Figure 3 ( A - D ) Succinate dehydrogenase staining was performed in WT and MCK - KO soleus at sedentary ( A , B ) and after a bout of low - intensity exercise for 50 min ( C , D ) ( 10X , 20X magnifications ) , and the staining intensity was quantified using ImageJ ( n = 6 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) . ( E - H ) The analogous set of data is shown with WT and MCK - KO GA muscles . ( n = 6 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) . ( I - L ) The analogous set of data is shown with WT and MCK - KO EDL muscles . ( n = 6 , means ± SEM , two - tailed student ' s t - test . EDL : Extensor digitorum longus ) ( M , N ) Mitochondrial respiration was measured in WT and MCK - KO soleus tissue after a bout of low - intensity exercise for 50 min under basal conditions and in response to 4 mM oligomycin ( complex V inhibitor ) , 4 mM FCCP ( uncoupler ) , or 4 mM each of rotenone and antimycin A ( complex I inhibitor ) ( n = 8 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) . ( O , P ) Mitochondrial respiration was measured in Dnmt3a knocked down L6 myotubes which were transduced with lentiviral under basal conditions and in response to 4 mM oligomycin ( complex V inhibitor ) , 4 mM FCCP ( uncoupler ) , or 4 mM rotenone and antimycin A ( complex I inhibitor ) ( n = 5 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A-D) Succinate dehydrogenase staining was performed in WT and MCK-KO soleus at sedentary (A, B) and after a bout of low-intensity exercise for 50 min (C, D) (10X, 20X magnifications), and the staining intensity was quantified using ImageJ (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test). (E-H) The analogous set of data is shown with WT and MCK-KO GA muscles. (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test). (I-L) The analogous set of data is shown with WT and MCK-KO EDL muscles. (n = 6, means ± SEM, two-tailed student's t-test. EDL: Extensor digitorum longus) (M, N) Mitochondrial respiration was measured in WT and MCK-KO soleus tissue after a bout of low-intensity exercise for 50 min under basal conditions and in response to 4 mM oligomycin (complex V inhibitor), 4 mM FCCP (uncoupler), or 4 mM each of rotenone and antimycin A (complex I inhibitor) (n = 8, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing). (O, P) Mitochondrial respiration was measured in Dnmt3a knocked down L6 myotubes which were transduced with lentiviral under basal conditions and in response to 4 mM oligomycin (complex V inhibitor), 4 mM FCCP (uncoupler), or 4 mM rotenone and antimycin A (complex I inhibitor) (n = 5, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


Figure 4 ( E , F ) ALDH1L1 protein expression and the quantification in WT vs . MCK - KO soleus , GA and EDL muscles at sedentary and normalizing to GAPDH using ImageJ .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(E, F) ALDH1L1 protein expression and the quantification in WT vs. MCK-KO soleus, GA and EDL muscles at sedentary and normalizing to GAPDH using ImageJ.


Figure 5Single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes were achieved by lentiviral transduction . NADPH levels ( H ) and NOX activity ( I ) were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes . ( n = 3 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) . ( J , K ) H2O2 levels were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes . ( J : n = 3 and K : n = 6 for Control , Aldh1l1 KD , Aldh1l1 KD + Dnmt3a KD , n = 5 for Dnmt3a KD , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes were achieved by lentiviral transduction. NADPH levels (H) and NOX activity (I) were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes. (n = 3, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).(J, K) H2O2 levels were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes. (J: n = 3 and K: n = 6 for Control, Aldh1l1 KD, Aldh1l1 KD + Dnmt3a KD, n = 5 for Dnmt3a KD, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


Figure 6 ( D , E ) mitochondrial respiration in Dnmt3a knockdown and control L6 myotubes treated with NAC ( 3mM ) or vehicle treatment for 24hrs ( n = 5 Control groups n = 3 Dnmt3a KD , n = 4 Dnmt3a KD with NAC treatment groups . means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(D, E) mitochondrial respiration in Dnmt3a knockdown and control L6 myotubes treated with NAC (3mM) or vehicle treatment for 24hrs (n = 5 Control groups n = 3 Dnmt3a KD, n = 4 Dnmt3a KD with NAC treatment groups. means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


Figure 7 ( D , E ) Exercise capacity of WT and MCK - KO with or without transfection of gCont vs . gAldh1l1 under the low intensity regimen ( n = 4 WT + gCont , n = 9 MCK - KO + gCont , and MCK - KO + gAldh1l1 , * p < 0 . 05 , means ± SEM , two - tailed student ' s t - test and one - way Anova ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(D, E) Exercise capacity of WT and MCK- KO with or without transfection of gCont vs. gAldh1l1 under the low intensity regimen (n = 4 WT + gCont, n = 9 MCK- KO + gCont, and MCK- KO + gAldh1l1, *p<0.05, means ± SEM, two-tailed student's t-test and one-way Anova).


Figure 1Number of genes detected on protein and mRNA level in each tissue . The colouring the bars indicates the fractions of transcripts and proteins that are expressed everywhere or enriched in certain tissues . The full classification is provided in the text . Abundance distribution of all transcripts detected in all tissues ( grey ) ; the fraction of detected proteins is shown in blue and the fraction of transcripts for which no protein was detected is shown in orange .

O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Number of genes detected on protein and mRNA level in each tissue. The colouring the bars indicates the fractions of transcripts and proteins that are expressed everywhere or enriched in certain tissues. The full classification is provided in the text.Abundance distribution of all transcripts detected in all tissues (grey); the fraction of detected proteins is shown in blue and the fraction of transcripts for which no protein was detected is shown in orange.


Figure 2Distribution of global transcript and protein abundance in all tissues . It is apparent that the dynamic range of protein expression ( iBAQ scale ) exceeds that of mRNA expression ( FPKM scale ; see Appendix Fig S10 for the corresponding plot for RNA and protein copy numbers ) . Protein to mRNA abundance plot for brain tissue . The slope of the regression line indicates that high abundance mRNAs give rise to more protein copies per mRNA than low abundance mRNAs . Ranked abundance plot of proteins and transcripts in human heart . While the 10 most abundant transcripts cover almost 70 % of all transcripts in this tissue , the corresponding proteins only represent about 20 % of the total protein . Analysis of the number of genes that are shared among the 100 most abundant transcripts and proteins . Regardless of the tissue , the fraction of shared genes rarely exceeds 20 % . Correlation analysis of protein to RNA abundance ( in log10 scale ) across tissues , resulting in almost 90 % positive correlations . The protein highlighted in the next panel are marked . Examples for proteins that show high ( SYK , left panel ) or no ( EIF4A3 , right panel ) correlation of protein / RNA ratios across tissues . While the former indicates that different tissues express different quantities of SYK , EIF4A3 expression appears to be similar in all tissues .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Distribution of global transcript and protein abundance in all tissues. It is apparent that the dynamic range of protein expression (iBAQ scale) exceeds that of mRNA expression (FPKM scale; see Appendix Fig S10 for the corresponding plot for RNA and protein copy numbers).Protein to mRNA abundance plot for brain tissue. The slope of the regression line indicates that high abundance mRNAs give rise to more protein copies per mRNA than low abundance mRNAs.Ranked abundance plot of proteins and transcripts in human heart. While the 10 most abundant transcripts cover almost 70% of all transcripts in this tissue, the corresponding proteins only represent about 20% of the total protein.Analysis of the number of genes that are shared among the 100 most abundant transcripts and proteins. Regardless of the tissue, the fraction of shared genes rarely exceeds 20%.Correlation analysis of protein to RNA abundance (in log10 scale) across tissues, resulting in almost 90% positive correlations. The protein highlighted in the next panel are marked.Examples for proteins that show high (SYK, left panel) or no (EIF4A3, right panel) correlation of protein/RNA ratios across tissues. While the former indicates that different tissues express different quantities of SYK, EIF4A3 expression appears to be similar in all tissues.


Figure 3Global correlation analysis of proteomes vs proteomes and transcriptomes vs transcriptomes across human tissues . It is apparent that proteomes correlate stronger across tissues than transcriptomes . Average cellular compositions of tissues highlighted in panel ( B ) showing that the molecular similarities in their transcriptomes and proteomes are driven by similarities in cell types .

O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Global correlation analysis of proteomes vs proteomes and transcriptomes vs transcriptomes across human tissues. It is apparent that proteomes correlate stronger across tissues than transcriptomes.Average cellular compositions of tissues highlighted in panel (B) showing that the molecular similarities in their transcriptomes and proteomes are driven by similarities in cell types.


