G . Number of molecules obtained by normalizing background - subtracted fluorescence intensities of individual transfected cells against the mean background intensity of untransfected cells , yielding relative expressions levels independent on the fluorophore ( methods ) . This value was then set into proportion to the known mean number of EGFR per MCF10A cell to yield absolute molecule count / cell . 1st column : EGFR - mCitrine in EmCit _ MCF7 cells ( N = 3 biological replicates , n = 102 cells ) ; 2nd column EGFR - mCitrine expressed in EmCit _ MCF7 RPTPγ - KO expressing RPTPγ - mTFP ( N = 3 biological replicates , n = 26 cells ) ; 3rd column : RPTPγ - mCitrine in MCF7 cells expressing additionally EGFR - mCherry ( N = 3 biological replicates , n = 253 cells ) ; 4th column : RPTPγ - mTFP expressed in EmCit _ MCF7 RPTPγ - KO ( N = 3 biological replicates , n = 26 cells ) ( individual cells with mean + SD ) . Color code in 2nd and 4th column is attribution to respective cluster ( H and EV2G ) .

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

G. Number of molecules obtained by normalizing background-subtracted fluorescence intensities of individual transfected cells against the mean background intensity of untransfected cells, yielding relative expressions levels independent on the fluorophore (methods). This value was then set into proportion to the known mean number of EGFR per MCF10A cell to yield absolute molecule count/cell. 1st column: EGFR-mCitrine in EmCit_MCF7 cells (N=3 biological replicates, n=102 cells); 2nd column EGFR-mCitrine expressed in EmCit_MCF7 RPTPγ-KO expressing RPTPγ-mTFP (N=3 biological replicates, n=26 cells); 3rd column: RPTPγ-mCitrine in MCF7 cells expressing additionally EGFR-mCherry (N=3 biological replicates, n=253 cells); 4th column: RPTPγ-mTFP expressed in EmCit_MCF7 RPTPγ-KO (N=3 biological replicates, n=26 cells) (individual cells with mean+SD). Color code in 2nd and 4th column is attribution to respective cluster (H and EV2G).


C - E ( C ) in cell EGF - dose response imaging for RPTPγ - mCitrine oxidation . Left panel : Representative confocal micrographs of RPTPγ - mCitrine in EmTFP _ MCF7 cells ( top row ) together with its oxidized fraction estimated using DyTo - FLIM ( αox , bottom row ) , upon 10 ' stimulation with EGF - Alexa647 ( 0 - 160 ng / ml ) including 5 ' together with 0 . 5 mM DyTo . Scale bar : 10 μm . Right panel : Quantification depicting the PM - proximal ( orange ) and PM - distal ( blue ) oxidized fractions as functions of receptor occupancy ( αL ) and corresponding EGF - Alexa647 , or H2O2 concentration in EmTFP MCF7 cells expressing RPTPγ - mCitrine ( WT ) or RPTPγC1060S - mCitrine ( C1060S ) as well as WT cells treated with 0 . 5 mM atto590 instead of DyTo ( atto590 ) . Mean of individual cells ( symbols ) with mean ± SD ( black lines ) , N = 3 biological replicates , n = 13 - 15 cells per EGF dose . P : unpaired two - tailed t - test , between PM ( serpentine peripheral structures ) and endosomal ( vesicular structures ) fractions . ( D ) Same as in ( C ) , for RPTPγ - mCitrine oxidation in p22phox - KO cells . N = 3 biological replicates , n = 14 - 26 cells per EGF dose . ( E ) Same as in ( C ) , for TCPTP - mCitrine or TCPTPC216S - mCitrine ( C216S ) oxidation in EmTFP _ MCF7 cells . N = 3 biological replicates , n = 18 - 21 cells per EGF dose .

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C-E (C) in cell EGF-dose response imaging for RPTPγ-mCitrine oxidation. Left panel: Representative confocal micrographs of RPTPγ-mCitrine in EmTFP_MCF7 cells (top row) together with its oxidized fraction estimated using DyTo-FLIM (αox, bottom row), upon 10' stimulation with EGF-Alexa647 (0-160 ng/ml) including 5' together with 0.5 mM DyTo. Scale bar: 10 μm. Right panel: Quantification depicting the PM-proximal (orange) and PM-distal (blue) oxidized fractions as functions of receptor occupancy (αL) and corresponding EGF-Alexa647, or H2O2 concentration in EmTFP MCF7 cells expressing RPTPγ-mCitrine (WT) or RPTPγC1060S-mCitrine (C1060S) as well as WT cells treated with 0.5 mM atto590 instead of DyTo (atto590). Mean of individual cells (symbols) with mean±SD (black lines), N=3 biological replicates, n=13-15 cells per EGF dose. P: unpaired two-tailed t-test, between PM (serpentine peripheral structures) and endosomal (vesicular structures) fractions. (D) Same as in (C), for RPTPγ-mCitrine oxidation in p22phox-KO cells. N=3 biological replicates, n=14-26 cells per EGF dose. (E) Same as in (C), for TCPTP-mCitrine or TCPTPC216S-mCitrine (C216S) oxidation in EmTFP_MCF7 cells. N=3 biological replicates, n=18-21 cells per EGF dose.


A . Representative confocal micrographs of MCF7 WT cells showing the co - localization of RPTPγ - mCitrine ( 1st column : green ; 3rd column : blue ) and EGFR - mCherry ( 2nd column : green ; 3rd column : yellow ) with recycling - endosome defined by immunostaining against Rab11a ( magenta ) , without ( top row ) or after 30 ' EGF - DyLight405 stimulus ( 160 ng / ml ; bottom row ) . Scale bar : 10 μm .

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A. Representative confocal micrographs of MCF7 WT cells showing the co-localization of RPTPγ-mCitrine (1st column: green; 3rd column: blue) and EGFR-mCherry (2nd column: green; 3rd column: yellow) with recycling-endosome defined by immunostaining against Rab11a (magenta), without (top row) or after 30' EGF-DyLight405 stimulus (160 ng/ml; bottom row). Scale bar: 10 μm.


B . Fraction of RPTPγ - mCitrine ( cyan ) or EGFR - mCherry ( green ) that spatially overlaps with Rab11a ( top left , N = 3 , n = 23 - 26 cells per timepoint ) , PM ( bottom left , N = 3 biological replicates , n = 15 - 17 cells ) , EEA1 - positive early endosomes ( top right , N = 3 biological replicates , n = 25 - 28 cells ) or Rab7 - positive late endosomes ( bottom right , N = 3 biological replicates , n = 23 - 27 cells ) in MCF7 cells as function of time after 160 ng / ml EGF - stimulus . Orange symbols / dotted line : same for EGFR - mCitrine in RPTPγ - KO cells ( N = 3 biological replicates , n = 17 - 21 cells ) . P : unpaired two - tailed t - test ; colored P values compare to the respective species before stimulation , black in between species .

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B. Fraction of RPTPγ-mCitrine (cyan) or EGFR-mCherry (green) that spatially overlaps with Rab11a (top left, N=3, n=23-26 cells per timepoint), PM (bottom left, N=3 biological replicates, n=15-17 cells), EEA1-positive early endosomes (top right, N=3 biological replicates, n=25-28 cells) or Rab7-positive late endosomes (bottom right, N=3 biological replicates, n=23-27 cells) in MCF7 cells as function of time after 160 ng/ml EGF-stimulus. Orange symbols/dotted line: same for EGFR-mCitrine in RPTPγ-KO cells (N=3 biological replicates, n=17-21 cells). P: unpaired two-tailed t-test; colored P values compare to the respective species before stimulation, black in between species.


C . Left : Representative confocal micrographs of MCF7 cells depicting the steady state localization of expressed RPTPγ - mCitrine ( cyan ) , without ( top ) or with co - expression of BFP - Rab11a ( yellow , bottom ) . Top right : Quantification of PM - localized fraction of RPTPγ - mCitrine without ( WT ) and with co - expression of BFP - Rab11a . Bottom right : Fraction of RPTPγ - mCitrine localized to the PM in individual cells as a function of BFP - Rab11a expression level , measured by mean BFP - fluorescence intensity . Scale bar : 10 μm . N = 2 biological replicates , n > 40 per condition , mean ± SD , ; P : unpaired two - tailed t - test .

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C. Left: Representative confocal micrographs of MCF7 cells depicting the steady state localization of expressed RPTPγ-mCitrine (cyan), without (top) or with co-expression of BFP-Rab11a (yellow, bottom). Top right: Quantification of PM-localized fraction of RPTPγ-mCitrine without (WT) and with co-expression of BFP-Rab11a. Bottom right: Fraction of RPTPγ-mCitrine localized to the PM in individual cells as a function of BFP-Rab11a expression level, measured by mean BFP-fluorescence intensity. Scale bar: 10 μm. N=2 biological replicates, n>40 per condition, mean±SD,; P: unpaired two-tailed t-test.


E . Upper panel : Dual - color widefield images ( 1st column ) , SRRF reconstructions ( 2nd column ) with magnifications of boxed areas ( 3rd column ) of Alexa647 - SNAP - EGFR ( green ) and RPTPγ - mCitrine ( magenta ) of cryo - arrested MCF7 cells , unstimulated ( top row ) or stimulated with 100 ng / ml EGF ( bottom row ) for 15 ' . Scale bar : 10 μm . Lower panel : corresponding Manders colocalization coefficients for Alexa647 - Snap - EGFR / RPTPγ - mCitrine from SRRF reconstructions on intracellular compartments or PM area for unstimulated ( n = 12 - 18 ) and 15 ' EGF - stimulated ( n = 13 - 14 ) cells . mean ± SD , P : unpaired two - tailed t - test .

