( B ) Akt was immunoprecipitated from soluble fraction of from fed or fasted ( 2d ) mice . Percipitated were analyzed by immunoblotting using an anti - ubiquitin conjugates . Mouse IgG was used as a control for non - specific binding .

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(B) Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted (2d) mice. Percipitated were analyzed by immunoblotting using an anti-ubiquitin conjugates. Mouse IgG was used as a control for non-specific binding.


) D ( Ubiquitinated Akt is a substrate for USP1 in vitro . Top : Akt was immunoprecipitated from soluble fraction of TA muscle from fed mice , was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi - well plate . dividing indicates removal of an intervening lane for presentation purposes . Bottom : Densitometric measurements of presented blots . Data is presented as between ubiquitinated Akt to total Akt in each well .

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

)D( Ubiquitinated Akt is a substrate for USP1 in vitro. Top: Akt was immunoprecipitated from the soluble fraction of TA muscle from fed mice, and was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi-well plate. The dividing line indicates the removal of an intervening lane for presentation purposes. Bottom: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each well.


( E ) shRNA - mediated knockdown of USP1 in Hela . Soluble extracts were analyzed by immunoblotting .

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(E) shRNA-mediated knockdown of USP1 in Hela cells. Soluble extracts were analyzed by immunoblotting.


( F ) USP1 deubiquitinates Akt during fasting in vivo . Left : Soluble fractions of TA transfected with shLacz control or shUSP1 from fed fasted mice were analyzed by SDS - PAGE immunoblotting using anti - Akt . actin blot serves as a loading control . Right : densitometric measurements of presented blots ( n = 3 ) . Data is presented as of ubiquitinated Akt to total Akt normalized to actin . * p < 0 . 05 vs . fed by one - t - . Data are represented as mean ± .

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(F) USP1 deubiquitinates Akt during fasting in vivo. Left: Soluble fractions of TA muscles transfected with shLacz control or shUSP1 from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using anti-Akt. The actin blot serves as a loading control. Right: densitometric measurements of presented blots (n=3). Data is presented as ratio of ubiquitinated Akt to total Akt normalized to actin. * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.


( G ) USP1 removes K63 - linked polyubiquitin chains on Akt in vivo . Akt was immunoprecipitated from soluble fraction of expressing USP1 ( C90S ) or control plasmids . Mouse IgG was used as a control for non - specific binding . precipitates were subjected to immunoblotting with an antibody against K63 - linked polyubiquitin conjugates .

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

(G) USP1 removes K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1(C90S) or control plasmids. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.


( H ) USP1 removes polyubiquitin chains linked to K8 on Akt . Akt was immunoprecipitated from soluble fraction of TA transfected with USP1 ( C90S ) , HA - Akt ( K8R ) or control from fasted mice , precipitates were analyzed by immunoblotting with anti - Akt antibody . Mouse IgG was used as a control for non - specific binding . Right : Densitometric measurements of presented blots . Data is presented as between ubiquitinated Akt to total Akt in each lane ( n = 2 ) .

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

(H) USP1 removes polyubiquitin chains linked to K8 on Akt. Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1(C90S) , HA-Akt(K8R) or control from fasted mice, and protein precipitates were analyzed by immunoblotting with anti-Akt antibody. Mouse IgG was used as a control for non-specific binding. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).


( A ) Inhibition of USP1 increases PI3K - Akt - FoxO signaling during fasting . Soluble fractions of normal atrophying expressing shLacz or USP1 ( C90S ) were analyzed by SDS - PAGE immunoblot . black indicate removal of intervening lanes for presentation purposes . Right : densitometric measurements of presented pAkt ( T308 ) , pAkt ( S473 ) , Akt blots . Data is presented as of phosphorylated Akt to total Akt ( n = 3 ) . * p < 0 . 05 vs . fed by one - t - . Data are represented as mean ± .

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(A) Inhibition of USP1 increases PI3K-Akt-FoxO signaling during fasting. Soluble fractions of normal and atrophying muscles expressing shLacz or USP1(C90S) were analyzed by SDS-PAGE and immunoblot. The black line indicate the removal of intervening lanes for presentation purposes. Right: densitometric measurements of presented pAkt (T308), pAkt (S473), and Akt blots. Data is presented as ratio of phosphorylated Akt to total Akt (n=3). * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.