Figure 1 ( A ) Growth assay of the indicated cells with and without doxycycline - inducible overexpression of PLK4 . Experiments were performed in wild - type or SAS6 monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m . ( D ) Top hits that emerged from the screens ranked by MaGeCK FDR value . Blue hits are genes required for centriole duplication or stability . Green hits are genes predicted to be required to arrest the growth of cells with extra centrosomes . ( G ) Top : Graph showing the efficiency of frameshifting INDELs measured using TIDE . N . D . = Not determined . Data shown is from n = 1 biological replicate . Bottom : Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes . Each dot displays measurements from a single experiment . Experiments were performed in polyclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m . ( H ) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes . Experiments were performed in polyclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in wild-type or SAS6 monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.(D) Top hits that emerged from the screens ranked by MaGeCK FDR value. Blue hits are genes required for centriole duplication or stability. Green hits are genes predicted to be required to arrest the growth of cells with extra centrosomes.(G) Top: Graph showing the efficiency of frameshifting INDELs measured using TIDE. N.D. = Not determined. Data shown is from n = 1 biological replicate. Bottom: Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.(H) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.


Figure 2 ( A ) Top , widefield image showing the localization of CEP164 and PIDD1 at the mature - mother centriole . Bottom : Representative 3D STORM image of the same centriole showing PIDD1 localization . STORM image colors correspond to the Z - depth with red being the closest to the coverslip and blue being the most distant . ( B ) 3D STORM image of PIDD1 at the mature mother centriole . The overlaid mask represents the inner and outer diameters of the PIDD1 signal . ( C ) Inner and outer diameter measurements for distal appendage proteins and PIDD1 . Measurements in the shaded region were previously reported in ( Bowler et al . , 2019 ) . Data displayed as box and whisker plots , where the box represents the upper and lower quartile and the whiskers represent the s . d . Scale bars : 1 μm for all wide - field images of centrioles and 500 nm for STORM images . Data acquired across n ≥ 6 cells . ( D ) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes . A dot displays measurements from each experiment . Experiments were performed in PLK4Dox polyclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m . ( F ) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes . Each dot displays measurements from a single experiment . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m . ( G ) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Top, widefield image showing the localization of CEP164 and PIDD1 at the mature-mother centriole. Bottom: Representative 3D STORM image of the same centriole showing PIDD1 localization. STORM image colors correspond to the Z-depth with red being the closest to the coverslip and blue being the most distant.(B) 3D STORM image of PIDD1 at the mature mother centriole. The overlaid mask represents the inner and outer diameters of the PIDD1 signal.(C) Inner and outer diameter measurements for distal appendage proteins and PIDD1. Measurements in the shaded region were previously reported in (Bowler et al., 2019). Data displayed as box and whisker plots, where the box represents the upper and lower quartile and the whiskers represent the s.d. Scale bars: 1 μm for all wide-field images of centrioles and 500 nm for STORM images. Data acquired across n ≥6 cells.(D) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes. A dot displays measurements from each experiment. Experiments were performed in PLK4Dox polyclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.(F) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.(G) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


Figure 3 ( A ) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m . ( B - D ) Representative images of WT or ANKRD26 - / - PLK4Dox cells immunostained with the indicated antibodies . Scale bar = 5 µm . ( E ) Quantification of the fraction of WT or ANKRD26 - / - hTERT RPE1 cells with cilia . Each dot displays measurements from a single experiment . Data acquired across n = 5 biological replicates . Mean ± s . e . m . ( F ) Growth assay of the indicated cells with and without doxycycline - inducible overexpression of PLK4 . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m . ( G ) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m . ( H ) Western blot showing expression of pro - CASP2 and P21 following treatment with dox for the specified number of days . Experiments were performed in PLK4Dox monoclonal knockout cells . ( I ) Quantification of pro - CASP2 levels following treatment with dox for the specified number of days . Experiments were performed in PLK4Dox monoclonal knockout cells . Each dot displays measurements from a single experiment . Data acquired across n = 6 biological replicates . Mean ± s . e . m .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.(B-D) Representative images of WT or ANKRD26-/- PLK4Dox cells immunostained with the indicated antibodies. Scale bar = 5 µm.(E) Quantification of the fraction of WT or ANKRD26-/- hTERT RPE1 cells with cilia. Each dot displays measurements from a single experiment. Data acquired across n = 5 biological replicates. Mean ± s.e.m.(F) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.(G) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.(H) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. (I) Quantification of pro-CASP2 levels following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. Each dot displays measurements from a single experiment. Data acquired across n = 6 biological replicates. Mean ± s.e.m.


Figure 4 ( A ) Top : schematic representation of PIDD1 and its various cleavage products . Bottom : HEK293FT cells were transfected with the indicated constructs , subjected to co - immunoprecipitation and immunoblotted with the indicated antibodies . ( B ) HEK293FT cells were transfected with the indicated constructs , subjected to co - immunoprecipitation and immunoblotted with the indicated antibodies . ( C ) Western blot showing the expression of ANKRD26 . Experiments were performed in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated ANKRD26 transgenes . ( D ) Quantification of the fraction of cells with ANKRD26 ( left ) and PIDD1 ( right ) localized to the mature mother centriole in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated transgenes . Each dot displays measurements from a single experiment . Data acquired across n = 3 biological replicates . Mean ± s . e . m . ( E ) Graph showing the relative growth of doxycycline - treated , monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated ANKRD26 transgenes . Each dot displays measurements from a single experiment . Data acquired across n = 3 biological replicates . Mean ± s . e . m . ( F ) Western blot showing expression of pro - CASP2 and P21 following treatment with dox for 4 days . Experiments were performed in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated transgenes .

O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Top: schematic representation of PIDD1 and its various cleavage products. Bottom: HEK293FT cells were transfected with the indicated constructs, subjected to co- immunoprecipitation and immunoblotted with the indicated antibodies.(B) HEK293FT cells were transfected with the indicated constructs, subjected to co- immunoprecipitation and immunoblotted with the indicated antibodies.(C) Western blot showing the expression of ANKRD26. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes.(D) Quantification of the fraction of cells with ANKRD26 (left) and PIDD1 (right) localized to the mature mother centriole in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.(E) Graph showing the relative growth of doxycycline-treated, monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.(F) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for 4 days. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes.


Figure 1 . ( A and B ) NIH3T3 cells stably expressing GFP - ULK1 ( A ) and - ULK2 ( B ) were cultured in complete or starvation medium for 30 min . They were then cultured in fresh complete medium for an additional 30 min ( starvation → complete ) . ( C and D ) NIH3T3 cells stably expressing GFP - ULK1 ( C ) and - ULK2 ( D ) were cultured in starvation medium for 120 min . The cells were fixed , permeabilized , and subjected to immunofluorescence microscopy using anti - Atg16L1 antibody and Alexa Fluor 660 - conjugated secondary antibody . More than 90 % of GFP - ULKs dots were positive for Atg16L1 . ( E - H ) Wild - type ( E and G ) and Atg5 − / − ( F and H ) MEFs were transfected with retroviral vectors encoding GFP - ULK1 and - ULK2 . MEFs stably expressing GFP - ULK1 ( E and F ) and - ULK2 ( G and H ) were cultured in complete or starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy . Bars , 20 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.(A and B) NIH3T3 cells stably expressing GFP-ULK1 (A) and -ULK2 (B) were cultured in complete or starvation medium for 30 min. They were then cultured in fresh complete medium for an additional 30 min (starvation→complete).(C and D) NIH3T3 cells stably expressing GFP-ULK1 (C) and -ULK2 (D) were cultured in starvation medium for 120 min. The cells were fixed, permeabilized, and subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 660-conjugated secondary antibody. More than 90% of GFP-ULKs dots were positive for Atg16L1.(E-H) Wild-type (E and G) and Atg5−/− (F and H) MEFs were transfected with retroviral vectors encoding GFP-ULK1 and -ULK2. MEFs stably expressing GFP-ULK1 (E and F) and -ULK2 (G and H) were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bars, 20 μm.