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E. Upper panel: Dual-color widefield images (1st column), SRRF reconstructions (2nd column) with magnifications of boxed areas (3rd column) of Alexa647-SNAP-EGFR (green) and RPTPγ-mCitrine (magenta) of cryo-arrested MCF7 cells, unstimulated (top row) or stimulated with 100 ng/ml EGF (bottom row) for 15'. Scale bar: 10 μm. Lower panel: corresponding Manders colocalization coefficients for Alexa647-Snap-EGFR/RPTPγ-mCitrine from SRRF reconstructions on intracellular compartments or PM area for unstimulated (n=12-18) and 15' EGF-stimulated (n=13-14) cells. mean±SD, P: unpaired two-tailed t-test.


F . Left : Representative IP - western blot showing co - IP of EGFR ( 2nd row ) upon RPTPγ - mCitrine ( 1st row : lanes 1 - 6 ) or RPTPγC1060S - mCitrine ( lane 7 ) pull - down by anti - GFP antibody from lysates of MCF7 cells co - transfected with EGFR and RPTPγ - mCitrine or RPTPγC1060S - mCitrine : without stimulus ( 0 ng / ml ) , upon 10 ' stimulus with EGF - Alexa647 ( 5 - 320 ng / ml , also displayed as corresponding receptor - occupancy αL , Fig 2D ) or 8 mM of H2O2 . 3rd and 4th row : total protein concentrations of RPTPγ - mCitrine and EGFR in the lysate measured by western blot as input control for the Co - IP . Right : corresponding ratiometric quantification of co - immunoprecipitated EGFR over pulled down RPTPg - mCitrine or RPTPγC1060S - mCitrine protein bands ( mean ± SD , N = 4 biological replicates , P : unpaired two - tailed t - test )

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F. Left: Representative IP-western blot showing co-IP of EGFR (2nd row) upon RPTPγ-mCitrine (1st row: lanes 1-6) or RPTPγC1060S-mCitrine (lane 7) pull-down by anti-GFP antibody from lysates of MCF7 cells co-transfected with EGFR and RPTPγ-mCitrine or RPTPγC1060S-mCitrine: without stimulus (0 ng/ml), upon 10' stimulus with EGF-Alexa647 (5-320 ng/ml, also displayed as corresponding receptor-occupancy αL, Fig 2D) or 8 mM of H2O2. 3rd and 4th row: total protein concentrations of RPTPγ-mCitrine and EGFR in the lysate measured by western blot as input control for the Co-IP. Right: corresponding ratiometric quantification of co-immunoprecipitated EGFR over pulled down RPTPg-mCitrine or RPTPγC1060S-mCitrine protein bands (mean±SD, N=4 biological replicates, P: unpaired two-tailed t-test)


. C . Catalytic ( ε1 − ε4 ) and autocatalytic ( α1 − α4 ) rate constants obtained from iterative global fitting the ODEs in ( B ) solved for steady state ( dEGFR p / T / dt = dEGF - EGFRp / T / dt = dRPTPγA / T / dt = 0 ) to EGF - dose response ( aL − aP ) data from all EGFR and PTP expression conditions ( Fig 2E , F , I ) . EGFRp , EGF - EGFRp : product of the corresponding

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. C. Catalytic (ε1 − ε4) and autocatalytic (α1 − α4) rate constants obtained from iterative global fitting the ODEs in (B) solved for steady state (dEGFR p/T/dt = dEGF-EGFRp/T/dt = dRPTPγA/T/dt = 0) to EGF-dose response (aL − aP) data from all EGFR and PTP expression conditions (Fig 2E, F, I). EGFRp, EGF-EGFRp: product of the corresponding reaction


. F . Change of the free parameter groups Γ1 = γ1RPTPγ / EGFRT and Β = β EGFRT / k1 in EmCit _ MCF7 RPTPγ - KO ( blue ) , EmCit _ MCF7 ( red ) , EmCit _ MCF7 RPTPγ - KO expressing RPTPγ - mTFP splitted in 3 clusters with increasing RPTPγ − mTFP / EGFR - mCitrine ratio ( yellow , green , purple ; Fig 2H , EV2G ) and WT MCF7 cells (

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. F. Change of the free parameter groups Γ1 = γ1RPTPγ/EGFRT and Β = β EGFRT/k1 in EmCit_MCF7 RPTPγ-KO (blue), EmCit_MCF7 (red), EmCit_MCF7 RPTPγ-KO expressing RPTPγ-mTFP splitted in 3 clusters with increasing RPTPγ−mTFP/EGFR-mCitrine ratio (yellow, green, purple; Fig 2H, EV2G) and WT MCF7 cells (black


C . Top : Representative Western blot against Erk and phosphorylated Erk ( pT202 and pY204 ) in WT ( red ) compared to p22phox - KO ( green ) MCF7 cells after the indicated times of sustained stimulation with 20 ng / ml EGF - Alexa647 . Bottom : Corresponding quantification of the fraction of phosphorylated ERK as a function of stimulation time . Mean ± SD , N = 4 biological replicates , P : unpaired two - tailed t - test .

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C. Top: Representative Western blot against Erk and phosphorylated Erk (pT202 and pY204) in WT (red) compared to p22phox-KO (green) MCF7 cells after the indicated times of sustained stimulation with 20 ng/ml EGF-Alexa647. Bottom: Corresponding quantification of the fraction of phosphorylated ERK as a function of stimulation time. Mean±SD, N=4 biological replicates, P: unpaired two-tailed t-test.


D . Quantification of cell proliferation using retinoblastoma ( Rb ) protein phosphorylation detected by immunofluorescence , for WT ( red ) , RPTPγ - KO ( blue ) and p22phox - KO ( green ) MCF7 cells without or post 24 h of EGF - Alexa647 treatment ( 1 , 20 , 160 ng / ml ) . Mean ± SEM , N = 3 biological replicates , n > 2000 cells per EGF stimulus per cell line , P : two - way ANOVA with Tukey multiple comparisons .

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D. Quantification of cell proliferation using retinoblastoma (Rb) protein phosphorylation detected by immunofluorescence, for WT (red), RPTPγ-KO (blue) and p22phox-KO (green) MCF7 cells without or post 24 h of EGF-Alexa647 treatment (1, 20, 160 ng/ml). Mean±SEM, N=3 biological replicates, n>2000 cells per EGF stimulus per cell line, P: two-way ANOVA with Tukey multiple comparisons.


E . Quantification of the culture - well area ( % ) occupied by proliferating cell - colonies , obtained from clonogenic assays of WT , RPTPγ - KO and p22phox - KO MCF7 cells , plated either in medium containing 20 ng / ml EGF and 0 . 5 % FCS ( left : orange bars , N = 3 - 4 biological replicates , 11 - 12 wells each ) or complete serum growth medium containing 10 % FCS ( right : pink bars , N = 4 biological replicates , 12 wells each ) . Mean ± SEM , P : unpaired two - tailed t - test with Welch ' s correction .

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E. Quantification of the culture-well area (%) occupied by proliferating cell-colonies, obtained from clonogenic assays of WT, RPTPγ-KO and p22phox-KO MCF7 cells, plated either in medium containing 20 ng/ml EGF and 0.5% FCS (left: orange bars, N=3-4 biological replicates, 11-12 wells each) or complete serum growth medium containing 10% FCS (right: pink bars, N=4 biological replicates, 12 wells each). Mean±SEM, P: unpaired two-tailed t-test with Welch's correction.


( a ) Relative quantification ( RQ ) of CG7630 transcript after RNAi with a 120 - bp inverted - repeat sequence ( act5c - gal4 > UAS - CG7630RNAi ) compared to control ( act5c - gal4 > + ) .

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(a) Relative quantification (RQ) of CG7630 transcript after RNAi with a 120-bp inverted-repeat sequence (act5c-gal4>UAS-CG7630RNAi) compared to control (act5c-gal4>+).


( b ) Relative percentage of egg to adult viability of CG7630 RNAi cross ( act5c - gal4 / CyO . GFP x UAS - CG7630RNAi ) and control cross ( act5c - gal4 / CyO . GFP x w1118 ) , calculated at three developmental stages ( eggs , pupae and adults ) , ( n > 250 , Chi - square test 9 . 920 df ( 2 ) , * * p = 0 . 007 ) .

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(b) Relative percentage of egg to adult viability of CG7630 RNAi cross (act5c-gal4/CyO.GFP x UAS-CG7630RNAi) and control cross (act5c-gal4/CyO.GFP x w1118), calculated at three developmental stages (eggs, pupae and adults), (n > 250, Chi-square test 9.920 df(2), **p = 0.007).


( d ) Kinetic enzyme activity of individual MRC complexes in CG7630 RNAi and control individuals normalized by citrate synthase ( CS ) activity .

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(d) Kinetic enzyme activity of individual MRC complexes in CG7630 RNAi and control individuals normalized by citrate synthase (CS) activity.


( e ) In gel - activity assays for MRC complexes II and IV in DDM - solubilized mitochondria from RNAi ( act5c - gal4 > UAS - CG7630RNAi ) and control larvae ( act5c - gal4 > + ) .

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(e) In gel-activity assays for MRC complexes II and IV in DDM-solubilized mitochondria from RNAi (act5c-gal4>UAS-CG7630RNAi) and control larvae (act5c-gal4>+).


( f ) BN - PAGE , Western blot immunodetection of MRC complexes from CG7630 RNAi ( act5c - gal4 > UAS - CG7630RNAi ) and control ( act5c - gal4 > + ) larvae using antibodies against specific subunits : anti - UQCRC2 ( complex III ) , anti - NDUFS3 ( complex I ) , anti - COX4 ( complex IV ) and anti - SDHA ( complex II ) .

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(f) BN-PAGE, Western blot immunodetection of MRC complexes from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae using antibodies against specific subunits: anti-UQCRC2 (complex III), anti-NDUFS3 (complex I), anti-COX4 (complex IV) and anti-SDHA (complex II).


