxon ( I ) Kruskall Wallis ( B ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , # p < 0 . 005 of aflibercept + AMG386 vs aflibercept ; data are mean ± ( A - K )

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

Flow cytometry of tumor infiltrating macrophages following enzymatic dissociation of mouse GL261 brain tumors plus/minus anti-angiogenic therapy. Percent of CD206+ (L) and MHC class IIHI cells (M) among CD45+CD11b+GR1-F4/80+ cells are displayed (L,M; control n=4; AMG386 n=4; aflibercept n=5; AMG386 + aflibercept n=5). Statistical analyses were performed using one-way ANOVA and Tukey's multiple comparison except for Log-rank and Wilcoxon (I) and Kruskall Wallis (B). * p < 0.05, ** p < 0.01, *** p < 0.005, # p < 0.005 of aflibercept + AMG386 vs aflibercept; data are mean ± SEM (A-K)


Figure 5 : Anti - VEGF therapy led to decreased infiltration of CD68 + macrophages in human GBM . Anti - CD68 ( A ) , anti - CSFR1 ( B ) anti - CD206 ( C ) immunohistochemistry of samples derived from treatment - naive GBM ( n = 24 ) , post - radiochemotherapy ( S / RTx / CTx ) ( n = 7 ) post - radiochemotherapy + bevacizumab ( S / RTx / CTx / Bev ) ( n = 29 ) therapy is 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 O O O O O O O O O O O O O O O O O O O O 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

Figure 5: Anti-VEGF therapy led to decreased infiltration of CD68+ macrophages in human GBM. Anti-CD68 (A), anti-CSFR1 (B) and anti-CD206 (C) immunohistochemistry of patient samples derived from treatment-naive GBM (n=24), post-radiochemotherapy (S/RTx/CTx) (n=7) and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) (n=29) therapy is shown.


Corresponding of infiltrating is displayed ( anti - CD68 , D ) , ( anti - CSF1R , E ) ( anti - CD206 , F ) . of CD206 + vs . CD68 + ( G ) .

Corresponding quantification of tumor infiltrating cells is displayed (anti-CD68, D), (anti-CSF1R, E) and (anti-CD206, F). Ratio of CD206+ vs. CD68+ cells (G).


Amount of CD206 + perivascular per blood vessel ( H )

Amount of CD206+ perivascular cells per blood vessel (H)


Double of Ang - 2 ( brown ) CD206 ( red ) of a S / RTx / CTx / Bev , arrowheads indicating perivascular CD206 + around Ang - 2 + vessels ( I ) . Arrows pointing on parenchymal CD206 + ( I ) .

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

Double staining of Ang-2 (brown) and CD206 (red) of a S / RTx / CTx / Bev patient, arrowheads indicating perivascular CD206+ cells around Ang-2+ vessels (I). Arrows pointing on parenchymal CD206+ cells (I).


) . CD206 + in relation to of hypoxic ( CAIX positive ) ( J ) . Statistical : Kruskall - Wallis ( Dunn ´ s post ) . * p < 0 . 05 , * * p < 0 . 01 ; data are mean ±

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

). CD206+ cells in relation to the percentage of hypoxic (CAIX positive) tumor area (J). Statistical analysis: Kruskall-Wallis (Dunn´s post test). * p < 0.05, ** p < 0.01; data are mean ± SEM


Figure 6 : Bevacizumab therapy leads to reduced vessel density increased Ang - 2 expression . Immunohistochemistry stainings with antibodies directed against CD31 ( A ) Ang - 2 ( B ) in treatment - naive GBM , post - radiochemotherapy ( S / RTx / CTx ) post - radiochemotherapy + bevacizumab ( S / RTx / CTx / Bev ) therapy are displayed .

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

Figure 6: Bevacizumab therapy leads to reduced vessel density and increased Ang-2 expression. Immunohistochemistry stainings with antibodies directed against CD31 (A) and Ang-2 (B) in treatment-naive GBM, post-radiochemotherapy (S/RTx/CTx) and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) therapy are displayed.