Figure 2 . ( A ) In vitro kinase assay of endogenous ULK1 . MEFs were cultured in complete or starvation medium for 60 min . ULK1 kinase activity was determined as described in Materials and methods . Relative kinase activity is shown . Data are the mean ± SE of five independent experiments . ( B and C ) NIH3T3 cells were transiently transfected with HA - ULK1 , HA - ULK2 , or their kinase - dead mutants and subjected to immunofluoresence microscopy using monoclonal anti - HA and polyclonal anti - Atg16L1 antibodies for primary staining and Alexa Fluor 488 - conjugated goat anti - mouse IgG and Alexa Fluor 568 - conjugated goat anti - rabbit IgG antibodies for secondary antibodies . Transfected cells are indicated by arrows . Bars , 20 μm . ( D ) NIH3T3 cells stably expressing GFP - ULK1K46N and GFP - ULK2K39T were cultured in starvation medium for 120 min . The cells were subjected to immunofluorescence microscopy using anti - Atg16L1 antibody . Bar , 20 μm . ( E ) NIH3T3 cells were transfected with the retroviral vectors encoding HA - ULK1K46N or with the corresponding empty retrovirus ( mock ) . They were cultured in complete or starvation medium for 120 min . Cell lysates were then analyzed by immunoblot analysis with the indicated antibodies .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.(A) In vitro kinase assay of endogenous ULK1. MEFs were cultured in complete or starvation medium for 60 min. ULK1 kinase activity was determined as described in Materials and methods. Relative kinase activity is shown. Data are the mean ± SE of five independent experiments.(B and C) NIH3T3 cells were transiently transfected with HA-ULK1, HA-ULK2, or their kinase-dead mutants and subjected to immunofluoresence microscopy using monoclonal anti-HA and polyclonal anti-Atg16L1 antibodies for primary staining and Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG antibodies for secondary antibodies. Transfected cells are indicated by arrows. Bars, 20 μm. (D) NIH3T3 cells stably expressing GFP-ULK1K46N and GFP-ULK2K39T were cultured in starvation medium for 120 min. The cells were subjected to immunofluorescence microscopy using anti-Atg16L1 antibody. Bar, 20 μm.(E) NIH3T3 cells were transfected with the retroviral vectors encoding HA-ULK1K46N or with the corresponding empty retrovirus (mock). They were cultured in complete or starvation medium for 120 min. Cell lysates were then analyzed by immunoblot analysis with the indicated antibodies.


Figure 3 . ( A ) HEK293T cells were cotransfected with FLAG - FIP200 and HA - ULKs . Cell lysates were subjected to immunoprecipitation ( IP ) using antibodies against FLAG . The resulting precipitates were examined by immunoblot ( IB ) analysis with the indicated antibodies . The asterisk indicates nonspecific band . ( B ) Lysates of MEFs were immunoprecipitated with anti - ULK1 or anti - FIP200 antibody or preimmune rabbit serum , and the resulting precipitates were subjected to immunoblot analysis with antibodies against ULK1 and FIP200 . ( D ) HEK293T cells were cotransfected with FLAG - FIP200 and various ULK1 mutants and analyzed as in A using anti - HA and anti - FLAG antibodies . ( E ) NIH3T3 cells stably expressing GFP - ULK1 ( left ) and GFP - ULK1ΔC ( right ) were cultured in starvation medium for 120 min . Bar , 20 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.(A) HEK293T cells were cotransfected with FLAG-FIP200 and HA-ULKs. Cell lysates were subjected to immunoprecipitation (IP) using antibodies against FLAG. The resulting precipitates were examined by immunoblot (IB) analysis with the indicated antibodies. The asterisk indicates nonspecific band.(B) Lysates of MEFs were immunoprecipitated with anti-ULK1 or anti-FIP200 antibody or preimmune rabbit serum, and the resulting precipitates were subjected to immunoblot analysis with antibodies against ULK1 and FIP200.(D) HEK293T cells were cotransfected with FLAG-FIP200 and various ULK1 mutants and analyzed as in A using anti-HA and anti-FLAG antibodies.(E) NIH3T3 cells stably expressing GFP-ULK1 (left) and GFP-ULK1ΔC (right) were cultured in starvation medium for 120 min. Bar, 20 μm.


Figure 4 . ( A ) NIH3T3 cells stably expressing GFP - FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed . ( B ) NIH3T3 cells stably expressing GFP - FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti - Atg16L1 antibody and Alexa Fluor 568 - conjugated secondary antibody . Bars , 20 μm . More than 90 % of GFP - FIP200 dots were positive for Atg16L1 . ( C ) NIH3T3 cells stably expressing GFP - ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti - FIP200 antibody and Alexa Fluor 568 - conjugated secondary antibody . Black squares indicate the enlarged areas shown in insets . Bar , 20 μm .

O O O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.(A) NIH3T3 cells stably expressing GFP-FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed.(B) NIH3T3 cells stably expressing GFP-FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 568-conjugated secondary antibody. Bars, 20 μm. More than 90% of GFP-FIP200 dots were positive for Atg16L1.(C) NIH3T3 cells stably expressing GFP-ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti-FIP200 antibody and Alexa Fluor 568-conjugated secondary antibody. Black squares indicate the enlarged areas shown in insets. Bar, 20 μm.


Figure 5 . ( A ) FIP200 + / + , FIP200 − / − , Atg5 + / + , and Atg5 − / − MEFs were cultured in complete or starvation medium for 60 min . In the recovery experiments , starved MEFs were cultured in fresh complete medium for an additional 60 min ( replenishment ) . The cell lysates were subjected to immunoblot analysis with the indicated antibodies . ( B ) Wild - type and FIP200 − / − MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A1 . The cell lysates were subjected to immunoblot analysis with anti - LC3 antibody . ( C ) Wild - type and FIP200 − / − MEFs were transfected with retroviral vectors encoding GFP - Atg5 or GFP - LC3 . Resulting cells were cultured in the starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy . Bar , 20 μm . ( D - G ) Wild - type ( D and E ) and FIP200 − / − ( F and G ) MEFs were cultured in complete ( D and F ) or starvation ( E and G ) medium for 120 min and then fixed and subjected to EM analysis . Autophagosome - like structures ( open arrowheads ) , and autolysosomes ( closed arrowheads ) are indicated . Bar , 1 μm . ( H ) The ratio of total area of autophagosomes ( AP ) and autolysosomes ( AL ) to total cytoplasmic area in D - G was determined by morphometric analysis .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5.(A) FIP200+/+, FIP200−/−, Atg5+/+, and Atg5−/− MEFs were cultured in complete or starvation medium for 60 min. In the recovery experiments, starved MEFs were cultured in fresh complete medium for an additional 60 min (replenishment). The cell lysates were subjected to immunoblot analysis with the indicated antibodies.(B) Wild-type and FIP200−/− MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.(C) Wild-type and FIP200−/− MEFs were transfected with retroviral vectors encoding GFP-Atg5 or GFP-LC3. Resulting cells were cultured in the starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm.(D-G) Wild-type (D and E) and FIP200−/− (F and G) MEFs were cultured in complete (D and F) or starvation (E and G) medium for 120 min and then fixed and subjected to EM analysis. Autophagosome-like structures (open arrowheads), and autolysosomes (closed arrowheads) are indicated. Bar, 1 μm. (H) The ratio of total area of autophagosomes (AP) and autolysosomes (AL) to total cytoplasmic area in D-G was determined by morphometric analysis.


Figure 6 . ( A ) FAK + / − and FAK − / − MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A1 . The cell lysates were subjected to immunoblot analysis with the indicated antibodies . ( B ) FAK + / − and FAK − / − MEFs stably expressing GFP - LC3 were cultured in complete or starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy . ( C ) Wild - type MEFs stably expressing GFP - FAK were cultured in complete or starvation medium for 120 min . The cells were fixed and subjected to immunofluorescence microscopy using anti - Atg16 antibody . Black squares indicate the enlarged areas shown in insets . Bars , 20 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6.(A) FAK+/− and FAK−/− MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with the indicated antibodies.(B) FAK+/− and FAK−/− MEFs stably expressing GFP-LC3 were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy.(C) Wild-type MEFs stably expressing GFP-FAK were cultured in complete or starvation medium for 120 min. The cells were fixed and subjected to immunofluorescence microscopy using anti-Atg16 antibody. Black squares indicate the enlarged areas shown in insets. Bars, 20 μm.


Figure 7 . ( A ) Wild - type and FIP200 − / − MEFs were treated with 100 ng / ml rapamycin ( rapa ) or vehicle ( DMSO ) for 120 min in the presence or absence of 100 nM bafilomycin A1 . The cell lysates were subjected to immunoblot analysis with anti - LC3 antibody . ( B ) Wild - type and FIP200 − / − MEFs stably expressing GFP - Atg5 or GFP - LC3 were cultured in the presence of 100 ng / ml rapamycin for 120 min . The formation of GFP - Atg5 ( top ) and GFP - LC3 ( bottom ) puncta was examined by fluorescence microscopy . Bar , 20 μm . ( C ) Wild - type and FIP200 − / − MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C2 - ceramide for 2 h .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7.(A) Wild-type and FIP200−/− MEFs were treated with 100 ng/ml rapamycin (rapa) or vehicle (DMSO) for 120 min in the presence or absence of 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.(B) Wild-type and FIP200−/− MEFs stably expressing GFP-Atg5 or GFP-LC3 were cultured in the presence of 100 ng/ml rapamycin for 120 min. The formation of GFP-Atg5 (top) and GFP-LC3 (bottom) puncta was examined by fluorescence microscopy. Bar, 20 μm.(C) Wild-type and FIP200−/− MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C2-ceramide for 2 h.