( g ) Western blot analysis of steady state levels ( SDS - PAGE ) of MRC subunits COX4 ( complex IV ) , SDHA ( complex II ) and UQCR - C2 ( complex III ) in whole lysates from CG7630 RNAi ( act5c - gal4 > UAS - CG7630RNAi ) and control ( act5c - gal4 > + ) larvae .

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(g) Western blot analysis of steady state levels (SDS-PAGE) of MRC subunits COX4 (complex IV), SDHA (complex II) and UQCR-C2 (complex III) in whole lysates from CG7630 RNAi (act5c-gal4>UAS-CG7630RNAi) and control (act5c-gal4>+) larvae.


( h ) Western blot analysis of anti - HA affinity purified samples from S2R + D . melanogaster cells . EV = samples from cells carrying the empty expression vector . CG7630 - HA = samples from cells expressing HA - tagged CG7630 . Tot = total mitochondrial - enriched fractions ; unb = unbound material ; IP : HA = immunoprecipitated material . Samples were probed with antibodies against MRC subunits of complexes I ( NDUFS3 ) , II ( SDHA ) , III ( UQCR - C2 ) , IV ( COX4 ) and V ( ATP5A ) .

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(h) Western blot analysis of anti-HA affinity purified samples from S2R+ D. melanogaster cells. EV = samples from cells carrying the empty expression vector. CG7630-HA = samples from cells expressing HA-tagged CG7630. Tot = total mitochondrial-enriched fractions; unb = unbound material; IP:HA = immunoprecipitated material. Samples were probed with antibodies against MRC subunits of complexes I (NDUFS3), II (SDHA), III (UQCR-C2), IV (COX4) and V (ATP5A).


( a ) Relative quantification ( RQ ) of CG7630 transcript in wild - type D . melanogaster S2R + cells treated with a dsRNA targeting CG7630 ( CG7630 KD ) and a mock control dsRNA ( mock ) .

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(a) Relative quantification (RQ) of CG7630 transcript in wild-type D. melanogaster S2R+ cells treated with a dsRNA targeting CG7630 (CG7630 KD) and a mock control dsRNA (mock).


( b ) Relative quantification ( RQ ) of CG7630 and COX7B transcripts in stable S2R + cell lines carrying the empty expression vector ( EV : GFP ; neo ) , and expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) and CG7630 ( CG7630 - HA ; neo ) . Cells were treated with a dsRNA targeting CG7630 ( striped bars , CG7630 KD ) and a mock control dsRNA ( solid bars , mock ) . N / D = not detected .

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(b) Relative quantification (RQ) of CG7630 and COX7B transcripts in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock). N/D = not detected.


( c ) Immunoblot analysis of stable S2R + cell lines carrying the empty expression vector ( EV : GFP ; neo ) , and expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) and CG7630 ( CG7630 - HA ; neo ) . The CG7630 transcript was knocked - down ( KD ) in cells by a dsRNA targeting CG7630 and compared with cells treated with a mock control dsRNA ( mock ) . Samples were probed with an antibody anti HA tag and with an antibody against Hsp70 as an endogenous control .

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(c) Immunoblot analysis of stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). The CG7630 transcript was knocked-down (KD) in cells by a dsRNA targeting CG7630 and compared with cells treated with a mock control dsRNA (mock). Samples were probed with an antibody anti HA tag and with an antibody against Hsp70 as an endogenous control.


( d ) Kinetic enzyme activity of COX normalized by citrate synthase activity ( CS ) in stable S2R + cell lines carrying the empty expression vector ( EV : GFP ; neo ) , and expression vectors carrying HA - tagged forms of human COX7B ( hCOX7B - HA ; neo ) and CG7630 ( CG7630 - HA ; neo ) . Cells were treated with a dsRNA targeting CG7630 ( striped bars , CG7630 KD ) and a mock control dsRNA ( solid bars , mock ) .

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(d) Kinetic enzyme activity of COX normalized by citrate synthase activity (CS) in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock).


( e ) Steady - state levels of MRC subunits in samples from rescue experiment performed as in ( d ) .

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(e) Steady-state levels of MRC subunits in samples from rescue experiment performed as in (d).


( B ) Representative immunoblot analysis of CALHM6 expression in BMDM and BMDC stimulated for 6 or 24 h with Poly ( I : C ) ( 50 μg / ml ) or LPS ( 100 ng / ml ) ( one representative experiment of two is shown , n = 2 mice ) .

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(B) Representative immunoblot analysis of CALHM6 expression in BMDM and BMDC stimulated for 6 or 24 h with Poly(I:C) (50 μg/ml) or LPS (100 ng/ml) (one representative experiment of two is shown, n = 2 mice).


( G and H ) Calhm6 mRNA expression measured by qPCR in naïve or IFN - γ ( 10 ng / ml ) primed ( 6 h ) BMDM stimulated with ( G ) Poly ( I : C ) ( 50 μg / ml ) ( one representative experiment of two is shown , n = 2 mice , biological replicates ) , or ( H ) Zymosan ( 100 μg / ml ) for 2 to 24h ( one representative experiment is shown , n = 3 mice , biological replicates ) .

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(G and H) Calhm6 mRNA expression measured by qPCR in naïve or IFN-γ (10 ng/ml) primed (6 h) BMDM stimulated with (G) Poly(I:C) (50 μg/ml) (one representative experiment of two is shown, n = 2 mice, biological replicates), or (H) Zymosan (100 μg/ml) for 2 to 24h (one representative experiment is shown, n = 3 mice, biological replicates).


CFU / g of spleen are shown ( B ) after 24 h ( pooled results from two independent experiments , WT mice = 9 , Calhm6 - / - mice = 12 , Man Whitney test ) , ( C ) 72 h ( pooled results from two independent experiments , WT mice = 16 , Calhm6 - / - mice = 17 , Man Whitney test )

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CFU/g of spleen are shown (B) after 24 h (pooled results from two independent experiments, WT mice = 9, Calhm6-/- mice = 12, Man Whitney test), (C) 72 h (pooled results from two independent experiments, WT mice = 16, Calhm6-/- mice = 17, Man Whitney test)


Serum collected from infected mice at peak infection ( day 3 ) and serum cytokines quantified using LEGENDplex Mouse Inflammation Panel assay to determine IFN - γ ( F ) , IL - 6 ( G ) and IL - 10 ( H ) concentration ( pooled results from two independent experiments , WT mice = 16 , Calhm6 - / - mice = 17 , Mann Whitney test ) .

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Serum collected from infected mice at peak infection (day 3) and serum cytokines quantified using LEGENDplex Mouse Inflammation Panel assay to determine IFN-γ (F), IL-6 (G) and IL-10 (H) concentration (pooled results from two independent experiments, WT mice = 16, Calhm6-/- mice = 17, Mann Whitney test).


( F - J ) WT and Calhm6 - / - mice were injected i . p . with Poly ( I : C ) ( 200 μg / mouse ) or PBS for 3 h ( F - H ) or 12 h ( I - J ) , spleens were collected and processed as in ( A - E ) . Representative contour plots ( F , I ) , pooled frequency of IFN - γ + in CD3 - NK1 . 1 + cells of WT vs Calhm6 - / - mice ( G , J ) and ratio of IFN - γ + cells that are NK1 . 1 + or CD3 + normalised as in C ( H ) ( For Poly ( I : C ) 3 h : pooled results from two independent experiments , Poly ( I : C ) WT mice = 10 , Calhm6 - / - mice = 10 , control WT mice = 2 , Calhm6 - / - mice = 2 , One way ANOVA with multiple comparisons . For Poly ( I : C ) 12 h : results from one experiment , Poly ( I : C ) WT mice = 6 , Calhm6 - / - mice = 5 , control WT mice = 1 , Calhm6 - / - mice = 1 mice , One - way ANOVA with multiple comparisons ) .

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(F-J) WT and Calhm6-/- mice were injected i.p. with Poly(I:C) (200 μg/mouse) or PBS for 3 h (F-H) or 12 h (I-J), spleens were collected and processed as in (A-E). Representative contour plots (F, I), pooled frequency of IFN-γ+ in CD3-NK1.1+ cells of WT vs Calhm6-/- mice (G,J) and ratio of IFN-γ+ cells that are NK1.1+ or CD3+ normalised as in C (H) (For Poly(I:C) 3 h: pooled results from two independent experiments, Poly(I:C) WT mice = 10, Calhm6-/- mice = 10, control WT mice = 2, Calhm6-/- mice = 2, One way ANOVA with multiple comparisons. For Poly(I:C) 12 h: results from one experiment, Poly(I:C) WT mice = 6, Calhm6-/- mice = 5, control WT mice = 1, Calhm6-/- mice = 1 mice, One-way ANOVA with multiple comparisons).


( A - B ) WT and Calhm6 - / - mice were injected i . p . with Poly ( I : C ) ( 200 μg / mouse ) or PBS . On day 3 spleens were collected , stained with antibodies against NK cell - maturation markers and analysed by flow cytometry . Representative contour plots ( A ) and pooled flow cytometry results ( B ) are shown ( results from one experiment , Poly ( I : C ) WT mice = 5 , Calhm6 - / - mice = 5 , control WT mice = 2 , Calhm6 - / - mice = 2 mice , One - way ANOVA with multiple comparisons ) .

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(A-B) WT and Calhm6-/- mice were injected i.p. with Poly(I:C) (200 μg/mouse) or PBS. On day 3 spleens were collected, stained with antibodies against NK cell-maturation markers and analysed by flow cytometry. Representative contour plots (A) and pooled flow cytometry results (B) are shown (results from one experiment, Poly(I:C) WT mice = 5, Calhm6-/- mice = 5, control WT mice = 2, Calhm6-/- mice = 2 mice, One-way ANOVA with multiple comparisons).