Quantitative analyses of microvessel densities Ang - 2 + vessels are shown in ( C ) ( D ) . of Ang - 2 + vs . CD31 + vessels was determined in ( E ) .

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

Quantitative analyses of microvessel densities and Ang-2+ vessels are shown in (C) and (D). The ratio of Ang-2+ vs. CD31+ vessels was determined in (E).


Western Blot corresponding of CD31 Ang - 2 in control aflibercept treated mice ( F ) .

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Western Blot and corresponding quantification of CD31 and Ang-2 in control and aflibercept treated mice (F).


CAIX of treatment - naive GBM S / RTx / CTx / Bev ( G ) . Analyses of hypoxic ( CAIX + ) with regard to whole in human GBM ( H ) . - infiltrating lymphocytes ( CD8 / CD3 ) in human ( I ) . Kruskall - Wallis ( Dunn ´ s post ) . * p < 0 . 05 , * * * p < 0 . 005 ; treatment - naive GBM n = 26 , S / RTx / CTx n = 7 , S / RTx / CTx / Bev n = 29 ; data are mean ±

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

CAIX staining of treatment-naive GBM and S / RTx / CTx / Bev patients (G). Analyses of hypoxic (CAIX+) area with regard to the whole tumor in human GBM (H). Tumor-infiltrating lymphocytes (CD8/CD3) in human patients (I). Kruskall-Wallis (Dunn´s post test). * p < 0.05, *** p < 0.005; treatment-naive GBM n=26, S/RTx/CTx n=7, S/RTx/CTx/Bev n=29; data are mean ± SEM


Whole - mount view of E14 . 5 - 15 . 5 hearts from Npr3 - sCreER ; R26 - tdTomato Npr3 - CreER ; R26 - tdTomato mice . Immunostaining for tdTomato , ESR CDH5 on heart sections . C . M . , compact myocardium ; T . M . , trabecular myocardium . Arrowheads , tdTomato + ESR - endocardial .

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

Whole-mount fluorescence view of E14.5-15.5 hearts from Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice. Immunostaining for tdTomato, ESR and CDH5 on heart sections. C.M., compact myocardium; T.M., trabecular myocardium. Arrowheads, tdTomato+ESR- endocardial cells.


of of tdTomato + or ESR + endothelial . Data are mean ± s . e . m . ; n = 5 .

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

Quantification of the percentage of tdTomato+ or ESR+ endothelial cells. Data are mean ± s.e.m.; n = 5.


Isolation of tdTomato - or tdTomato + from E14 . 5 Npr3 - sCreER ; R26 - tdTomato Npr3 - CreER ; R26 - tdTomato mice by FACS .

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Isolation of tdTomato- or tdTomato+ cells from E14.5 Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice by FACS.


qRT - PCR of Cre , ESR , Npr3 Cdh5 from tdTomato + . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 .

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

qRT-PCR of Cre, ESR, Npr3 and Cdh5 from tdTomato+ cells. Data are mean ± s.e.m.; n = 5; *P < 0.05.


Western blotting of ESR GAPDH in tdTomato - tdTomato + . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 ; n . s . , non - significant . Scale bars , 500 µm . Each figure is representative of 5 biological 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

Western blotting of ESR and GAPDH in tdTomato- and tdTomato+ cells. Data are mean ± s.e.m.; n = 5; *P < 0.05; n.s., non-significant. Scale bars, 500 µm. Each figure is representative of 5 individual biological samples.


Whole - mount of E14 . 5 Npr3 - CreER ; R26 - Confetti Npr3 - sCreER ; R26 - Confetti mouse hearts . No Tam is used as control for leakiness of Npr3 - sCreER .

O 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

Whole-mount fluorescence images of E14.5 Npr3-CreER;R26-Confetti and Npr3-sCreER;R26-Confetti mouse hearts. No Tam is used as control for leakiness of Npr3-sCreER.