Figure 8 . ( A ) FIP200 + / − and FIP200 − / − MEFs stably expressing GFP - ULK1 or - ULK2 were cultured in starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy . Bar , 20 μm . ( B ) FIP200 + / + and FIP200 − / − MEFs were cultured in complete medium . The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 ( loading control ) . ( C ) Phosphatase sensitivity of ULK1 . FIP200 + / + and FIP200 − / − MEFs were cultured in complete medium . ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30 ° C in the absence or presence of phosphatase inhibitors ( 1 mM Na2VO4 , 50 mM KF , 15 mM Na4P2O7 , and 1 mM EGTA ) .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8.(A) FIP200+/− and FIP200−/− MEFs stably expressing GFP-ULK1 or -ULK2 were cultured in starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm.(B) FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 (loading control).(C) Phosphatase sensitivity of ULK1. FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30°C in the absence or presence of phosphatase inhibitors (1 mM Na2VO4, 50 mM KF, 15 mM Na4P2O7, and 1 mM EGTA).


Figure 1Example images showing the localization of SOX2 - SNAP and OCT4 - HALO by fluorescence microscopy of SBROS cells . Scale bar : 50µm . Correlation between OCT4 - HALO and total OCT4 levels determined by immunofluorescence and HALO labelling . R is Pearson ' s correlation coefficient . Distributions of OCT4 ( n = 2682 ) , SOX2 ( n = 1236 ) and NANOG ( n = 2416 ) levels in WT E14 and SBROS cell lines as determined by quantitative immunofluorescence . Dashed lines : mean protein levels ; Dotted lines : median protein levels . Protein half - lives of OCT4 - HALO and SOX2 - SNAP in SBROS cells . Whiskers : minimum and maximum values ; Box : lower and upper quartiles ; Solid line : median . Black dots : outliers . Cell cycle duration of E14 WT ( n = 6 ) and SBROS ( n = 8 ) cells . Error bars : SE . p - value : two - sided t - test with unequal variance .

O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Example images showing the localization of SOX2-SNAP and OCT4-HALO by fluorescence microscopy of SBROS cells. Scale bar: 50µm.Correlation between OCT4-HALO and total OCT4 levels determined by immunofluorescence and HALO labelling. R is Pearson's correlation coefficient.Distributions of OCT4 (n=2682), SOX2 (n=1236) and NANOG (n=2416) levels in WT E14 and SBROS cell lines as determined by quantitative immunofluorescence. Dashed lines: mean protein levels; Dotted lines: median protein levels.Protein half-lives of OCT4-HALO and SOX2-SNAP in SBROS cells. Whiskers: minimum and maximum values; Box: lower and upper quartiles; Solid line: median. Black dots: outliers.Cell cycle duration of E14 WT (n=6) and SBROS (n=8) cells. Error bars: SE. p-value: two-sided t-test with unequal variance.


Figure 2Endogenous SOX2 levels in single cells ( turquoise ) normalized to the value at t = 0 in SNSF cells upon induction of SOX2 - SNAP ( n = 37 , left ) or a truncated SOX2 version missing the DNA binding domain ( YPET - SOX2 - delDBD , n = 47 , right ) . Black line : average SOX2 - SNAP levels after induction normalized to the maximum level ( n = 22 ) ; Yellow line : average YPET - SOX2 - delDBD levels after induction , normalized to the maximum level ( n = 20 ) . Sox2 and Oct4 mRNA levels upon overexpression of YPet - SOX2 or YPET - SOX2 - delDBD ( n = 3 ) . p - values : two - sided t - test with unequal variance . Changes of OCT4 half - life upon SOX2 - SNAP or YPET - SOX2 - delDBD overexpression ( n = 20 cells each ) . p - values : Mann - Whitney U test . SBROS cells harbouring inducible SOX2 - SNAP or YPET - SOX2 - delDBD were treated with 0 or 500 ng / ml dox for 6 - 8 hours followed by pulse - labelling of OCT4 - HALO and live - cell imaging . Sorting strategy to evaluate the impact of endogenous fluctuations of SOX2 and OCT4 on OCT4 and SOX2 protein levels , respectively . Changes of SOX2 and OCT4 levels after sorting for high and low SOX2 levels in cells expressing intermediate OCT4 levels ( n = 4 ) . Error bars : SE ; p - values : one - sided t - test with unequal variance . Changes of SOX2 and OCT4 levels after sorting for high and low OCT4 levels in cells expressing intermediate SOX2 levels ( n = 4 ) . p - values : one - sided t - test with unequal variance . Representative traces of the absolute number of SOX2 - NLUC molecules ( top ) and inferred concentration in nM ( bottom ) in in - silico synchronized cells ( n = 10 ) . Single cells were ranked according to their SOX2 expression at t = 0 and assigned a color code for their initial rank from low ( blue ) to high ( red ) levels , and changes in ranks over time are shown ( n = 59 ) . Rank - based autocorrelation function of the SOX2 ranks ( n = 59 ) . Error bars : SE estimated by bootstrapping . Representative single cell traces of the integrated intensity of OCT4 - HALO in single cells ( top ) and the corresponding inferred concentration ( bottom ) in in - silico synchronized cells ( n = 10 ) . Representation of the ranks as in G . Rank - based autocorrelation function as in I ( n = 47 ) . Error bars : SE estimated by bootstrapping .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Endogenous SOX2 levels in single cells (turquoise) normalized to the value at t=0 in SNSF cells upon induction of SOX2-SNAP (n=37, left) or a truncated SOX2 version missing the DNA binding domain (YPET-SOX2-delDBD, n=47, right). Black line: average SOX2-SNAP levels after induction normalized to the maximum level (n=22); Yellow line: average YPET-SOX2-delDBD levels after induction, normalized to the maximum level (n=20).Sox2 and Oct4 mRNA levels upon overexpression of YPet-SOX2 or YPET-SOX2-delDBD (n=3). p-values: two-sided t-test with unequal variance.Changes of OCT4 half-life upon SOX2-SNAP or YPET-SOX2-delDBD overexpression (n=20 cells each). p-values: Mann-Whitney U test. SBROS cells harbouring inducible SOX2-SNAP or YPET-SOX2-delDBD were treated with 0 or 500 ng/ml dox for 6-8 hours followed by pulse-labelling of OCT4-HALO and live-cell imaging.Sorting strategy to evaluate the impact of endogenous fluctuations of SOX2 and OCT4 on OCT4 and SOX2 protein levels, respectively.Changes of SOX2 and OCT4 levels after sorting for high and low SOX2 levels in cells expressing intermediate OCT4 levels (n=4). Error bars: SE; p-values: one-sided t-test with unequal variance.Changes of SOX2 and OCT4 levels after sorting for high and low OCT4 levels in cells expressing intermediate SOX2 levels (n=4). p-values: one-sided t-test with unequal variance.Representative traces of the absolute number of SOX2-NLUC molecules (top) and inferred concentration in nM (bottom) in in-silico synchronized cells (n=10). Single cells were ranked according to their SOX2 expression at t=0 and assigned a color code for their initial rank from low (blue) to high (red) levels, and changes in ranks over time are shown (n=59). Rank-based autocorrelation function of the SOX2 ranks (n=59). Error bars: SE estimated by bootstrapping.Representative single cell traces of the integrated intensity of OCT4-HALO in single cells (top) and the corresponding inferred concentration (bottom) in in-silico synchronized cells (n=10). Representation of the ranks as in G. Rank-based autocorrelation function as in I (n=47). Error bars: SE estimated by bootstrapping.