( F ) NO production in WT and Calhm6 - / - BMDM treated overnight with LPS or Poly ( I : C ) quantified with a Griess test on cell supernatant ( results from one experiment , WT mice = 3 , Calhm6 - / - mice = 3 , Kruskal Wallis test with multiple comparisons ) .

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(F) NO production in WT and Calhm6-/- BMDM treated overnight with LPS or Poly(I:C) quantified with a Griess test on cell supernatant (results from one experiment, WT mice = 3, Calhm6-/- mice = 3, Kruskal Wallis test with multiple comparisons).


( G ) BMDM with or without IFN − γ priming were grown overnight in antibiotic free medium . Cells were infected with L . monocytogenes ( MOI 1 ) for 30 min . Gentamicin ( 5 ng / ml ) was then added to the medium to kill extracellular bacteria . At 0 , 4 and 8 h cells were harvested , lysed with 0 . 1 % Triton X - 100 and the lysate was plated on antibiotic free BHI plates for 24 h , and CFU were quantified ( pooled results from three independent experiments , WT mice = 3 , Calhm6 - / - mice = 3 , Two - way ANOVA test ) .

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(G) BMDM with or without IFN−γ priming were grown overnight in antibiotic free medium. Cells were infected with L. monocytogenes (MOI 1) for 30 min. Gentamicin (5 ng/ml) was then added to the medium to kill extracellular bacteria. At 0, 4 and 8 h cells were harvested, lysed with 0.1% Triton X-100 and the lysate was plated on antibiotic free BHI plates for 24 h, and CFU were quantified (pooled results from three independent experiments, WT mice = 3, Calhm6-/- mice = 3, Two-way ANOVA test).


( D - G ) BMDM were transduced with a retroviral vector expressing CALHM6 - eGFP . BMDM ( 3x105 ) were plated on coverslips and pre - treated with IFN - γ and LPS for 6 h . ( D ) Freshly isolated primary CD3 - NK1 . 1 + cells isolated from mouse spleens were added for 0 , 10 , 20 , or 40 min on top of plated BMDMs that were generated from bone marrow cells transduced with a retroviral vector expressing CALHM6 - eGFP . The interacting cells were then fixed and stained with phalloidin to identify actin and DAPI to identify nucleus . Synaptic interaction between BMDM and NK cell is indicated by white arrowheads ; entire collapsed imaging Z - stack is shown . ( E ) Images were blinded and CALHM6 - eGFP polarisation towards CD3 - NK1 . 1 + cells manually scored only in Z - slices where BMDM - NK interaction was identified ( p - value calculated by two - way ANOVA of polarisation and time points ) . Numbers on top of each bar indicate number of synapses scored for that time point . ( F ) IgG2a - opsonised sRBC labelled with PHK26 were added on top of plated CALHM - eGFP - expressing BMDM . One single Z - slice containing BMDM - sRBC interaction plane is shown . Synaptic interaction between BMDM and sRBC cell is indicated by white arrowheads ( G ) Quantified results of experiment in F . Numbers on top of each bar indicate number of synapses scored for that time point . Longer time points were not possible to score for opsonised sRBC because of the speed of the phagocytosis as sRBC were quickly internalised . Images taken with 60x objective ( p - value calculated by two - way ANOVA of polarisation and time points ) .

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(D-G) BMDM were transduced with a retroviral vector expressing CALHM6-eGFP. BMDM (3x105) were plated on coverslips and pre-treated with IFN-γ and LPS for 6 h. (D) Freshly isolated primary CD3-NK1.1+ cells isolated from mouse spleens were added for 0, 10, 20, or 40 min on top of plated BMDMs that were generated from bone marrow cells transduced with a retroviral vector expressing CALHM6-eGFP. The interacting cells were then fixed and stained with phalloidin to identify actin and DAPI to identify nucleus. Synaptic interaction between BMDM and NK cell is indicated by white arrowheads; entire collapsed imaging Z-stack is shown. (E) Images were blinded and CALHM6-eGFP polarisation towards CD3-NK1.1+ cells manually scored only in Z-slices where BMDM-NK interaction was identified (p-value calculated by two-way ANOVA of polarisation and time points). Numbers on top of each bar indicate number of synapses scored for that time point. (F) IgG2a-opsonised sRBC labelled with PHK26 were added on top of plated CALHM-eGFP-expressing BMDM. One single Z-slice containing BMDM-sRBC interaction plane is shown. Synaptic interaction between BMDM and sRBC cell is indicated by white arrowheads (G) Quantified results of experiment in F. Numbers on top of each bar indicate number of synapses scored for that time point. Longer time points were not possible to score for opsonised sRBC because of the speed of the phagocytosis as sRBC were quickly internalised. Images taken with 60x objective (p-value calculated by two-way ANOVA of polarisation and time points).


( B ) WT or Calhm6 - / - BMDM stimulated with IFN - γ ( 10 ng / ml ) for 24 h were lysed with 1 % Triton X100 - containing buffer with cOmplete protease inhibitor cocktail and treated with increasing concentrations of DTT . The samples , without boiling , were resolved by SDS - PAGE gel ( one representative experiment of two is shown , n = 4 mice ) . Lack of DTT influences solubilisation of GADPH monomer , and its detection for immunoblotting .

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(B) WT or Calhm6-/- BMDM stimulated with IFN-γ (10 ng/ml) for 24 h were lysed with 1% Triton X100-containing buffer with cOmplete protease inhibitor cocktail and treated with increasing concentrations of DTT. The samples, without boiling, were resolved by SDS-PAGE gel (one representative experiment of two is shown, n = 4 mice). Lack of DTT influences solubilisation of GADPH monomer, and its detection for immunoblotting.


( G ) Current - voltage ( I - V ) relations for WT - mCALHM6 , E119R - mCALHM6 and control ( ASO ) in 2 - mM Ba2 + bath solution ( pooled individual whole - cell patches , WT - mCALHM6 n = 13 , E119R - mCALHM6 n = 4 , control = 4 , SEM error bars ) .

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(G) Current-voltage (I-V) relations for WT-mCALHM6, E119R-mCALHM6 and control (ASO) in 2-mM Ba2+ bath solution (pooled individual whole-cell patches, WT-mCALHM6 n = 13, E119R-mCALHM6 n = 4, control = 4, SEM error bars).


( A ) Fura - 2 fluorescence ratio ( F340 / 380 ) recorded in N2a cells expressing human CALHM1 ( hCALHM1 ) , mouse CALHM6 ( mCALHM6 ) or control ( vector ) in response to removal and subsequent add - back of extracellular Ca2 + . Traces representative of four independent experiments for each , normalized to baseline ratio . Right : Resting F340 / 380 ratio in N2a cells expressing hCALHM1 , mCALHM6 or vector in 2 mM Ca2 + ( the error bars represent SEM ) .

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(A) Fura-2 fluorescence ratio (F340/380) recorded in N2a cells expressing human CALHM1 (hCALHM1), mouse CALHM6 (mCALHM6) or control (vector) in response to removal and subsequent add-back of extracellular Ca2+. Traces representative of four independent experiments for each, normalized to baseline ratio. Right: Resting F340/380 ratio in N2a cells expressing hCALHM1, mCALHM6 or vector in 2 mM Ca2+ (the error bars represent SEM).


( E ) Steady - state ATP release from activated macrophages : BMDMs were plated at 150 , 000 cells / well in a 48 - well plate , and incubated for 3 h with Poly ( I : C ) ( 50 μg / ml ) , LPS ( 100 ng / ml ) and ATPase inhibitor ARL67156 ( 1 mM ) in culture medium . Control cells were incubated with 1 mM ARL67156 only . ATP release ( nM ) measured using Sigma ATP Bioluminescent Assay : WT without stimulation : 9 . 12 ± 0 . 76 ; WT with stimulation : 22 . 16 ± 1 . 83 ; Calhm6 - / - cells without stimulation : 8 . 76 ± 0 . 55 ; KO cells with stimulation : 12 . 19 ± . 75 . ( pooled results from three independent experiments , WT mice = 3 , Calhm6 - / - mice = 3 , Two - tailed unpaired Student ' s T test , error bars represent SEM ) .

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(E) Steady-state ATP release from activated macrophages: BMDMs were plated at 150,000 cells/well in a 48-well plate, and incubated for 3 h with Poly(I:C) (50 μg/ml), LPS (100 ng/ml) and ATPase inhibitor ARL67156 (1 mM) in culture medium. Control cells were incubated with 1 mM ARL67156 only. ATP release (nM) measured using Sigma ATP Bioluminescent Assay: WT without stimulation: 9.12 ± 0.76; WT with stimulation: 22.16 ± 1.83; Calhm6-/- cells without stimulation: 8.76 ± 0.55; KO cells with stimulation: 12.19 ± .75. (pooled results from three independent experiments, WT mice = 3, Calhm6-/- mice = 3, Two-tailed unpaired Student's T test, error bars represent SEM).


( left ) Luciferase expression in HEK293 cells driven by deletion constructs of POLG proximal promoter . ( right ) Luciferase expression with mutated CAAT - boxes in POLG promoter . The error bars indicate standard deviation in 3 biological replicates . AU ; arbitrary units .

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(left) Luciferase expression in HEK293 cells driven by deletion constructs of POLG proximal promoter. (right) Luciferase expression with mutated CAAT-boxes in POLG promoter. The error bars indicate standard deviation in 3 biological replicates. AU; arbitrary units.


Expression pattern driven by 500 bp Polg proximal promoter in E12 . 5 mouse embryo . LacZ - positive cell populations in the developing midbrain ( black arrows ) , dorsal root ganglia ( grey arrows ) and motoneuron progenitors ( arrowhead ) of the neural tube . Somites show some expression ( white arrow ) . Sectioning planes indicated by red lines . Scale bars 100 µm .

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Expression pattern driven by 500 bp Polg proximal promoter in E12.5 mouse embryo. LacZ-positive cell populations in the developing midbrain (black arrows), dorsal root ganglia (grey arrows) and motoneuron progenitors (arrowhead) of the neural tube. Somites show some expression (white arrow). Sectioning planes indicated by red lines. Scale bars 100 µm.