( B ) Downregulation of USP1 reduces MuRF1 Atrogin1 expression during fasting . Quantitative RT - PCR of mRNA preparations from atrophying control expressing shLacz or shUSP1 ( 80 % transfection efficiency ) using primers for MuRF1 Atrogin1 . Data are plotted as mean fold change relative to fed control . n = 4 . * , P < 0 . 05 vs . shLacz in fed . # , P < 0 . 05 vs . shLacz in fasting by one - t - . Data are represented as mean ± .

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(B) Downregulation of USP1 reduces MuRF1 and Atrogin1 expression during fasting. Quantitative RT-PCR of mRNA preparations from atrophying and control muscles expressing shLacz or shUSP1 (80% transfection efficiency) using primers for MuRF1 and Atrogin1. Data are plotted as the mean fold change relative to fed control. n = 4. *, P < 0.05 vs. shLacz in fed. #, P < 0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.


( C ( Downregulation of USP1 markedly reduces muscle fiber atrophy . Cross - sectional areas of 500 transfected with shUSP1 ( that express GFP , green bars ) vs . 500 nontransfected ( black bars ) in same muscle . n = 5 mice . A representative image is shown ; Laminin is in red ; Bar , 50μm .

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(C( Downregulation of USP1 markedly reduces muscle fiber atrophy. Cross-sectional areas of 500 fibers transfected with shUSP1 (that express GFP, green bars) vs. 500 nontransfected fibers (black bars) in the same muscle. n = 5 mice. A representative image is shown; Laminin staining is in red; Bar, 50μm.


( D ) Downregulation of USP1attenuates loss of muscle mass during fasting . shUSP1 was delivered to more than 60 % of muscle . Mean weights of electroporated are plotted as loss . n = 6 . # , P < 0 . 05 vs . shLacz in fed ; * , P < 0 . 05 vs . shLacz in fasting by one - t - . Data are represented as mean ± .

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(D) Downregulation of USP1attenuates the loss of muscle mass during fasting. shUSP1 was delivered to more than 60% of muscle fibers. Mean weights of electroporated muscles are plotted as the percent weight loss. n = 6. #, P < 0.05 vs. shLacz in fed; *, P < 0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.


( E ) USP1 inhibition significantly increases glucose tolerance in mice during fasting . Left : mice injected i . p . with specific USP1 Inhibitor ( 12ug / gr ) or saline during fasting ( 2d ) were subjected to glucose tolerance . Blood glucose were measured at indicated time points following glucose injection ( 1 mg / gr ) . Data is depicted in a graph as mg / dL glucose ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 vs . mice injected with saline by one - t - . Data are represented as mean ± . Right : soluble fractions of from injected mice were analyzed by SDS - PAGE immunoblot .

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(E) USP1 inhibition significantly increases glucose tolerance in mice during fasting. Left: mice injected i.p. with specific USP1 Inhibitor (12ug/gr body weight) or saline during fasting (2d) were subjected to glucose tolerance test. Blood glucose levels were measured at the indicated time points following glucose injection (1 mg/gr body weight). Data is depicted in a graph as mg/dL glucose (n=3). * p<0.05, ** p<0.005, *** p<0.0005 vs. mice injected with saline by one-tailed t-test. Data are represented as mean ±SEM. Right: soluble fractions of muscles from injected mice were analyzed by SDS-PAGE and immunoblot.


( A ) During fasting , downregulation of PHLPP1 enhances Akt phosphorylation at S473 . Soluble fractions of transfected with shLacz or shPHLPP1 from fasted mice ( 2d ) were analyzed by immunoblotting . Right : densitometric measurements of presented blots . Data is presented as of shPHLPP1 to shLacz ( n = 3 for shLacz n = 4 for shPHLPP1 ) . * p < 0 . 05 vs . shLacz in fasting by one - t - . Data are represented as mean ± .

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(A) During fasting, downregulation of PHLPP1 enhances Akt phosphorylation at S473. Soluble fractions of muscles transfected with shLacz or shPHLPP1 from fasted mice (2d) were analyzed by immunoblotting. Right: densitometric measurements of presented blots. Data is presented as ratio of shPHLPP1 to shLacz (n=3 for shLacz and n=4 for shPHLPP1). * p<0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.


( B ) PHLPP1 downregulation increases rates of synthesis during fasting . Mice were injected with puromycin , soluble fractions of electroporated were analyzed by immunoblotting using puromycin antibody . of right ( R ) left ( L ) limbs for each mouse is shown .

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(B) PHLPP1 downregulation increases rates of protein synthesis during fasting. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Analysis of right (R) and left (L) limbs for each mouse is shown.


( C ) Downregulation of PHLPP1 markedly reduces muscle fiber atrophy . Cross - sectional areas of 500 transfected with shPHLPP1 ( that express GFP , green bars ) vs . 500 nontransfected ( black bars ) in same muscle . n = 4 mice . A representative image is shown ; Laminin is in red ; Bar , 50μm .

(C) Downregulation of PHLPP1 markedly reduces muscle fiber atrophy. Cross-sectional areas of 500 fibers transfected with shPHLPP1 (that express GFP, green bars) vs. 500 nontransfected fibers (black bars) in the same muscle. n = 4 mice. A representative image is shown; Laminin staining is in red; Bar, 50μm.


( D ) USP1 / UAF1 interact with PHLPP1 in normal muscle . USP1 was immunoprecipitated from soluble fraction of normal . Mouse IgG was used as a control for non - specific binding . Precipitates were analyzed by immunoblotting .

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(D) USP1/UAF1 interact with PHLPP1 in normal muscle. USP1 was immunoprecipitated from the soluble fraction of normal muscles. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting.


( C ( In fasting , Akt sediments to glycerol gradient fractions containing USP1 / UAF1 , PHLPP1 , TSC1 / TSC2 . Muscle homogenates from fed or fasted mice were separated on 10 % - 40 % glycerol gradient , alternate fractions were analyzed by SDS - PAGE immunoblot . Red rectangle marks fractions containing USP1 / UAF1 , PHLPP1 , TSC1 TSC2 in fed conditions .

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(C( In fasting, Akt sediments to glycerol gradient fractions containing USP1/UAF1, PHLPP1, and TSC1/TSC2. Muscle homogenates from fed or fasted mice were separated on 10%-40% glycerol gradient, and alternate fractions were analyzed by SDS-PAGE and immunoblot. Red rectangle marks fractions containing USP1/UAF1, PHLPP1, TSC1 and TSC2 in fed conditions.


( D ) During fasting , Akt is recruited to USP1 - PHLPP1 - TSC1 . TSC1 was immunoprecipitated from soluble fraction of expressing shLacz or USP1 ( C90S ) from fed or fasted mice . Mouse IgG was used as a control for non - specific binding . Precipitates were analyzed by immunoblotting . Black lines indicate removal of intervening lanes for presentation purposes .

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

(D) During fasting, Akt is recruited to USP1-PHLPP1-TSC1 complex. TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or USP1(C90S) from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Black lines indicate the removal of intervening lanes for presentation purposes.


( E ) During fasting , inhibition of USP1 reduces rates of synthesis . Mice were injected with puromycin , soluble fractions of electroporated were analyzed by immunoblotting using puromycin antibody . Right : Densitometric measurement of presented blot . n = 3 . * , P < 0 . 05 vs . shLacz by one - t - . Data are represented as mean ± .

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(E) During fasting, inhibition of USP1 reduces rates of protein synthesis. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.


( F ) Inhibition of USP1 results in TSC1 accumulation . Soluble fraction of expressing shLacz or USP1 ( C90S ) from fasted mice were analyzed by SDS - PAGE immunoblotting . Right : Densitometric measurement of presented blot . n = 3 . * , P < 0 . 05 vs . shLacz . Data is presented as content ( AU ) in USP1 ( C90S ) vs . shLacz by one - t - . Data are represented as mean ± .

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(F) Inhibition of USP1 results in TSC1 accumulation. Soluble fraction of muscles expressing shLacz or USP1(C90S) from fasted mice were analyzed by SDS-PAGE and immunoblotting. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz. Data is presented as protein content (AU) in USP1(C90S) vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.


( G ) mTOR inhibition with rapamycin results in reduced USP1 content enhanced Akt phosphorylation at T308 . However , a simultaneous downregulation of USP1 is required to promote TSC1 accumulation . Soluble fraction of expressing shLacz or gUSP1 ( in vivo CRISPR ) from fed fasted mice injected i . p . with rapamycin ( 6mg / kg ) or saline were analyzed by SDS - PAGE immunoblot .

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(G) mTOR inhibition with rapamycin results in reduced USP1 protein content and enhanced Akt phosphorylation at T308. However, a simultaneous downregulation of USP1 is required to promote TSC1 accumulation. Soluble fraction of muscles expressing shLacz or gUSP1 (in vivo CRISPR) from fed and fasted mice injected i.p. with rapamycin (6mg/kg body weight) or saline were analyzed by SDS-PAGE and immunoblot.


( A ) During fasting , inhibition of Dab2 - Akt association reduces interaction of Akt with USP1 - PHLPP1 - TSC1 . Left : TSC1 was immunoprecipitated from soluble fraction of expressing shLacz or Dab2 - DN from fed or fasted mice . Mouse IgG was used as a control for non - specific binding . Precipitates were analyzed by immunoblotting . Right : densitometric measurement of PHLPP1 TSC1 blots , data is presented as PHLPP1 / TSC1 .

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(A) During fasting, inhibition of Dab2-Akt association reduces the interaction of Akt with USP1-PHLPP1-TSC1 complex. Left: TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Right: densitometric measurement of PHLPP1 and TSC1 blots, and data is presented as PHLPP1/TSC1 ratio.


( B ) Dab2 promotes Akt deubiquitination in vivo . Left : Akt was immunoprecipitated from soluble fraction of expressing shLacz or Dab2 - DN from fed fasted mice using a specific antibody . Mouse IgG was used as a control for non - specific binding . precipitates were analyzed by Western blot using Akt antibody . Right : Densitometric measurements of presented blots . Data is presented as between ubiquitinated Akt to total Akt in each lane ( n = 2 ) .

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(B) Dab2 promotes Akt deubiquitination in vivo. Left: Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were analyzed by Western blot analysis using Akt antibody. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).


( C ( Dab2 promotes cleavage of K63 - linked polyubiquitin chains on Akt in vivo . Akt was immunoprecipitated from soluble fraction of expressing shLacz or Dab2 - DN from fed fasted mice using a specific antibody . Mouse IgG was used as a control for non - specific binding . precipitates were subjected to immunoblotting with an antibody against K63 - linked polyubiquitin conjugates .

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(C( Dab2 promotes cleavage of K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.


( D ) Inhibition of Dab2 - Akt association enhances Akt phosphorylation in fasting . Soluble fraction of TA transfected with shLacz or Dab2 - DN from fed fasted mice were analyzed by SDS - PAGE immunoblotting using indicated antibodies . Right : densitometric measurement of presented blots . Data are mean ± , n = 3 . * p < 0 . 05 . vs . shLacz in fed conditions , by one - t - .

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(D) Inhibition of Dab2-Akt association enhances Akt phosphorylation in fasting. Soluble fraction of TA muscles transfected with shLacz or Dab2-DN from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Right: densitometric measurement of presented blots. Data are mean ±SEM, n=3. * p<0.05. vs. shLacz in fed conditions, by one-tailed t-test.


( b ) Experimental validation of predicted synergistic SR - combinational therapies in head neck cancer : A table summarizing experimentally observed synergism between drugs their predicted rescuer - targeting treatments in 5 HNSC lines , on drug treatment experiments . Synergism was estimated using standard Fa - CI . table displays average combination index ( CI ; synergism CI < 1 , additivity effect CI = 1 , antagonism CI > 1 , NAN indeterminate CI ) at 50 % growth inhibition ( Fraction affected ) . Combinations that are synergistic are colored blue ( black otherwise ) for each lines tested . inset shows an example of CI calculation for BYL719 Dasatinib combination in HN12 lines on corresponding dose matrix ( number indicates % viability at 48h , n = 3 ) , Fa - CI curve .

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(b) Experimental validation of predicted synergistic SR-based combinational therapies in head and neck cancer: A table summarizing the experimentally observed synergism between primary drugs and their predicted rescuer-targeting treatments in 5 HNSC cell lines, based on drug treatment experiments. Synergism was estimated using standard Fa-CI analysis. The table displays the average combination index (CI; synergism CI < 1, additivity effect CI = 1, antagonism CI > 1, NAN indeterminate CI) at 50% growth inhibition (Fraction affected). Combinations that are synergistic are colored blue (black otherwise) for each cell lines tested. The inset shows an example of CI calculation for BYL719 and Dasatinib combination in HN12 cell lines based on the corresponding dose matrix (number indicates % cell viability at 48h, n = 3), and Fa-CI curve.


( 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 .

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 B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O

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 .

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

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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 .

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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 ) .

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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 .

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

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 B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_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

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

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).