Figure 3Snapshot from luminescence movie of SNSF cells in differentiation . White arrowheads : cells starting to express SOX1 - P2A - FLUC ; Orange arrowheads : cells staying SOX1 - negative throughout the experiment . Red : SOX2 - NLUC , turquoise : Sox1 - P2A - FLUC . Scale bar : 50µm . Single cell traces of SOX2 levels in differentiating cells . Turquoise traces : cells becoming SOX1 - positive in the middle cell cycle , population average is shown in bold black ; Red traces : cells remaining SOX1 - negative throughout the experiment , population average is shown in bold ; Green line : population average FLUC signal in cells that become SOX1 - positive . * p < 0 . 05 determined by two - sided t - test with unequal variance . Strategies to sort SBROS cell populations with different SOX2 and OCT4 levels . Differentiation outcome for cells sorted for different SOX2 levels ( n = 5 for low and high , n = 4 for medium ) . p - values : two - sided t - test with unequal variance . Differentiation outcome for cells sorted for different OCT4 levels ( n = 4 ) . p - values : two - sided t - test with unequal variance . Differentiation outcome of uninduced cells or cells induced 12 hours for OCT4 - SNAP overexpression before sorting in G1 phase ( n = 6 ) . p - values : two - sided t - test with unequal variance . Sorting strategy for SBROS cells in G1 or S phase and into the four following subpopulations : SOX2 high & OCT4 high ( SHOH ) , SOX2 high & OCT4 low ( SHOL ) , SOX2 low & OCT4 high ( SLOH ) or SOX2 low & OCT4 low ( SLOL ) . Differentiation outcome for cells sorted in G1 phase ( n = 5 ) . p - values : two - sided t - test with unequal variance ( SOX1 ) and Mann - Whitney U test ( BRA ) . Differentiation outcome for cells sorted in S phase ( n = 5 ) . p - values : Mann - Whitney U test ( SOX1 ) and two - sided t - test with unequal variance ( BRA ) . Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 3μM CHIR ( n = 7 ) . p - values : two - sided t - test with unequal variance . Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 1 μM SB431542 and 25 ng / ml bFGF ( n = 7 ) . p - values : two - sided t - test with unequal variance .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Snapshot from luminescence movie of SNSF cells in differentiation. White arrowheads: cells starting to express SOX1-P2A-FLUC; Orange arrowheads: cells staying SOX1-negative throughout the experiment. Red: SOX2-NLUC, turquoise: Sox1-P2A-FLUC. Scale bar: 50µm.Single cell traces of SOX2 levels in differentiating cells. Turquoise traces: cells becoming SOX1-positive in the middle cell cycle, population average is shown in bold black; Red traces: cells remaining SOX1-negative throughout the experiment, population average is shown in bold; Green line: population average FLUC signal in cells that become SOX1-positive. * p<0.05 determined by two-sided t-test with unequal variance.Strategies to sort SBROS cell populations with different SOX2 and OCT4 levels. Differentiation outcome for cells sorted for different SOX2 levels (n=5 for low and high, n=4 for medium). p-values: two-sided t-test with unequal variance. Differentiation outcome for cells sorted for different OCT4 levels (n=4). p-values: two-sided t-test with unequal variance.Differentiation outcome of uninduced cells or cells induced 12 hours for OCT4-SNAP overexpression before sorting in G1 phase (n=6). p-values: two-sided t-test with unequal variance.Sorting strategy for SBROS cells in G1 or S phase and into the four following subpopulations: SOX2 high & OCT4 high (SHOH), SOX2 high & OCT4 low (SHOL), SOX2 low & OCT4 high (SLOH) or SOX2 low & OCT4 low (SLOL).Differentiation outcome for cells sorted in G1 phase (n=5). p-values: two-sided t-test with unequal variance (SOX1) and Mann-Whitney U test (BRA). Differentiation outcome for cells sorted in S phase (n=5). p-values: Mann-Whitney U test (SOX1) and two-sided t-test with unequal variance (BRA).Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 3μM CHIR (n=7). p-values: two-sided t-test with unequal variance.Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 1 μM SB431542 and 25 ng/ml bFGF (n=7). p-values: two-sided t-test with unequal variance.


Figure 4Example tracks of the ATAC - seq signal in SHOH , SHOL , SLOH and SLOL populations for loci where chromatin accessibility is unaffected , affected by OCT4 levels , SOX2 levels , or affected by both . Heatmap of relative chromatin accessibility in SHOH / SHOL / SLOH / SLOL samples for all loci with significantly altered accessibility , with examples of genes close to affected regulatory regions . Groups are defined by their significance ( OHvsOL / SHvsSL / SHOHvsSLOL ) and fold - change direction . Percentage of regions in each group showing an overlap with OCT4 , SOX2 and H3K4me3 ChIP - seq peaks .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 4Example tracks of the ATAC-seq signal in SHOH, SHOL, SLOH and SLOL populations for loci where chromatin accessibility is unaffected, affected by OCT4 levels, SOX2 levels, or affected by both.Heatmap of relative chromatin accessibility in SHOH/SHOL/SLOH/SLOL samples for all loci with significantly altered accessibility, with examples of genes close to affected regulatory regions. Groups are defined by their significance (OHvsOL/SHvsSL/SHOHvsSLOL) and fold-change direction.Percentage of regions in each group showing an overlap with OCT4, SOX2 and H3K4me3 ChIP-seq peaks.


Figure 1B . Comparison of ΔPrP expression levels . Representative immunoblot is shown for detection of ( 3F4 ) MoPrP and ΔPrPs in transiently transfected N2a cells using mAb 3F4 . ΔPrPs are similar in expression level glycosylation appearance , except for Δ159 - 167 which has an extra fragment larger than the diglycoform ( lane 7 , arrow ) . C . ΔPrPs are not converted into PK - resistant PrP in prion - infected cells . 22L - ScN2a cells were transiently transfected with ( 3F4 ) MoPrP , Δ159 or Δ159 - 175 , and cell lysates digested with PK or not . Immunoblot was done using mAb 3F4 . . D . Pilot study for dominant - negative inhibition ( DNI ) of Δ159 and Δ159 - 175 . DNI was assessed by co - transfecting 22L - ScN2a cells with ( 3F4 ) MoPrP and Δ159 or Δ159 - 175 , respectively , and testing for PK - resistant ( 3F4 ) MoPrP . Left panel shows representative immunoblot ( mAb 3F4 ) and right panel the statistical analysis of quantified PK - res levels of a triplicate experiment . Empty pcDNA3 . 1 plasmid was used as control for co - transfection in lane 1 . The error bars indicate standard deviation . * , p < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Comparison of ΔPrP expression levels. Representative immunoblot is shown for detection of (3F4)MoPrP and ΔPrPs in transiently transfected N2a cells using mAb 3F4. ΔPrPs are similar in expression level glycosylation appearance, except for Δ159-167 which has an extra fragment larger than the diglycoform (lane 7, arrow).C. ΔPrPs are not converted into PK-resistant PrP in prion-infected cells. 22L-ScN2a cells were transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, and cell lysates digested with PK or not. Immunoblot was done using mAb 3F4.. D. Pilot study for dominant-negative inhibition (DNI) of Δ159 and Δ159-175. DNI was assessed by co-transfecting 22L-ScN2a cells with (3F4)MoPrP and Δ159 or Δ159-175, respectively, and testing for PK-resistant (3F4)MoPrP. Left panel shows representative immunoblot (mAb 3F4) and right panel the statistical analysis of quantified PK-res levels of a triplicate experiment. Empty pcDNA3.1 plasmid was used as control for co-transfection in lane 1. The error bars indicate standard deviation. *, p<0.05.


Figure 2A . N - linked glycans of ΔPrPs are not complex - type . Immunoblot comparing EndoH - and non - digested lysates of ΔPrP159 and Δ159 - 175 transfected cells . Left panel was probed with mAb 3F4 mAb , detecting transiently expressed ΔPrPs . On the right panel , the membrane was re - probed with mAb 4H11 , detecting total PrP . Arrowheads depict deglycosylated ΔPrPs . B . The additional band of Δ159 - 167 represents a third N - glycan sensitive to EndoH . Upon EndoH digestion , all bands converge into a non - glycoform band ( lane 3 and 4 ) . In Δ159 - 167 ( 169 ) the third glycosylation site is not present anymore . C . ΔPrPs are mainly distributed in intracellular compartments rather than on the cell surface . Confocal microscopy images of N2a cells transiently transfected with ( 3F4 ) MoPrP , Δ159 or Δ159 - 175 , either permeabilized ( right ) or non - permeabilized ( left ) with 0 . 2 % TX100 after fixation . Cells were immuno - labeled with mAb 3F4 . ( 3F4 ) MoPrP - expressing cells show bright immunopositivity on the cell surface , whereas ΔPrPs are not observed on the cell surface . Also intracellular staining differs strongly between ( 3F4 ) MoPrP and ΔPrP expressing cells Scale bars , 25 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. N-linked glycans of ΔPrPs are not complex-type. Immunoblot comparing EndoH- and non-digested lysates of ΔPrP159 and Δ159-175 transfected cells. Left panel was probed with mAb 3F4 mAb, detecting transiently expressed ΔPrPs. On the right panel, the membrane was re-probed with mAb 4H11, detecting total PrP. Arrowheads depict deglycosylated ΔPrPs.B. The additional band of Δ159-167 represents a third N-glycan sensitive to EndoH. Upon EndoH digestion, all bands converge into a non-glycoform band (lane 3 and 4). In Δ159-167(169) the third glycosylation site is not present anymore.C. ΔPrPs are mainly distributed in intracellular compartments rather than on the cell surface. Confocal microscopy images of N2a cells transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, either permeabilized (right) or non-permeabilized (left) with 0.2% TX100 after fixation. Cells were immuno-labeled with mAb 3F4. (3F4)MoPrP-expressing cells show bright immunopositivity on the cell surface, whereas ΔPrPs are not observed on the cell surface. Also intracellular staining differs strongly between (3F4)MoPrP and ΔPrP expressing cells Scale bars, 25 µm.


Figure 3A . ΔPrPs are insoluble in detergent solution . Immunoblots showing partition of Δ159 and Δ159 - 175 in supernatant ( Sup ) and pellet fraction after ultracentrifugation of lysates supplemented with 4 % sarcosyl . Upper panel is mAb 3F4 , lower panel is membrane re - probed with mAb 4H11 detecting also endogenous wild - type PrP besides ΔPrPs . The majority of ΔPrPs is found in the insoluble fraction in both infected and non - infected cells ( upper panel , lanes 3 , 4 , 7 and 8 ) . Wild - type PrP almost completely partitions into the soluble fraction ( Sup ) in non - infected N2a cells with present conditions . PrP in pellet fractions of non - infected N2a cells is from ΔPrPs as can be seen from the banding pattern ( Lane 7 and 8 , square bracket ) . PrP in lanes 3 and 4 ( lower panel ) is PrP27 - 30 . B . ΔPrPs are moderately resistant to chymotrypsin digestion . Immunoblots showing chymotrypsin - resistant fragments of Δ159 and Δ159 - 175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin ( Chy ) . Note that full - length diglycoform of ΔPrP still remains at 8 µg / ml Chy ( lanes 5 and 10 , upper panel , closed arrowhead ) , whereas that of endogenous wild - type PrP is completely digested ( lanes 5 and 10 , lower panel , open arrowhead ) and only small amounts of truncated fragments remained ( curly bracket ) . Other smaller fragments in lane 5 and 10 ( square - brackets ) represent ΔPrPs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. ΔPrPs are insoluble in detergent solution. Immunoblots showing partition of Δ159 and Δ159-175 in supernatant (Sup) and pellet fraction after ultracentrifugation of lysates supplemented with 4% sarcosyl. Upper panel is mAb 3F4, lower panel is membrane re-probed with mAb 4H11 detecting also endogenous wild-type PrP besides ΔPrPs. The majority of ΔPrPs is found in the insoluble fraction in both infected and non-infected cells (upper panel, lanes 3, 4, 7 and 8). Wild-type PrP almost completely partitions into the soluble fraction (Sup) in non-infected N2a cells with present conditions. PrP in pellet fractions of non-infected N2a cells is from ΔPrPs as can be seen from the banding pattern (Lane 7 and 8, square bracket). PrP in lanes 3 and 4 (lower panel) is PrP27-30.B. ΔPrPs are moderately resistant to chymotrypsin digestion. Immunoblots showing chymotrypsin-resistant fragments of Δ159 and Δ159-175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin (Chy). Note that full-length diglycoform of ΔPrP still remains at 8 µg/ml Chy (lanes 5 and 10, upper panel, closed arrowhead), whereas that of endogenous wild-type PrP is completely digested (lanes 5 and 10, lower panel, open arrowhead) and only small amounts of truncated fragments remained (curly bracket). Other smaller fragments in lane 5 and 10 (square-brackets) represent ΔPrPs.


Figure 4A . ΔPrPs are substantially soluble in detergent without GdnHCl denaturation . Immunoblots probed with mAbs 3F4 ( left ) or 4H11 ( right , membrane re - probed ) showing ΔPrPs and endogenous PrP in detergent and aqueous phases of TX114 lysates prepared from N2a cells transfected with Δ159 or Δ159 - 175 . Det denotes detergent phase , Aq aqueous phase . B . ΔPrPs are highly hydrophobic after denaturation with GdnHCl . Representative immunoblot of TX114 - treated lysates after denaturation with 3 M GdnHCl and developed with mAb 3F4 showing Δ159 and Δ159 - 175 almost exclusively in detergent phase ( lanes 1 - 4 ) . PIPLC treatment ( + , even lanes ) was done after denaturation with GdnHCl or not ( − , uneven lanes ) . PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions . C . Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion . After further diluting out GdnHCl by phase - separation and removal of the aqueous phase containing GdnHCl , PIPLC was able to digest ΔPrPs . Left two panels ( upper mAb 3F4 , lower 4H11 ) are shorter expositions , right panels longer expositions to show also difference in PrP levels in the detergent phase . Under these conditions a substantial reduction of ΔPrPs in the detergent phase was achieved by PIPLC digestion ( e . g . upper - left panel , lanes 1 , 3 vs . 2 , 4 ) . Migration of ΔPrPs in the aqueous phase of PIPLC - digested samples was slightly slower than that of PIPLC - undigested samples ( − ) ( upper - right panel , lane 9 vs . 10 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. ΔPrPs are substantially soluble in detergent without GdnHCl denaturation. Immunoblots probed with mAbs 3F4 (left) or 4H11 (right, membrane re-probed) showing ΔPrPs and endogenous PrP in detergent and aqueous phases of TX114 lysates prepared from N2a cells transfected with Δ159 or Δ159-175. Det denotes detergent phase, Aq aqueous phase.B. ΔPrPs are highly hydrophobic after denaturation with GdnHCl. Representative immunoblot of TX114-treated lysates after denaturation with 3 M GdnHCl and developed with mAb 3F4 showing Δ159 and Δ159-175 almost exclusively in detergent phase (lanes 1-4). PIPLC treatment (+, even lanes) was done after denaturation with GdnHCl or not (−, uneven lanes). PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions.C. Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion. After further diluting out GdnHCl by phase-separation and removal of the aqueous phase containing GdnHCl, PIPLC was able to digest ΔPrPs. Left two panels (upper mAb 3F4, lower 4H11) are shorter expositions, right panels longer expositions to show also difference in PrP levels in the detergent phase. Under these conditions a substantial reduction of ΔPrPs in the detergent phase was achieved by PIPLC digestion (e.g. upper-left panel, lanes 1, 3 vs. 2, 4). Migration of ΔPrPs in the aqueous phase of PIPLC-digested samples was slightly slower than that of PIPLC-undigested samples (−) (upper-right panel, lane 9 vs. 10).


Figure 5A . Degradation of ΔPrPs by lysosomal and proteasomal systems . ( Left ) N2a cells transiently transfected with Δ159 or Δ159 - 175 were treated for 7 hours with inhibitors either for lysosomal degradation ( bafilomycin A1 , Baf , 120 nM ) , autophagy ( 3 - methyladenine , 3MA , 10 mM ) , or the proteasome ( MG132 , 10 µM ) and cell lysates analyzed in immunoblot for expression of Δ159 and Δ159 - 175 ( mAb 3F4 ) . Square bracket and bracket denote diglycoforms and nonglycoforms of ΔPrPs , respectively . An increment in diglycoforms is observed in Baf - and 3MA - treated cells , whereas in MG132 - treated cells also the non - glycoforms are increased . ( Right ) A graph showing quantification of all PrP bands by densitometric analysis . Data from 3 independent ( one in duplicate ) experiments for Δ159 and 2 independent ( one in duplicate ) for Δ159 - 175 were statistically analyzed for mean and standard deviation ( error bars ) . B . ΔPrPs can be localized in LAMP1 - immunopositive vesicles . Immunofluorescence analysis shows distribution of ΔPrP ( mAb 3F4 ) and LAMP1 in cells with or without 6M GdnHCl antigen - retrieval treatment . N2a cells transfected with ΔPrP159 were fixed , permeabilized and incubated without ( upper panel ) or with 6M GdnHCl ( lower panel ) . With GndHCl treatment ΔPrP is diffusely distributed ( arrowheads ) and co - localizes poorly with LAMP1 . After GdnHCl treatment ( lower panel ) , bright 3F4 - immunopositive puncta ( arrows ) are observed which partly co - localize with LAMP1 - positive structures ( merge ) . Scale bars , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Degradation of ΔPrPs by lysosomal and proteasomal systems. (Left) N2a cells transiently transfected with Δ159 or Δ159-175 were treated for 7 hours with inhibitors either for lysosomal degradation (bafilomycin A1, Baf, 120 nM), autophagy (3-methyladenine, 3MA, 10 mM), or the proteasome (MG132, 10 µM) and cell lysates analyzed in immunoblot for expression of Δ159 and Δ159-175 (mAb 3F4). Square bracket and bracket denote diglycoforms and nonglycoforms of ΔPrPs, respectively. An increment in diglycoforms is observed in Baf- and 3MA-treated cells, whereas in MG132-treated cells also the non-glycoforms are increased. (Right) A graph showing quantification of all PrP bands by densitometric analysis. Data from 3 independent (one in duplicate) experiments for Δ159 and 2 independent (one in duplicate) for Δ159-175 were statistically analyzed for mean and standard deviation (error bars).B. ΔPrPs can be localized in LAMP1-immunopositive vesicles. Immunofluorescence analysis shows distribution of ΔPrP (mAb 3F4) and LAMP1 in cells with or without 6M GdnHCl antigen-retrieval treatment. N2a cells transfected with ΔPrP159 were fixed, permeabilized and incubated without (upper panel) or with 6M GdnHCl (lower panel). With GndHCl treatment ΔPrP is diffusely distributed (arrowheads) and co-localizes poorly with LAMP1. After GdnHCl treatment (lower panel), bright 3F4-immunopositive puncta (arrows) are observed which partly co-localize with LAMP1-positive structures (merge). Scale bars, 10 µm.


Figure 6A . DNI efficiencies of ΔPrPs are inversely correlated to deletion size . Representative immunoblot showing PK - resistant PrPSc moiety of ( 3F4 ) MoPrP co - expressed with indicated ΔPrPs in 22L - ScN2a cells . Quantitative analysis is shown below as scheme . Relative PrPSc levels were calculated as proportion of PK - resistant ( 3F4 ) MoPrP co - transfected with each ΔPrP to that of co - transfected with empty vector control in the same experiment . Data from 4 ( for Δ159 - 165 , Δ159 - 167 and Δ159 - 167 ( 169 ) only 3 ) independent experiments were statistically analyzed for mean and standard deviation ( error bars ) . B . DNI of ΔPrP with a deletion in the C - terminal part of H1 ∼ H2 . Δ171 - 175 ( see Fig . 7A ) was transiently expressed in N2a cells ( left panel ) and DNI efficiency evaluated in 22L - ScN2a cells ( right panel ) ( mAb 3F4 ) . Despite the much shorter deletion size , Δ171 - 175 exerted only inefficient DNI , similar to Δ159 - 175 ( right panel ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. DNI efficiencies of ΔPrPs are inversely correlated to deletion size. Representative immunoblot showing PK-resistant PrPSc moiety of (3F4)MoPrP co-expressed with indicated ΔPrPs in 22L-ScN2a cells. Quantitative analysis is shown below as scheme. Relative PrPSc levels were calculated as proportion of PK-resistant (3F4)MoPrP co-transfected with each ΔPrP to that of co-transfected with empty vector control in the same experiment. Data from 4 (for Δ159-165, Δ159-167 and Δ159-167(169) only 3) independent experiments were statistically analyzed for mean and standard deviation (error bars).B. DNI of ΔPrP with a deletion in the C-terminal part of H1∼H2. Δ171-175 (see Fig. 7A) was transiently expressed in N2a cells (left panel) and DNI efficiency evaluated in 22L-ScN2a cells (right panel) (mAb 3F4). Despite the much shorter deletion size, Δ171-175 exerted only inefficient DNI, similar to Δ159-175 (right panel).


Figure 7 . B . Δ31 - 160 variants are expressed at comparable levels and have EndoH - sensitive and - resistant N - glycans . Immunoblot comparing EndoH - digested and non - digested samples from N2a cells transiently transfected with Δ31 - 160 variants , probed with anti - PrP mAb C16 - S raised against the C - terminal portion of H3 . Non - Tf , samples prepared from N2a cells without transfection . Arrowhead denotes deglycosylated Δ159 ( lane 11 ) , bracket deglycosylated fragments , square bracket EndoH - resistant fragments . Asterisks indicate non - specific bands . Δ31 - 160 variants are not completely deglycosylated by EndoH , resulting in EndoH - resistant fragments ( square bracket ) which are observed also in reducing conditions ( right panel ; + / − dithiothreitol ( DTT ) treatment ) . Note that endogenous wild - type PrPc was not detected under used conditions . C . DNI of Δ31 - 160 variants is similarly inversely related to size of deletion and dependent on intact C - terminal H1 - H2 portion . Immunoblot probed with mAb 3F4 showing PK - resistant ( 3F4 ) MoPrP co - transfected with indicated constructs into 22L - ScN2a cells ( left panel ) . Δ31 - 160 was slightly more effective than Δ159 . Right panel shows quantification of results as obtained from a triplicate experiment , using densitometry on ImageJ . Bars illustrate mean ± standard deviation . * , p < 0 . 05 . D . Δ31 - 160 forms spontaneous aggregates with moderate chymotrypsin ( Chy ) resistance . Immunoblots probed with mAbs C16 - S ( upper panel ) or 3F4 ( lower panel ) for comparing Chy - resistant fragments of Δ31 - 160 with that of Δ159 . Lysates from N2a cells expressing Δ159 or Δ31 - 160 were digested with indicated concentrations of Chy for 30 minutes and then digested with PNGaseF . A sample from non - transfected N2a cells is shown in the lane 5 . Asterisks indicate non - specific C16 - S bands . Arrowhead denotes deglycoform of Δ159 , bracket deglycoform of Δ31 - 160 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7. B. Δ31-160 variants are expressed at comparable levels and have EndoH-sensitive and -resistant N-glycans. Immunoblot comparing EndoH-digested and non-digested samples from N2a cells transiently transfected with Δ31-160 variants, probed with anti-PrP mAb C16-S raised against the C-terminal portion of H3. Non-Tf, samples prepared from N2a cells without transfection. Arrowhead denotes deglycosylated Δ159 (lane 11), bracket deglycosylated fragments, square bracket EndoH-resistant fragments. Asterisks indicate non-specific bands. Δ31-160 variants are not completely deglycosylated by EndoH, resulting in EndoH-resistant fragments (square bracket) which are observed also in reducing conditions (right panel; +/− dithiothreitol (DTT) treatment). Note that endogenous wild-type PrPc was not detected under used conditions.C. DNI of Δ31-160 variants is similarly inversely related to size of deletion and dependent on intact C-terminal H1-H2 portion. Immunoblot probed with mAb 3F4 showing PK-resistant (3F4)MoPrP co-transfected with indicated constructs into 22L-ScN2a cells (left panel). Δ31-160 was slightly more effective than Δ159. Right panel shows quantification of results as obtained from a triplicate experiment, using densitometry on ImageJ. Bars illustrate mean ± standard deviation. *, p<0.05.D. Δ31-160 forms spontaneous aggregates with moderate chymotrypsin (Chy) resistance. Immunoblots probed with mAbs C16-S (upper panel) or 3F4 (lower panel) for comparing Chy-resistant fragments of Δ31-160 with that of Δ159. Lysates from N2a cells expressing Δ159 or Δ31-160 were digested with indicated concentrations of Chy for 30 minutes and then digested with PNGaseF. A sample from non-transfected N2a cells is shown in the lane 5. Asterisks indicate non-specific C16-S bands. Arrowhead denotes deglycoform of Δ159, bracket deglycoform of Δ31-160.