- ( G ) POLG enhancer elements are functional in vivo and drive expression in E12 . 5 transgenic mice . Sectioning planes indicated by red lines . Black line in ( E ) marks the expression in the dorsal neural tube , whereas rostral and caudal regions lack dorsal expression ( black arrows ) .

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- (G) POLG enhancer elements are functional in vivo and drive expression in E12.5 transgenic mice. Sectioning planes indicated by red lines. Black line in (E) marks the expression in the dorsal neural tube, whereas rostral and caudal regions lack dorsal expression (black arrows).


( H ) - ( J ) Neural tube lacZ expression driven by ( H ) EE1 : immature neuronal precursors ( grey arrow ) , ( I ) EE2 : dorsal neural tube ( grey arrow ) and dorsal root ganglia ( black arrow ) , and ( J ) EE3 : dorsal neural tube ( grey arrow ) . Scale bars 100 µm .

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(H)- (J) Neural tube lacZ expression driven by (H) EE1: immature neuronal precursors (grey arrow), (I) EE2: dorsal neural tube (grey arrow) and dorsal root ganglia (black arrow), and (J) EE3: dorsal neural tube (grey arrow). Scale bars 100 µm.


K ) - ( M ) Midbrain lacZ expression , driven by ( K ) EE1 , ( L ) EE2 and ( M ) EE3 . Black arrow indicates neuronal population from EE1 embryo stained in ( N ) . Scale bars 100 µm .

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K)- (M) Midbrain lacZ expression, driven by (K) EE1, (L) EE2 and (M) EE3. Black arrow indicates neuronal population from EE1 embryo stained in (N). Scale bars 100 µm.


( N ) EE1 drives expression in oculomotor complex ; immunofluorescent co - staining with antibodies against ISL1 / 2 ( motoneurons of oculomotor complex ; red ) and β - Gal ( green ) . LacZ - staining of the region in ( k ) ; black arrow . Scale bars 50 µm .

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(N) EE1 drives expression in oculomotor complex; immunofluorescent co-staining with antibodies against ISL1/2 (motoneurons of oculomotor complex; red) and β-Gal (green). LacZ-staining of the region in (k); black arrow. Scale bars 50 µm.


Sagittal section of adult mouse brain showing lacZ expression driven by EE2 . HC , hippocampus .

Sagittal section of adult mouse brain showing lacZ expression driven by EE2. HC, hippocampus.


EE2 - driven expression in rostral migratory stream ( black arrow ) and in subventricular zone ( white arrow ) . Scale bar 760 µm .

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EE2-driven expression in rostral migratory stream (black arrow) and in subventricular zone (white arrow). Scale bar 760 µm.


Adult mouse spinal cord , EE2 and ( D ) EE3 expression pattern . Dorsal horns , laminae I - III ( black arrows ) and central canal ( arrowheads ) . LacZ staining . Dashed lines in ( C ) indicates the region of dorsal horn shown in ( D ) , ( F ) and ( G ) . Inlets in ( C ) and ( E ) show POLG immunohistochemistry of dorsal horn and central canal ( grey arrow ) , respectively . Scale bars : 100 µm if not indicated otherwise .

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Adult mouse spinal cord, EE2 and (D) EE3 expression pattern. Dorsal horns, laminae I-III (black arrows) and central canal (arrowheads). LacZ staining. Dashed lines in (C) indicates the region of dorsal horn shown in (D), (F) and (G). Inlets in (C) and (E) show POLG immunohistochemistry of dorsal horn and central canal (grey arrow), respectively. Scale bars: 100 µm if not indicated otherwise.


( F ) - ( G ) Calbindin ( red ) and β - Gal ( green ) expression in interneurons of the dorsal horn laminae I - III of ( F ) EE2 and ( G ) EE3 transgenic adult mice . Immunofluorescence . Scale bars 20 µm .

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(F)-(G) Calbindin (red) and β-Gal (green) expression in interneurons of the dorsal horn laminae I-III of (F) EE2 and (G) EE3 transgenic adult mice. Immunofluorescence. Scale bars 20 µm.


The number of distal DNAse I hypersensitive sites ( DHSs ) of mtDNA maintenance genes and genes in the genomic POLG locus correlating > 0 . 85 with target promoter DHS across 125 cell lines . Abbreviations : TWNK , Twinkle protein ; POLG , DNA Polymerase gamma , catalytic subunit ; POLG2 , DNA Polymerase gamma , accessory subunit ; SSBP1 , Single stranded DNA binding protein 1 ; TFAM , mitochondrial transcription factor A ; FANCI , Fanconi anemia group I protein ; RHCG , Ammonium transporter Rh type C ; TICRR , Treslin .

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The number of distal DNAse I hypersensitive sites (DHSs) of mtDNA maintenance genes and genes in the genomic POLG locus correlating >0.85 with target promoter DHS across 125 cell lines. Abbreviations: TWNK, Twinkle protein; POLG, DNA Polymerase gamma, catalytic subunit; POLG2, DNA Polymerase gamma, accessory subunit; SSBP1, Single stranded DNA binding protein 1; TFAM, mitochondrial transcription factor A; FANCI, Fanconi anemia group I protein; RHCG, Ammonium transporter Rh type C; TICRR, Treslin.


Distribution histogram of DHSs for mtDNA maintenance genes .

Distribution histogram of DHSs for mtDNA maintenance genes.


Genomic distribution of POLG DHSs . Black arrow shows a cluster upstream from POLG coding region .

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Genomic distribution of POLG DHSs. Black arrow shows a cluster upstream from POLG coding region.


Expression of LINC00925 and POLG in different human tissues and cell types . Quantitative PCR amplification of cDNA . Abbreviations : iPS , induced pluripotent stem cell ; SH5Y , neuroblastoma line ; HepG2 , liver hepatocellular carcinoma line ; U2OS bone osteosarcoma line . Error bars indicate standard deviation in 3 technical replicates . AU ; arbitrary units .

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Expression of LINC00925 and POLG in different human tissues and cell types. Quantitative PCR amplification of cDNA. Abbreviations: iPS, induced pluripotent stem cell; SH5Y, neuroblastoma line; HepG2, liver hepatocellular carcinoma line; U2OS bone osteosarcoma line. Error bars indicate standard deviation in 3 technical replicates. AU; arbitrary units.


Polg and long - non - coding RNA Ai854517 ( mouse homologue of human LINC00925 ) correlate tightly in mouse cerebellar development . Time points : E18 , postnatal days 0 , 3 , 6 , 9 ; three mice per time point . Expression calculated as cap analysis of gene expression ( CAGE ) hits in the transcription start site ( CTSS ) .

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Polg and long-non-coding RNA Ai854517 (mouse homologue of human LINC00925) correlate tightly in mouse cerebellar development. Time points: E18, postnatal days 0, 3, 6, 9; three mice per time point. Expression calculated as cap analysis of gene expression (CAGE) hits in the transcription start site (CTSS).


Ai854517 and Polg transcripts colocalize in adult mouse brain ; in situ hybridization . HC , hippocampus ; cerebellar molecular layer ( M ) , Purkinje cell layer ( PC ) , granular cell layer ( GC ) . Scale bars : hippocampus 760 µm , cerebellum 300 µm .

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Ai854517 and Polg transcripts colocalize in adult mouse brain; in situ hybridization. HC, hippocampus; cerebellar molecular layer (M), Purkinje cell layer (PC), granular cell layer (GC). Scale bars: hippocampus 760 µm, cerebellum 300 µm.


Expression levels of MIR9 - 3 and Ai854517 correlate in mouse cerebellar development . Time points : E18 , postnatal day 0 , 3 , 6 , 9 ; three mice per time point . . Expression calculated as cap analysis of gene expression ( CAGE ) hits in the transcription start site ( CTSS ) .

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Expression levels of MIR9-3 and Ai854517 correlate in mouse cerebellar development. Time points: E18, postnatal day 0, 3, 6, 9; three mice per time point.. Expression calculated as cap analysis of gene expression (CAGE) hits in the transcription start site (CTSS).


RNA expressions of MIR9 , NR2E2 , LDLDRAP1 and MTHFD2 in HEK293 untransfected controls and in cells transfected with pre - MIR9 or scrambled RNA . Shown is mean with standard error of the mean . Statistical testing was performed using 1 - way ANOVA with Dunnet ' s correction for multiple comparisons . Abbreviations : AU ; arbitrary units .

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RNA expressions of MIR9, NR2E2, LDLDRAP1 and MTHFD2 in HEK293 untransfected controls and in cells transfected with pre-MIR9 or scrambled RNA. Shown is mean with standard error of the mean. Statistical testing was performed using 1-way ANOVA with Dunnet's correction for multiple comparisons. Abbreviations: AU; arbitrary units.


Spinal cord ( C8 - level ) of MIRAS patient , neurofilament staining . Dorsal columns , especially the gracile , show pallor ( star ) ; motoneurons in the anterior horns are preserved ( black arrow ) . Scale bar 1 mm .

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Spinal cord (C8-level) of MIRAS patient, neurofilament staining. Dorsal columns, especially the gracile, show pallor (star); motoneurons in the anterior horns are preserved (black arrow). Scale bar 1 mm.


Oculomotor complex of POLG patient with progressive ophthalmoplegia ; midbrain level . Hematoxylin - eosin staining . The whole area shows spongiotic degeneration . Scale bar 50 µm .

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Oculomotor complex of POLG patient with progressive ophthalmoplegia; midbrain level. Hematoxylin-eosin staining. The whole area shows spongiotic degeneration. Scale bar 50 µm.


Seedling phenotype under GA treatment . After germination , Five - day - old seedlings with the same growth stage were transferred to 0 . 5 MS liquid media without or with different concentrations of GA3 . PA from soybean was added to the media at a final concentration of 20 μM . Pictures were taken 7 days after transfer . The horizontal red line separates different plants and the vertical red scale bar represents 2 cm .

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Seedling phenotype under GA treatment. After germination, Five-day-old seedlings with the same growth stage were transferred to 0.5 MS liquid media without or with different concentrations of GA3. PA from soybean was added to the media at a final concentration of 20 μM. Pictures were taken 7 days after transfer. The horizontal red line separates different plants and the vertical red scale bar represents 2 cm.


Immunoblotting of His - tagged PLDα6 ( arrow ) expressed in E . coli as separated on an 10 % SDS - PAGE and blotted to a membrane .

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Immunoblotting of His-tagged PLDα6 (arrow) expressed in E. coli as separated on an 10% SDS-PAGE and blotted to a membrane.


Lipid hydrolyzing activity assayed in the presence of different phospholipids using purified PLDα6 from E . coli . Solid bars are activities assayed using purified PLDα6 whereas open bars were empty vector control that used an equal volume of eluents from bacteria containing the empty vector that was identically processed as those expressing PLDα6 . Values are means ± SD ( n = 3 biological replicates ) .

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Lipid hydrolyzing activity assayed in the presence of different phospholipids using purified PLDα6 from E. coli. Solid bars are activities assayed using purified PLDα6 whereas open bars were empty vector control that used an equal volume of eluents from bacteria containing the empty vector that was identically processed as those expressing PLDα6. Values are means ± SD (n = 3 biological replicates).


Total lipid levels in WT , pldα6 and COM without and with 10 μM GA3 . Leaf samples ( 15 seedling each ) from 4 - leaf stage rice ( Dongjin background ) were collected and lipids were extracted and profiled using ESI - tandem mass spectrometry . Values are means ± SD ( n = 3 biological replicates ) ) . MGDG , monogalactosyldiacylglycerol ; DGDG , digalactosyldiacylglycerol ; PC , phosphatidylcholine ; PE , phosphatidylethanolamine ; PG , phosphatidylglycerol ; PS , phosphatidylserine ; PA , phosphatidic acid .

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Total lipid levels in WT, pldα6 and COM without and with 10 μM GA3. Leaf samples (15 seedling each) from 4-leaf stage rice (Dongjin background) were collected and lipids were extracted and profiled using ESI-tandem mass spectrometry. Values are means ± SD (n = 3 biological replicates)). MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; PA, phosphatidic acid.


Phospholipid species in WT , pldα6 and COM . Leaf samples ( 15 seedling each ) from 4 - leaf stage rice ( Dongjin background ) were collected and the different phospholipid species including carbon number and double bond number were determined . Values are means ± SD ( n = 3 biological replicates ) . GA3 was dissolved in ethanol and seedlings treated with the same ethanol concentration were used as control .

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Phospholipid species in WT, pldα6 and COM. Leaf samples (15 seedling each) from 4-leaf stage rice (Dongjin background) were collected and the different phospholipid species including carbon number and double bond number were determined. Values are means ± SD (n = 3 biological replicates). GA3 was dissolved in ethanol and seedlings treated with the same ethanol concentration were used as control.


Immunoblotting of His - GID1 proteins using constructs shown in ( D ) . Proteins were separated by SDS - PAGE , followed by immunoblotting with anti - His - tag antibodies . The PA binding activity of different truncation mutations were analyzed by fat - immunoblotting . The red arrowheads indicate truncated proteins with different molecular weights .

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Immunoblotting of His-GID1 proteins using constructs shown in (D). Proteins were separated by SDS-PAGE, followed by immunoblotting with anti-His-tag antibodies. The PA binding activity of different truncation mutations were analyzed by fat-immunoblotting. The red arrowheads indicate truncated proteins with different molecular weights.


Immunoblotting of His - GID1 mutants and lipid - immunoblotting of PA binding by GID1 mutant proteins on a filter .

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Immunoblotting of His-GID1 mutants and lipid-immunoblotting of PA binding by GID1 mutant proteins on a filter.


PLDα6 - GFP distribution in rice protoplasts with different concentrations of GA3 or IAA . Protoplasts from 12 - day old rice ( ZH11 background ) leaf sheath tissue were collected . Rice protoplasts were transfected with pM999 - PLDα6 for 12 hours . After that , GA3 or IAA was added to protoplasts and 2 hours later , confocal images of protoplasts are shown . pM999 - GFP refers to the empty vector with GFP only that was transformed as control and Ghd7 - RFP was a nucleus marker . Scale bar = 10 μm .

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PLDα6-GFP distribution in rice protoplasts with different concentrations of GA3 or IAA. Protoplasts from 12-day old rice (ZH11 background) leaf sheath tissue were collected. Rice protoplasts were transfected with pM999-PLDα6 for 12 hours. After that, GA3 or IAA was added to protoplasts and 2 hours later, confocal images of protoplasts are shown. pM999-GFP refers to the empty vector with GFP only that was transformed as control and Ghd7-RFP was a nucleus marker. Scale bar = 10 μm.


Immunoblotting of PLDα6 in subcellular fractions . Total ( T ) , soluble ( S ) and nuclear ( N ) proteins were isolated from PLDα6 : GFP expressed in rice protoplasts treated with 10 μM GA3 or IAA . Equal amount of each sample was loaded for SDS - PAGE and blotting .

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Immunoblotting of PLDα6 in subcellular fractions. Total (T), soluble (S) and nuclear (N) proteins were isolated from PLDα6:GFP expressed in rice protoplasts treated with 10 μM GA3 or IAA. Equal amount of each sample was loaded for SDS-PAGE and blotting.


Subcellular location of GID1 in WT and pldα6 protoplasts with or without GA3 treatments . Cells for 12 hours after transformation were treated with or without 10 μM GA3 for two hours . Bars = 10 μm .

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Subcellular location of GID1 in WT and pldα6 protoplasts with or without GA3 treatments. Cells for 12 hours after transformation were treated with or without 10 μM GA3 for two hours. Bars = 10 μm.


Immunoblotting of subcellular fractions of GID1 expressed in WT and pldα6 protoplasts . After 2 - hour treatment with 10 μM GA3 , total ( T ) , soluble ( S ) and nuclear ( N ) protein fractions were isolated from 10 samples of protoplasts . Equal amount of each sample was loaded for SDS - PAGE and immunoblotting .

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Immunoblotting of subcellular fractions of GID1 expressed in WT and pldα6 protoplasts. After 2-hour treatment with 10 μM GA3, total (T), soluble (S) and nuclear (N) protein fractions were isolated from 10 samples of protoplasts. Equal amount of each sample was loaded for SDS-PAGE and immunoblotting.


Protoplasts of WT and pldα6 from 12 - day old stage leaf sheath were collected and transfected with a pM999 - SLR1 construct , incubated for 12 hours and then treated with 10 μM GA3 for 0 , 3 , and 9 hours . The first column , fluorescence from GFP ; the second , red fluorescence from the nucleus marker Ghd7 ; the third , bright field , and the fourth , overlay of the three channels . All confocal images were scanned using similar laser gain and offset settings . Bars = 10 μm .

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Protoplasts of WT and pldα6 from 12-day old stage leaf sheath were collected and transfected with a pM999-SLR1 construct, incubated for 12 hours and then treated with 10 μM GA3 for 0, 3, and 9 hours. The first column, fluorescence from GFP; the second, red fluorescence from the nucleus marker Ghd7; the third, bright field, and the fourth, overlay of the three channels. All confocal images were scanned using similar laser gain and offset settings. Bars = 10 μm.


Immunoblotting of SLR1 - GFP proteins from protoplast cells . Equal amounts of proteins from each sample were used for SDS - PAGE , followed by immunoblotting with anti - GFP antibodies .

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Immunoblotting of SLR1-GFP proteins from protoplast cells. Equal amounts of proteins from each sample were used for SDS-PAGE, followed by immunoblotting with anti-GFP antibodies.


( A ) Graph derived from two published data available in the PubMed GEO database ( GSE6919 and GSE35988 ) . The box charts depict the relative expression of ESM1 in benign , primary , and metastatic prostate cancer patients . The centre line donates the median value while the box contains the 25th to 75th percentiles of dataset . The whiskers mark the 5th and 95th percentiles , and values beyond these upper and lower bounds are considered outliers .

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(A) Graph derived from two published data available in the PubMed GEO database (GSE6919 and GSE35988). The box charts depict the relative expression of ESM1 in benign, primary, and metastatic prostate cancer patients. The centre line donates the median value while the box contains the 25th to 75th percentiles of dataset. The whiskers mark the 5th and 95th percentiles, and values beyond these upper and lower bounds are considered outliers.


( B ) Relative ESM1 mRNA expression and ESM1 protein expression in two sets of human metastatic PCa cell lines . P : parental cells , M : metastatic cells .

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(B) Relative ESM1 mRNA expression and ESM1 protein expression in two sets of human metastatic PCa cell lines. P: parental cells, M: metastatic cells.


( C ) Cell invasion assays of 22Rv1 - M and PC3 - M cells transfected with either Scramble - shRNA or ESM1 - shRNA . The data are shown as the relative fold change of invasive cells compared with the shScramble group . Scale bar : 100 μm .

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(C) Cell invasion assays of 22Rv1-M and PC3-M cells transfected with either Scramble-shRNA or ESM1-shRNA. The data are shown as the relative fold change of invasive cells compared with the shScramble group. Scale bar: 100 μm.


( D ) Representative images of lungs , livers , intestines , and kidneys from mice 8 weeks after orthotopical injection of 22Rv1 - M cells transfected with either Scramble - shRNA or ESM1 - shRNA . Scale bar : 0 . 5 cm . Tumors were removed and weighed . Quantitative summary of tumor luminescence in different metastases measured in photons / second are shown .