Figure 1A , A targeted MAPPIT screen identifies several DUBs as putative RAS interactors . A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait . pSEL ( + 2L ) - HRAS G12V was expressed in HEK293T cells together with the indicated prey . BRAF served as a positive control . Each measurement was done in triplicate . The results are expressed as a mean of normalized luciferase activity ( leptin - treated cells vs leptin - untreated cells ) . The overall mean value + 2 s . d . served as a threshold . B , MAPPIT assay confirms the interaction between OTUB1 and RAS proteins . pSEL ( + 2L ) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey . Empty vector and two random baits , MAL and eDHFR , were used as negative controls . REM2 and EFHA1 preys that bind to the bait receptor itself was used to evaluate expression of the RAS baits . The results are expressed as a mean of normalized luciferase activity ± s . e . m ( leptin - treated cells vs leptin - untreated cells ) , n = 3 . C , NRAS Q61K mutant co - immunoprecipitates with OTUB1 . At 48 hours post - transfection with Flag - tagged NRAS Q61K and HA - tagged OTUB1 expression constructs , HA - tagged OTUB1 was immunoprecipitated with anti - HA - agarose followed by immunoblotting using anti - Flag or anti - HA antibodies . D , OTUB1 interacts with wt NRAS and active NRAS mutant . Flag - tagged NRAS proteins were immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells overexpressing HA - tagged OTUB1 or empty vector ( V ) . E , GTP binding does not affect the complex formation between NRAS and OTUB1 . Recombinant Flag - tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA - tagged OTUB1 in the excess of GTP - γ - S or GDP , followed by immunoblotting with the indicated antibodies . F , OTUB1 interacts with wt KRAS . Flag - tagged KRAS was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells overexpressing HA - tagged OTUB1 or empty vector ( V ) . C , D , E , F , Immunoprecipitates , IP . Whole cell lysate , WCL . G , OTUB1 co - localizes with KRAS at the plasmamembrane . At 24 hours after co - transfection with GFP - tagged KRAS and HA - tagged OTUB1 , HeLa cells were immunostained with anti - HA antibody . The outlined areas are shown at higher magnification at the top of each image . Scale bar , 10 micron .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, A targeted MAPPIT screen identifies several DUBs as putative RAS interactors. A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait. pSEL(+2L)-HRAS G12V was expressed in HEK293T cells together with the indicated prey. BRAF served as a positive control. Each measurement was done in triplicate. The results are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells). The overall mean value + 2 s.d. served as a threshold.B, MAPPIT assay confirms the interaction between OTUB1 and RAS proteins. pSEL(+2L) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey. Empty vector and two random baits, MAL and eDHFR, were used as negative controls. REM2 and EFHA1 preys that bind to the bait receptor itself was used to evaluate expression of the RAS baits. The results are expressed as a mean of normalized luciferase activity ± s.e.m (leptin-treated cells vs leptin-untreated cells), n=3.C, NRAS Q61K mutant co-immunoprecipitates with OTUB1. At 48 hours post-transfection with Flag-tagged NRAS Q61K and HA-tagged OTUB1 expression constructs, HA-tagged OTUB1 was immunoprecipitated with anti-HA-agarose followed by immunoblotting using anti-Flag or anti-HA antibodies.D, OTUB1 interacts with wt NRAS and active NRAS mutant. Flag-tagged NRAS proteins were immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V).E, GTP binding does not affect the complex formation between NRAS and OTUB1. Recombinant Flag-tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA-tagged OTUB1 in the excess of GTP-γ-S or GDP, followed by immunoblotting with the indicated antibodies.F, OTUB1 interacts with wt KRAS. Flag-tagged KRAS was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). C, D, E, F, Immunoprecipitates, IP. Whole cell lysate, WCL.G, OTUB1 co-localizes with KRAS at the plasmamembrane. At 24 hours after co-transfection with GFP-tagged KRAS and HA-tagged OTUB1, HeLa cells were immunostained with anti-HA antibody. The outlined areas are shown at higher magnification at the top of each image. Scale bar, 10 micron.


Figure 2A , Suppression of OTUB1 expression increases NRAS mono - and di - ubiquitination . 6xHis - tagged ubiquitin and Flag - NRAS were introduced into HEK293T cells expressing shGFP or shRNAs against OTUB1 . Ubiquitinated NRAS was purified by Co2 + metal affinity chromatography and detected by anti - Flag antibody . B , C , D , Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination . 6xHis - tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA - OTUB1 , the catalytically dead mutant HA - OTUB1 C91S , or empty vector ( V ) . Ubiquitinated RAS was purified by Co2 + metal affinity chromatography and detected by anti - Flag antibody . E , OTUB1 induces membrane RAS re - localization . Confocal imaging of HeLa cells expressing the indicated constructs . For each sample , z - stacks obtained by scanning the sample from the apical to the basal layer . Step - size , 2 micron . F , RAS cellular distribution expressed as percentage of cells with specific RAS localization . For quantification of RAS localization , cells were randomly imaged using IN Cell Analyzer . RAS localization ( > 200 cells ) was scored as intracellular and diffused ( IN ) , mostly at the plasmamembrane ( PM ) , or both intracellular and plasmamembrane ( PM / IN ) . p - value = 0 . 0005 as determined by Chi2 - test , n = 3 . Representative images of HeLa cells expressing GFP - tagged KRAS are shown in Appendix Figure S1 . Pull - down , PD . Whole cell lysate , WCL . Not modified , n . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, Suppression of OTUB1 expression increases NRAS mono- and di-ubiquitination. 6xHis-tagged ubiquitin and Flag-NRAS were introduced into HEK293T cells expressing shGFP or shRNAs against OTUB1. Ubiquitinated NRAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.B, C, D, Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination. 6xHis-tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA-OTUB1, the catalytically dead mutant HA-OTUB1 C91S, or empty vector (V). Ubiquitinated RAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.E, OTUB1 induces membrane RAS re-localization. Confocal imaging of HeLa cells expressing the indicated constructs. For each sample, z-stacks obtained by scanning the sample from the apical to the basal layer. Step-size, 2 micron.F, RAS cellular distribution expressed as percentage of cells with specific RAS localization. For quantification of RAS localization, cells were randomly imaged using IN Cell Analyzer. RAS localization (>200 cells) was scored as intracellular and diffused (IN), mostly at the plasmamembrane (PM), or both intracellular and plasmamembrane (PM/IN). p-value=0.0005 as determined by Chi2-test, n=3. Representative images of HeLa cells expressing GFP-tagged KRAS are shown in Appendix Figure S1. Pull-down, PD. Whole cell lysate, WCL. Not modified, n.m.


Figure 3A , B , OTUB1 overexpression promotes serum - induced activation of endogenous wt RAS ( A ) or wt NRAS ( B ) . GTP - bound RAS was pulled down from HEK293T cells expressing HA - tagged OTUB1 or empty vector ( V ) using recombinant RAF1 RBD conjugated to agarose beads . Input was controlled by immunoblotting using anti - panRAS or anti - Flag antibodiesC , D OTUB1 enhances serum - induced ERK1 / 2 phosphorylation in endogenous wt RAS ( C ) or wt KRAS ( D ) . Levels of phosphorylated ERK1 / 2 and total ERK1 / 2 in HEK293T cells expressing HA - tagged OTUB1 or empty vector ( V ) were analyzed by Meso Scale assay . The results are expressed as a mean of pERK1 / 2 levels relative to total ERK1 / 2 ± s . e . m . n = 2 . . E , F , Overexpression of wt OTUB1 ( E ) or the catalytically dead mutant OTUB1 C91S mutant ( F ) promotes serum - induced MAPK activation in cell expressing wt NRAS . Whole cell lysates were analyzed by immunoblotting using the indicated antibodies . G , H , Overexpression of OTUB1 has no effect on serum - induced MAPK activation in cell expressing mutant NRAS Q61K or KRAS G12V . Whole cell lysates were analyzed by immunoblotting using antibodies as indicated . A , B , C , D , E , F , G , H Serum - starved HEK293T cells expressing the indicated constructs were stimulated with 10 % serum for the indicated time periods . I , Immunoblot of OTUB1 overexpression in immortalized human embryonic kidney epithelial cells ( HEK TEST ) , expressing empty vector ( V ) , myristoylated AKT1 ( myr - AKT ) , MEK1 D218 , D222 mutant ( MEKDD ) . J , K , OTUB1 cooperates with active AKT1 to promote anchorage - independent growth . Representative images of soft - agar colonies formed by HEK TEST cells expressing the indicated constructs . The number of soft agar colonies formed by cells expressing OTUB1 compared to cells expressing an empty vector . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 2 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, B, OTUB1 overexpression promotes serum-induced activation of endogenous wt RAS (A) or wt NRAS (B). GTP-bound RAS was pulled down from HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) using recombinant RAF1 RBD conjugated to agarose beads. Input was controlled by immunoblotting using anti-panRAS or anti-Flag antibodiesC, D OTUB1 enhances serum-induced ERK1/2 phosphorylation in endogenous wt RAS (C) or wt KRAS (D). Levels of phosphorylated ERK1/2 and total ERK1/2 in HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m. n=2. . E, F, Overexpression of wt OTUB1 (E) or the catalytically dead mutant OTUB1 C91S mutant (F) promotes serum-induced MAPK activation in cell expressing wt NRAS. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies. G, H, Overexpression of OTUB1 has no effect on serum-induced MAPK activation in cell expressing mutant NRAS Q61K or KRAS G12V. Whole cell lysates were analyzed by immunoblotting using antibodies as indicated. A,B,C,D,E,F,G,H Serum-starved HEK293T cells expressing the indicated constructs were stimulated with 10% serum for the indicated time periods.I, Immunoblot of OTUB1 overexpression in immortalized human embryonic kidney epithelial cells (HEK TEST), expressing empty vector (V), myristoylated AKT1 (myr-AKT), MEK1 D218,D222 mutant (MEKDD).J, K, OTUB1 cooperates with active AKT1 to promote anchorage-independent growth. Representative images of soft-agar colonies formed by HEK TEST cells expressing the indicated constructs. The number of soft agar colonies formed by cells expressing OTUB1 compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=2.