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(D) Representative images of lungs, livers, intestines, and kidneys from mice 8 weeks after orthotopical injection of 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Scale bar: 0.5 cm. Tumors were removed and weighed. Quantitative summary of tumor luminescence in different metastases measured in photons/second are shown.


( E ) Formalin - fixed , paraffin - embedded tissue microarray sections of 59 normal prostate tissues and 65 prostate adenocarcinoma tissues with and without metastasis ( n = 16 and n = 49 , respectively ) were stained with an anti - ESM1 antibody . Tissue - bound ESM1 is shown in brown . Scale bar : 50 μm . Arrowheads indicate positively stained nucleus of cancer cells . Plot representation of scores according to immunohistochemical expression of ESM1 in normal prostate tissue related to the prostate adenocarcinoma tissue with and without metastasis . The scores are calculated by intensity ( score 1 - 3 ) x percentage ( score 1 - 4 ) of stained cells . The line in the middle of the box is plotted at the median . The box extends from the 25th to 75th percentiles . Whiskers above and below the box indicate the 5th and 95th percentiles .

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(E) Formalin-fixed, paraffin-embedded tissue microarray sections of 59 normal prostate tissues and 65 prostate adenocarcinoma tissues with and without metastasis (n=16 and n=49, respectively) were stained with an anti-ESM1 antibody. Tissue-bound ESM1 is shown in brown. Scale bar: 50 μm. Arrowheads indicate positively stained nucleus of cancer cells. Plot representation of scores according to immunohistochemical expression of ESM1 in normal prostate tissue related to the prostate adenocarcinoma tissue with and without metastasis. The scores are calculated by intensity (score 1-3) x percentage (score 1-4) of stained cells. The line in the middle of the box is plotted at the median. The box extends from the 25th to 75th percentiles. Whiskers above and below the box indicate the 5th and 95th percentiles.


( A ) Immunoblotting analysis of ESM1 in supernatant ( S ) , nucleus ( N ) , and cytoplasm ( C ) was performed . Ponceau S , Lamin A / C , and α - tubulin were used as loading control in supernatant , nucleus , and cytoplasm , respectively . P : parental cells , M : metastatic cells .

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(A) Immunoblotting analysis of ESM1 in supernatant (S), nucleus (N), and cytoplasm (C) was performed. Ponceau S, Lamin A/C, and α-tubulin were used as loading control in supernatant, nucleus, and cytoplasm, respectively. P: parental cells, M: metastatic cells.


( B ) Left , representative images of cells in the transwell invasion assay . Right , quantification of cells that invaded through the matrigel - coated membrane following treatment with PBS or human recombinant ESM1 ( rhESM1 ) or normal goat IgG control or anti - ESM1 neutralizing antibody . Scale bar : 100 μm .

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(B) Left, representative images of cells in the transwell invasion assay. Right, quantification of cells that invaded through the matrigel-coated membrane following treatment with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody. Scale bar: 100 μm.


( D ) Elicitation of ESM1 overexpression using HA - tag antibody results in detectable ESM1 expression in the nucleus and cytoplasm of 22Rv1 - P cells . Lamin A / C and α - tubulin were used as loading control in the nucleus and cytoplasm , respectively .

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(D) Elicitation of ESM1 overexpression using HA-tag antibody results in detectable ESM1 expression in the nucleus and cytoplasm of 22Rv1-P cells. Lamin A/C and α-tubulin were used as loading control in the nucleus and cytoplasm, respectively.


( E ) Immunoblotting of ESM1 expression with different expressing patterns in the supernatant ( S ) of 22Rv1 - P cells .

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(E) Immunoblotting of ESM1 expression with different expressing patterns in the supernatant (S) of 22Rv1-P cells.


( F ) Left , representative images of 22Rv1 - P cells in the transwell invasion assay . Right , statistic data of invasive ability of ESM1 expressing cells . Scale bar : 100 μm .

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(F) Left, representative images of 22Rv1-P cells in the transwell invasion assay. Right, statistic data of invasive ability of ESM1 expressing cells. Scale bar: 100 μm.


( G ) Left , representative bioluminescence images of lung metastasis in mice after tail vein injection of 22Rv1 - Luc cells with different ESM1 expression patterns . Scale bar : 0 . 5 cm . Right , quantification of the lung bioluminescence by photons / seconds .

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(G) Left, representative bioluminescence images of lung metastasis in mice after tail vein injection of 22Rv1-Luc cells with different ESM1 expression patterns. Scale bar: 0.5 cm. Right, quantification of the lung bioluminescence by photons/ seconds.


( A ) Tumor spheroid formation . Representative images of tumor spheres formed and quantitative data comparing the average number of spheres formed in the indicated cells treated with PBS or human recombinant ESM1 ( rhESM1 ) or normal goat IgG control or anti - ESM1 neutralizing antibody .

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(A) Tumor spheroid formation. Representative images of tumor spheres formed and quantitative data comparing the average number of spheres formed in the indicated cells treated with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody.


( B ) Representative images of tumor spheres . Spheres were cultured for 14 days before counting . Histogram shows the mean numbers of spheres cultured . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments .

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(B) Representative images of tumor spheres. Spheres were cultured for 14 days before counting. Histogram shows the mean numbers of spheres cultured. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.


( C ) 22Rv1 - P cells stably expressing ESM1 were treated with docetaxel at the indicated concentrations for 48 h . * *

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(C) 22Rv1-P cells stably expressing ESM1 were treated with docetaxel at the indicated concentrations for 48 h. **


( D ) Cells with or without ESM1 - NLS overexpression were subcutaneously injected ( 10 , 100 , 1000 cells per mouse ) into NOD / SCID mice . Tumor formation ability and tumor weight were analyzed . Scale bar : 1 cm . Bars are the mean ± SD of indicated ( n ) independent experiments .

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(D) Cells with or without ESM1-NLS overexpression were subcutaneously injected (10, 100, 1000 cells per mouse) into NOD/SCID mice. Tumor formation ability and tumor weight were analyzed. Scale bar: 1 cm. Bars are the mean ± SD of indicated (n) independent experiments.


( E ) Flow cytometry analysis of the ratio of CD44 + and CD133 + cells in the indicated cells with different subcellular localization types of ESM1 overexpression were performed .

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(E) Flow cytometry analysis of the ratio of CD44+ and CD133+ cells in the indicated cells with different subcellular localization types of ESM1 overexpression were performed.


( F ) qRT - PCR analysis of the mRNA levels of the indicated genes in cells with different types of ESM1 overexpression . Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments . *

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(F) qRT-PCR analysis of the mRNA levels of the indicated genes in cells with different types of ESM1 overexpression. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments. *


( A ) IPA analysis of RNA - seq data showing the 14 top - predicted upstream regulators of differentially expressed genes . Positive Z - scores ( orange ) represent activated upstream regulators , negative Z - scores ( blue ) represent inhibited upstream regulators .

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(A) IPA analysis of RNA-seq data showing the 14 top-predicted upstream regulators of differentially expressed genes. Positive Z-scores (orange) represent activated upstream regulators, negative Z-scores (blue) represent inhibited upstream regulators.


( E ) The transcriptional activity of β - catenin in 22Rv1 - P cells with different patterns of ESM1 overexpression . Bars are the mean ± SD of three independent experiments .

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(E) The transcriptional activity of β-catenin in 22Rv1-P cells with different patterns of ESM1 overexpression. Bars are the mean ± SD of three independent experiments.


( F ) Manipulating ESM1 expression impacts transcriptional activity of NFκB . Bars are the mean ± SD of three independent experiments .

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(F) Manipulating ESM1 expression impacts transcriptional activity of NFκB. Bars are the mean ± SD of three independent experiments.


( A ) qRT - PCR analysis of the mRNA levels of the genes related to β - catenin downstream signaling pathway . Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments .

(A) qRT-PCR analysis of the mRNA levels of the genes related to β-catenin downstream signaling pathway. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments.


( B ) qRT - PCR analysis of the mRNA levels of NFκB targeting genes . Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments .

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(B) qRT-PCR analysis of the mRNA levels of NFκB targeting genes. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments.


( C ) Immunoblotting analysis of 22Rv1 - P cells with different ESM1 overexpression in the nucleus and cytoplasm were performed with the antibodies indicated .

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(C) Immunoblotting analysis of 22Rv1-P cells with different ESM1 overexpression in the nucleus and cytoplasm were performed with the antibodies indicated.


( D ) Suppression of β - catenin or NFκB in 22Rv1 - P cells stably expressing ESM1 - NLS were treated with docetaxel at the indicated concentrations for 48 h . * * P < 0 . 01 when compared to vector - shScramble cells by two - tailed Student ' s t test and error bars represent the standard deviation of three independent experiments .

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(D) Suppression of β-catenin or NFκB in 22Rv1-P cells stably expressing ESM1-NLS were treated with docetaxel at the indicated concentrations for 48 h. ** P < 0.01 when compared to vector-shScramble cells by two-tailed Student's t test and error bars represent the standard deviation of three independent experiments.


( E ) Tumor spheres formed by 22Rv1 - P cells stably expressing ESM1 - NLS with either β - catenin or NFκB shRNAs . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments . # # P < 0 . 01 when compared to vector - shScramble cells , * * P < 0 . 01 when compared to ESM1 - NLS - shScramble cells by two - tailed Student ' s t test .

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(E) Tumor spheres formed by 22Rv1-P cells stably expressing ESM1-NLS with either β-catenin or NFκB shRNAs. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.


( F ) Representative images and quantitative data comparing cell invasion of the indicated cells . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments . # # P < 0 . 01 when compared to vector - shScramble cells , * * P < 0 . 01 when compared to ESM1 - NLS - shScramble cells by two - tailed Student ' s t test .

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(F) Representative images and quantitative data comparing cell invasion of the indicated cells. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.


( A ) Nuclear and cytosolic extracts of 22Rv1 - M cells transfected with either shScramble or shCTNNB1 were prepared and the levels of indicated proteins were detected by immunoblotting .

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(A) Nuclear and cytosolic extracts of 22Rv1-M cells transfected with either shScramble or shCTNNB1 were prepared and the levels of indicated proteins were detected by immunoblotting.


( B ) Nuclear or cytosolic extracts of 22Rv1 - M cells were immunoprecipitated with an ESM1 antibody .

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(B) Nuclear or cytosolic extracts of 22Rv1-M cells were immunoprecipitated with an ESM1 antibody.


( C ) Human recombinant His - ESM1 and GST - β - catenin protein were pull down with either Ni sepharose or Glutathione sepharose .

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(C) Human recombinant His-ESM1 and GST-β-catenin protein were pull down with either Ni sepharose or Glutathione sepharose.


( E ) HEK293T cells were co - transfected with the indicated β - catenin - Flag and ESM1 - HA constructs . Cell extracts were immunoprecipitated with Flag - M2 agarose beads . Light chain was labeled as l . c .

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(E) HEK293T cells were co-transfected with the indicated β-catenin-Flag and ESM1-HA constructs. Cell extracts were immunoprecipitated with Flag-M2 agarose beads. Light chain was labeled as l.c.


( F ) HEK293T cells were transfected with ESM1 - HA constructs and pull - down was carried out with different purified GST - ARM fragments .

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(F) HEK293T cells were transfected with ESM1-HA constructs and pull-down was carried out with different purified GST-ARM fragments.


( G ) HEK293T cells were transfected with the indicated ESM1 - GFP constructs . Cell extracts were pulled down with purified GST - ARM .

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(G) HEK293T cells were transfected with the indicated ESM1-GFP constructs. Cell extracts were pulled down with purified GST-ARM.


( B ) HEK293T cells were transfected with the indicated ESM1 point mutant constructs . Cell extracts were pulled down with purified GST - ARM .

(B) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM.


( C ) Cell extracts of 22Rv1 - M transfected with either Scramble - shRNA or ESM1 - shRNA were immunoprecipitated with an β - catenin antibody and blotted with anti - TCF4 and anti - BCL9 antibody .

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(C) Cell extracts of 22Rv1-M transfected with either Scramble-shRNA or ESM1-shRNA were immunoprecipitated with an β-catenin antibody and blotted with anti-TCF4 and anti-BCL9 antibody.


( E ) Upper , Schematic diagram of ESM1 - targeting short peptides . Lower , HEK293T cells were transfected with ESM1 - HA constructs . Cell extracts were treated with the short peptides , pulled down with purified GST - ARM and blotted with anti - HA and anti - TCF4 antibody .

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(E) Upper, Schematic diagram of ESM1-targeting short peptides. Lower, HEK293T cells were transfected with ESM1-HA constructs. Cell extracts were treated with the short peptides, pulled down with purified GST-ARM and blotted with anti-HA and anti-TCF4 antibody.


( F ) HEK293T cells were transfected with the indicated ESM1 point mutant constructs . Cell extracts were pulled down with purified GST - ARM and blotted with an anti - TCF4 antibody .

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(F) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM and blotted with an anti-TCF4 antibody.


( G ) ChIP - qPCR validation of β - catenin binding to the CCND1 and c - Myc promoter in 22Rv1 - M cells transfected with either Scramble - shRNA or ESM1 - shRNA . Bars are the mean ± SD of three independent experiments .

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(G) ChIP-qPCR validation of β-catenin binding to the CCND1 and c-Myc promoter in 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Bars are the mean ± SD of three independent experiments.


( H ) Upper , Schematic diagram of ChIP - qPCR primers targeting regions upstream of the CCND1 and c - Myc transcription start sites ( TSSs ) . Bar graphs show β - catenin and ESM1 binding at the CCND1 and c - Myc promoter regions . Differences in DNA levels compared with IgG control are shown as fold changes presented as the mean ± SD of three independent experiments .

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(H) Upper, Schematic diagram of ChIP-qPCR primers targeting regions upstream of the CCND1 and c-Myc transcription start sites (TSSs). Bar graphs show β-catenin and ESM1 binding at the CCND1 and c-Myc promoter regions. Differences in DNA levels compared with IgG control are shown as fold changes presented as the mean ± SD of three independent experiments.


( I ) The effects of ESM1 targeting peptides on tumor invasion and spheroid formation . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments .

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(I) The effects of ESM1 targeting peptides on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.


( J ) The effects of ESM1 point mutants on tumor invasion and spheroid formation . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments .

(J) The effects of ESM1 point mutants on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.


( B ) Correlation between replicate mock infections . Path - seq reads were recovered from samples of macrophage RNA spiked with 0 . 1 % MTB RNA . Scatter plot of log2 RPKM values is shown with Pearson correlation , P - value < 0 . 0001 . Inset summarizes the mock infection replicates and their fraction of MTB reads ( of all aligned reads ) from Path - seq and standard RNA - seq methods .

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(B) Correlation between replicate mock infections. Path-seq reads were recovered from samples of macrophage RNA spiked with 0.1% MTB RNA. Scatter plot of log2 RPKM values is shown with Pearson correlation, P-value < 0.0001. Inset summarizes the mock infection replicates and their fraction of MTB reads (of all aligned reads) from Path-seq and standard RNA-seq methods.


( C ) Correlation between MTB RNA sequenced by RNA - seq ( Illumina TruSeq library prep ) and the same MTB RNA sample combined with macrophage RNA at 1 : 1000 ratio and processed using Path - seq method . Scatter plot of log2 RPKM values is shown with Pearson correlation , P - value < 0 . 0001 .

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(C) Correlation between MTB RNA sequenced by RNA-seq (Illumina TruSeq library prep) and the same MTB RNA sample combined with macrophage RNA at 1:1000 ratio and processed using Path-seq method. Scatter plot of log2 RPKM values is shown with Pearson correlation, P-value < 0.0001.


( A ) Path - seq profiles of desA1 , desA2 in MTB infected BMDMs . Error bars show the standard deviation from three biological samples . Representative results from two experiments are presented .

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(A) Path-seq profiles of desA1, desA2 in MTB infected BMDMs. Error bars show the standard deviation from three biological samples. Representative results from two experiments are presented.


( B ) Upper panel : dissolved oxygen curve over 120 h time course showing controlled depletion ( 1 ) , sustained hypoxia ( 2 ) and controlled reaeration ( 3 ) . Points represent the average of three biological replicates and were measured via fiber optic technology that non - invasively probes oxygen levels in the culture ( PreSens Precision Sensing GmbH ) . Lower panel : expression profiles ( RNA - seq ) of desA1 and desA2 over the time course and oxygen levels . Error bars show the standard deviation from three biological samples .

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(B) Upper panel: dissolved oxygen curve over 120 h time course showing controlled depletion (1), sustained hypoxia (2) and controlled reaeration (3). Points represent the average of three biological replicates and were measured via fiber optic technology that non-invasively probes oxygen levels in the culture (PreSens Precision Sensing GmbH). Lower panel: expression profiles (RNA-seq) of desA1 and desA2 over the time course and oxygen levels. Error bars show the standard deviation from three biological samples.


( B ) Heatmap of TFs with decreased ( purple ) or increased ( green ) activity at specific time points during in vitro or in vivo infection .

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(B) Heatmap of TFs with decreased (purple) or increased (green) activity at specific time points during in vitro or in vivo infection.


( C ) Rv0681 regulon genes differentially expressed in vitro and the evidence for predicted high Rv0681 activity ( induced expression of target genes ) . ( D ) Zur regulon genes differentially expressed in vitro and the evidence for decreased Zur activity ( de - represses target genes ) . ( E ) Rv0691c regulon genes differentially expressed in vitro and in vivo ; evidence for increased Rv0691c activity ( increased up - and down - regulation of target genes ) at 24 h from both in vitro and in vivo infection .

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(C) Rv0681 regulon genes differentially expressed in vitro and the evidence for predicted high Rv0681 activity (induced expression of target genes). (D) Zur regulon genes differentially expressed in vitro and the evidence for decreased Zur activity (de-represses target genes). (E) Rv0691c regulon genes differentially expressed in vitro and in vivo; evidence for increased Rv0691c activity (increased up- and down-regulation of target genes) at 24 h from both in vitro and in vivo infection.


( B ) Plot of read pile - ups from MSM with inducible overexpression of MSMEG _ 0916 shows ChIP - binding in the promoters of desA1 and desA2 .

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(B) Plot of read pile-ups from MSM with inducible overexpression of MSMEG_0916 shows ChIP-binding in the promoters of desA1 and desA2.


Boxplots representing RPKM values from RNA - seq of MSM with inducible overexpression of MSMEG _ 0916 . Significant log2 fold change ( FC ) between uninduced ( - ATc ) and induced ( + ATc ) samples for MSMEG _ 0916 ( log2 FC = 4 . 99 ) , desA1 ( log2 FC = - 1 . 32 ) and desA2 ( log2 FC = - 1 . 8 ) with multiple hypothesis adjusted P - values < 0 . 0001 . Data is from three biological samples ( induced ) and nine biological samples ( uninduced ) .

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Boxplots representing RPKM values from RNA-seq of MSM with inducible overexpression of MSMEG_0916. Significant log2 fold change (FC) between uninduced (-ATc) and induced (+ATc) samples for MSMEG_0916 (log2 FC = 4.99), desA1 (log2 FC = -1.32) and desA2 (log2 FC = -1.8) with multiple hypothesis adjusted P-values < 0.0001. Data is from three biological samples (induced) and nine biological samples (uninduced).


( A ) Serial ten - fold dilutions of MTB H37Rv wild type ( wt ) and MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc .

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(A) Serial ten-fold dilutions of MTB H37Rv wild type (wt) and MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc.