( F ) Quantitative histomorphometry of melanin production . N = 9 - 10 anagen VI HFs from 4 different donors treated with siTSC2 or non - targeting oligos for 6 days . ( G ) Representative bright field microscopic images of Masson - Fontana histochemistry . Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mean ± SEM , Unpaired Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . Samples from each donor are represented by a different color .

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(F) Quantitative histomorphometry of melanin production. N = 9-10 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos for 6 days. (G) Representative bright field microscopic images of Masson-Fontana histochemistry. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color.


( H ) Quantitative analysis of tyrosinase activity . N = 8 - 9 anagen VI HFs from 4 different donors treated with siTSC2 or non - targeting oligos for 6 days . ( I ) Representative images of tyrosinase activity immunofluorescence . : Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mean ± SEM , Unpaired Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . Samples from each donor are represented by a different color . Nuclei stained with DAPI .

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(H) Quantitative analysis of tyrosinase activity. N = 8-9 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos for 6 days. (I) Representative images of tyrosinase activity immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color. Nuclei stained with DAPI.


( J ) Quantitative analysis of α - MSH expression . N = 8 - 9 anagen VI HFs from 4 different donors treated with siTSC2 or non - targeting oligos for 6 days . ( K ) Representative images of α - MSH immunofluorescence . Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mean ± SEM , Unpaired Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . Samples from each donor are represented by a different color . Nuclei stained with DAPI .

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(J) Quantitative analysis of α-MSH expression. N = 8-9 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos for 6 days. (K) Representative images of α-MSH immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color. Nuclei stained with DAPI.


( A ) Quantitative analysis of phosphorylated S6 ( p - S6 ) ( mTORC1 activity read - out ) immunoreactivity . N = 9 - 10 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( B ) Representative fluorescence images of p - S6 immunofluorescence . : Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Nuclei stained with DAPI .

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(A) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 9-10 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (B) Representative fluorescence images of p-S6 immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.


( C ) Hair cycle staging was performed using Ki - 67 and Masson - Fontana histochemistry . Mean ± SEM ; N = 16 - 17 grey HFs per groups from 3 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( D ) Representative fluorescence images of Ki - 67 and bright field microscopic images of Masson - Fontana . for C - D where all HFs were analyzed ) were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Nuclei stained with DAPI .

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(C) Hair cycle staging was performed using Ki-67 and Masson-Fontana histochemistry. Mean ± SEM; N = 16-17 grey HFs per groups from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (D) Representative fluorescence images of Ki-67 and bright field microscopic images of Masson-Fontana. for C-D where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.


( E ) Quantitative histomorphometry of melanin production by Masson - Fontana histochemistry . N = 7 - 9 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( F ) Representative pictures of Masson - Fontana histochemistry . Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mann - Whitney U -

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(E) Quantitative histomorphometry of melanin production by Masson-Fontana histochemistry. N = 7-9 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (F) Representative pictures of Masson-Fontana histochemistry. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mann-Whitney U-test


( G ) Quantitative analysis of tyrosinase activity . N = 7 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( H ) Representative images of tyrosinase activity immunofluorescence . : Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mann - Whitney U - test Nuclei stained with DAPI .

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(G) Quantitative analysis of tyrosinase activity. N = 7 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (H) Representative images of tyrosinase activity immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mann-Whitney U-test Nuclei stained with DAPI.


( I ) Quantitative analysis of α - MSH expression in defined reference area within the bulb . N = 5 - 7 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng / ml or untreated ( vehicle ) for 7 days . ( J ) Representative images of α - MSH immunofluorescence . Only anagen VI HFs were investigated and analyses performed in defined reference areas ( dotted areas ) in the HFPU . Mann - Whitney U - test Nuclei stained with DAPI .

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(I) Quantitative analysis of α-MSH expression in defined reference area within the bulb. N = 5-7 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (J) Representative images of α-MSH immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mann-Whitney U-test Nuclei stained with DAPI.


C - E . Western blot analysis for CCNA , CCNB , Cdk1 and GAPDH ( loading control ) protein expression in lysates from control ( si - GFP ) and p53 - depleted ( si - p53 ; si - p53 _ 1 ) ( C ) MDA - MB - 231 and CAL27 cell lines . Representative images are shown .

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C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and p53-depleted (si-p53; si-p53_1) (C) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


C - E . Western blot analysis for CCNA , CCNB , Cdk1 and GAPDH ( loading control ) protein expression in lysates from control ( si - GFP ) and YAP - depleted ( si - YAP ; si - YAP _ 1 ) ( D ) MDA - MB - 231 and CAL27 cell lines . Representative images are shown .

C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and YAP-depleted (si-YAP; si-YAP_1) (D) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


C - E . Western blot analysis for CCNA , CCNB , Cdk1 and GAPDH ( loading control ) protein expression in lysates from control ( si - GFP ) and TAZ - depleted ( si - TAZ ) ( E ) MDA - MB - 231 and CAL27 cell lines . Representative images are shown .

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C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and TAZ-depleted (si-TAZ) (E) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


A . Primary human breast cancers of the METABRIC dataset were stratified according to high or low YAP activity signature [ 47 ] and by TP53 mutational status , and then the levels of the cycline signature score were determined in the four groups . Cyclin activity is significantly higher in mut - p53 tumors with high levels of the YAP signature , as visualized by box - plot . Signature scores have been obtained summarizing the standardized expression levels of signature genes into a combined score with zero mean [ 7 ] . The values shown in graphs are thus adimensional . The bottom and top of the box are the first and third quartiles , and the band inside the box is the median ; whiskers represent 1st and 99th percentiles ; values lower and greater are shown as circles ( p < 0 . 0001 , n = 701 ) .

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A. Primary human breast cancers of the METABRIC dataset were stratified according to high or low YAP activity signature [47] and by TP53 mutational status, and then the levels of the cycline signature score were determined in the four groups. Cyclin activity is significantly higher in mut-p53 tumors with high levels of the YAP signature, as visualized by box-plot. Signature scores have been obtained summarizing the standardized expression levels of signature genes into a combined score with zero mean [7]. The values shown in graphs are thus adimensional. The bottom and top of the box are the first and third quartiles, and the band inside the box is the median; whiskers represent 1st and 99th percentiles; values lower and greater are shown as circles (p<0.0001, n=701).


B . Same as in A ) using the YAP / TAZ activity signature of Zhang et al . ( 2009 ) ( p < 0 . 0001 , n = 701 ) .

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B. Same as in A) using the YAP/TAZ activity signature of Zhang et al. (2009) (p<0.0001, n=701).


C . Kaplan - Meier analysis representing the probability of disease - specific survival in mutant and wild - type p53 breast cancer patients from the METABRIC dataset stratified according high or low YAP / TAZ signature score . The log - rank test p - value reflects the significance of the association between high levels of the YAP / TAZ signature score and shorter survival in in mutant - p53 as compared to wild - type p53 patients ( p < 0 . 0001 , n = 251 ) . All p - values were calculated with two - tailed Student ' s t - test .

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C. Kaplan-Meier analysis representing the probability of disease-specific survival in mutant and wild-type p53 breast cancer patients from the METABRIC dataset stratified according high or low YAP/TAZ signature score. The log-rank test p-value reflects the significance of the association between high levels of the YAP/TAZ signature score and shorter survival in in mutant-p53 as compared to wild-type p53 patients (p<0.0001, n=251). All p-values were calculated with two-tailed Student's t-test.


A . SKBr3 and MDA - MB - 468 cells knocked - down as indicated in the figure , were transiently transfected with pCCAAT - B2LUC ( 100 ng ) or pmutCCAAT - B2LUC ( 100 ng ) luciferase reporter vectors . Values are means + s . d . of three replicates from three independent experiments .

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A. SKBr3 and MDA-MB-468 cells knocked-down as indicated in the figure, were transiently transfected with pCCAAT-B2LUC (100 ng) or pmutCCAAT-B2LUC (100 ng) luciferase reporter vectors. Values are means + s.d. of three replicates from three independent experiments.


B . RT - qPCR of CCNA , CCNB2 and cdk1 mRNA levels in SKBr3 cells upon transduction with si - GFP and si - YAP ( left graph ) or si - p53 ( right graph ) . Values are means + s . d . of three replicates from three independent experiments .

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B. RT-qPCR of CCNA, CCNB2 and cdk1 mRNA levels in SKBr3 cells upon transduction with si-GFP and si-YAP (left graph) or si-p53 (right graph). Values are means + s.d. of three replicates from three independent experiments.


C . H1299 transfected with the indicated siRNAs , were transiently transfected with pCCAAT - B2LUC ( 100 ng ) luciferase reporter vector together with empty pcDNA3 or mutant p53R175H . Representative western blotting to control the transfections presented in the right panel . Values are means + s . d . of three replicates from three independent experiments .

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C. H1299 transfected with the indicated siRNAs, were transiently transfected with pCCAAT-B2LUC (100 ng) luciferase reporter vector together with empty pcDNA3 or mutant p53R175H. Representative western blotting to control the transfections presented in the right panel. Values are means + s.d. of three replicates from three independent experiments.


D . RT - qPCR of CCNA and cdk1 mRNA levels in H1299 cells upon transfection with si - GFP or si - YAP oligos and empty pCDNA3 or mutp53R280H expression vectors as indicated in the figure . Error bars represent mean + s . d . , from three biological replicates . P - values are indicated in the figures ; two - tailed Student ' s t - test was applied throughout .

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D. RT-qPCR of CCNA and cdk1 mRNA levels in H1299 cells upon transfection with si-GFP or si-YAP oligos and empty pCDNA3 or mutp53R280H expression vectors as indicated in the figure. Error bars represent mean + s.d., from three biological replicates. P-values are indicated in the figures; two-tailed Student's t-test was applied throughout.


A and B . Coimmunoprecipitation and western blot analysis from H1299 cell lysates showing endogenous YAP ( A ) and overexpressed GFP - YAP ( B ) bound to mutp53R273H ( A ) or to mutp53R175H ( B ) . In ( A ) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and rabbit IgG was used as negative control of IP . In ( B ) p53 protein was immunoprecipitated with a sheep polyclonal antibody and the total lysate from H1299 transfected with empty pCDNA3 vector was used in as negative control of IP .

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A and B. Coimmunoprecipitation and western blot analysis from H1299 cell lysates showing endogenous YAP (A) and overexpressed GFP-YAP (B) bound to mutp53R273H (A) or to mutp53R175H (B). In (A) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and rabbit IgG was used as negative control of IP. In (B) p53 protein was immunoprecipitated with a sheep polyclonal antibody and the total lysate from H1299 transfected with empty pCDNA3 vector was used in as negative control of IP.


C and D . Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins ( described in Materials and Methods ) ( C ) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP .

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C and D. Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins (described in Materials and Methods) (C) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP.


C and D . Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins ( described in Materials and Methods ) ( C ) and from HCT116 cells expressing wild type p53 protein ( D ) . YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP .

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C and D. Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins (described in Materials and Methods) (C) and from HCT116 cells expressing wild type p53 protein (D). YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP.


E . Cellular extracts from H1299 cells transiently transfected with 2 μg of each indicated plasmids were immunoprecipitated with rabbit polyclonal YAP antibody . Cell lysates derived from cells transfected with the empty vector were used as negative control of immunoprecipitation .

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E. Cellular extracts from H1299 cells transiently transfected with 2 μg of each indicated plasmids were immunoprecipitated with rabbit polyclonal YAP antibody. Cell lysates derived from cells transfected with the empty vector were used as negative control of immunoprecipitation.


F . Cellular extracts from MDA - MB - 468 were immunoprecipitated with rabbit polyclonal YAP , sheep polyclonal p53 and rabbit polyclonal NF - YB antibodies . Rabbit ( for YAP and NF - YB IPs ) and sheep ( for p53 IP ) IgGs were used as negative control of IPs .

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F. Cellular extracts from MDA-MB-468 were immunoprecipitated with rabbit polyclonal YAP, sheep polyclonal p53 and rabbit polyclonal NF-YB antibodies. Rabbit (for YAP and NF-YB IPs) and sheep (for p53 IP) IgGs were used as negative control of IPs.


G . Cell lysates from CAL27 cells transfected with si - GFP and si - NF - YB oligonucleotides were immunoprecipitated with rabbit polyclonal YAP . As specificity control , immunoprecipitations were performed with rabbit IgG .

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G. Cell lysates from CAL27 cells transfected with si-GFP and si-NF-YB oligonucleotides were immunoprecipitated with rabbit polyclonal YAP. As specificity control, immunoprecipitations were performed with rabbit IgG.


H and I . ChIP analysis of mutant p53 , YAP and H4 acetylated histone - bound chromatin from MDA - MB - 468 cells on transduction with siRNAs oligos targeting endogenous YAP ( siYAP ) ( H ) , NF - Y ( siNF - YB ) ( I ) , or siGFP as a negative control ( H and I ) . The experiment was performed in biological triplicates . The CCNA , CCNB2 and Cdk1 promoter occupancy was analyzed by RT - qPCR . Normalization was performed to the amount of input chromatin . The ChIP results were further normalised on the RT - qPCR of a region resulted negative for the recruitment of mutant p53 ( Appendix Table S5 ) . P - values were calculated with two tailed Student ' s t - test . Statistically significant results were referred with p - value < 0 . 05 .

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H and I. ChIP analysis of mutant p53, YAP and H4 acetylated histone-bound chromatin from MDA-MB-468 cells on transduction with siRNAs oligos targeting endogenous YAP (siYAP) (H), NF-Y (siNF-YB) (I), or siGFP as a negative control (H and I). The experiment was performed in biological triplicates. The CCNA, CCNB2 and Cdk1 promoter occupancy was analyzed by RT-qPCR. Normalization was performed to the amount of input chromatin. The ChIP results were further normalised on the RT-qPCR of a region resulted negative for the recruitment of mutant p53 (Appendix Table S5). P-values were calculated with two tailed Student's t-test. Statistically significant results were referred with p-value<0.05.


A and B . Proliferation curves of SKBr3 cell line knocked - down for YAP ( A ) or p53 ( B ) . si - GFP oligos are used as negative control . Cell viability analysis in A and B was determined by trypan blue dye exclusion staining , in C was determined by ATPlite luminescence analysis . All the values are means + s . d . of six replicates from three independent experiments .

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A and B. Proliferation curves of SKBr3 cell line knocked-down for YAP (A) or p53 (B). si-GFP oligos are used as negative control.Cell viability analysis in A and B was determined by trypan blue dye exclusion staining, in C was determined by ATPlite luminescence analysis. All the values are means + s.d. of six replicates from three independent experiments.


C . Viability of CAL27 cell line transfected with si - GFP or si - p53 oligos and empty vector or GFP - YAP construct , as indicated in the legend to the graph . Cell viability analysis in A and B was determined by trypan blue dye exclusion staining , in C was determined by ATPlite luminescence analysis . All the values are means + s . d . of six replicates from three independent experiments .

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C. Viability of CAL27 cell line transfected with si-GFP or si-p53 oligos and empty vector or GFP-YAP construct, as indicated in the legend to the graph.Cell viability analysis in A and B was determined by trypan blue dye exclusion staining, in C was determined by ATPlite luminescence analysis. All the values are means + s.d. of six replicates from three independent experiments.


A . CCNA and CCNB2 expression transcripts were analyzed by RT - qPCR in cDNA derived from tumours of control ( saline ) or zoledronic acid ( ZA ) - treated mice ( ref . 31 ) . N = 3 mice per group . P - values were calculated with two - tailed t - test .

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A. CCNA and CCNB2 expression transcripts were analyzed by RT-qPCR in cDNA derived from tumours of control (saline) or zoledronic acid (ZA)-treated mice (ref. 31). N=3 mice per group. P-values were calculated with two-tailed t-test.


B . ChIP analysis of mutant p53 - bound chromatin from MDA - MB - 231 cells treated or not with 1 µM cerivastatin ( Cer ) for 24h . The CCNB2 promoter occupancy was analyzed by RT - qPCR . Normalization was performed to the amount of input chromatin . The experiment was performed in biological triplicate . P - values were calculated with two - tailed t - test .

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

B. ChIP analysis of mutant p53 -bound chromatin from MDA-MB-231 cells treated or not with 1 µM cerivastatin (Cer) for 24h. The CCNB2 promoter occupancy was analyzed by RT-qPCR. Normalization was performed to the amount of input chromatin. The experiment was performed in biological triplicate. P-values were calculated with two-tailed t-test.


C . MDA - MB - 231 cells treated with the indicated concentrations of Cer for 24h were transfected with pCCAAT - B2LUC ( 100 ng ) luciferase reporter vector . Error bars represent mean + s . d . , from three biological replicates . P - values are indicated in the figures ; p - values were calculated with two - tailed t - test .

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

C. MDA-MB-231 cells treated with the indicated concentrations of Cer for 24h were transfected with pCCAAT-B2LUC (100 ng) luciferase reporter vector. Error bars represent mean + s.d., from three biological replicates. P-values are indicated in the figures;p-values were calculated with two-tailed t-test.


D . CCNA , CCNB2 and Cdk1 expression transcripts were analyzed by RT - qPCR in cDNA derived from MDA - MB - 231 ( left panel ) and SKBr3 ( right panel ) cell lines treated or not with 1 µM Cer for 48h . Error bars represent mean + s . d . , from three biological replicates . P - values were calculated with two - tailed t - test . P - value < 0 . 01 .

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

D. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231 (left panel) and SKBr3 (right panel) cell lines treated or not with 1 µM Cer for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01.


E . Western blot analysis for CCNA , CCNB and Actin ( loading control ) expression in lysates from MDA - MB - 231 cells treated or not with 1 µM Cer .

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

E. Western blot analysis for CCNA, CCNB and Actin (loading control) expression in lysates from MDA-MB-231 cells treated or not with 1 µM Cer.


F . CCNA , CCNB2 and Cdk1 expression transcripts were analyzed by RT - qPCR in cDNA derived from MDA - MB - 231cell line treated or not with 30µM ZA for 48h . Error bars represent mean + s . d . , from three biological replicates . P - values were calculated with two - tailed t - test . P - value < 0 . 01 .

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

F. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231cell line treated or not with 30µM ZA for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01.


G . Viability assay of siGFP and sip53 - MDA - MB - 231 cells after treatment with increasing amounts of cerivastatin ( 0 , 0 . 01 , 0 . 1 , 1 and 10 µM ) for 48 h . Data are normalized to untreated . Error bars represent mean + s . d . , from three biological replicates .

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G. Viability assay of siGFP and sip53 -MDA-MB-231 cells after treatment with increasing amounts of cerivastatin (0, 0.01, 0.1, 1 and 10 µM) for 48 h. Data are normalized to untreated. Error bars represent mean + s.d., from three biological replicates.


( B ) GALpr - Dfm1 - BirA - Flag and GALpr - BirA - Flag levels were measured by western blotting with α - FLAG at 0 ( uninduced ) vs . 5 hours post - galactose induction ( 3 biological replicates ; n = 3 ) .

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(B) GALpr-Dfm1-BirA-Flag and GALpr-BirA-Flag levels were measured by western blotting with α -FLAG at 0 (uninduced) vs. 5 hours post-galactose induction (3 biological replicates; n=3).


( C ) Dfm1 - BirA is still functional and able to degrade Hmg2 - GFP . dfm1Δ + Hmg2 - GFP strains containing DFM1 - BIRA , empty vector , or BIRA only addbacks were grown to log phase and degradation was measured by CHX . After CHX addition , cells were lysed at the indicated times and analyzed by SDS - PAGE and immunoblotted for Hmg2 - GFP with α - GFP .

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

(C) Dfm1-BirA is still functional and able to degrade Hmg2-GFP. dfm1Δ+Hmg2-GFP strains containing DFM1-BIRA, empty vector, or BIRA only addbacks were grown to log phase and degradation was measured by CHX. After CHX addition, cells were lysed at the indicated times and analyzed by SDS-PAGE and immunoblotted for Hmg2-GFP with α-GFP.


( D ) Yeast strains expressing Dfm1 - Bira and BirA only negative control were incubated with different amounts of biotin : 0 , 0 . 1 , and 1 mM . dfm1Δ + Hmg2 - GFP . Microsomes were isolated from each strain and subjected to streptavidin pulldown ( 3 biological replicates ; n = 3 ) . Flow - through and pull - down fractions were detected by western blotting for Dfm1 - BirA with α - Flag and Cdc48 with α - Cdc48 antibodies .

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

(D) Yeast strains expressing Dfm1-Bira and BirA only negative control were incubated with different amounts of biotin: 0, 0.1, and 1 mM. dfm1Δ+Hmg2-GFP. Microsomes were isolated from each strain and subjected to streptavidin pulldown (3 biological replicates; n=3). Flow-through and pull-down fractions were detected by western blotting for Dfm1-BirA with α-Flag and Cdc48 with α-Cdc48 antibodies.


( A ) Dfm1 - GFP binding to Lcb2 - RFP and Orm2 - RFP were analyzed by co - IP . As negative control , cells not expressing Dfm1 - GFP were used ( 3 biological replicates ; n = 3 ) .

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

(A) Dfm1-GFP binding to Lcb2-RFP and Orm2-RFP were analyzed by co-IP. As negative control, cells not expressing Dfm1-GFP were used (3 biological replicates; n=3).


( A ) Indicated strains were spotted 5 - fold dilutions on synthetic complete ( SC ) plates , and plates were incubated at room temperature , 30oC , and 37oC ( 3 biological replicates , 2 technical replicates ; n = 5 ) . WT , dfm1 ∆ , tsc3 ∆ , and dfm1 ∆ tsc3 ∆ were compared for growth in the dilution assay . Arrowhead indicates growth phenotype of tsc3 ∆ cells ; open circle indicates growth phenotype of dfm1 ∆ tsc3 ∆ cells . ( B ) dfm1 ∆ tsc3 ∆ confers resistance to myrocin . WT , dfm1 ∆ , tsc3 ∆ , and dfm1 ∆ tsc3 ∆ strains were grown to log - phase in YPD medium , and 5 - fold serial dilutions of cultures were spotted on ( SC ) plates containing either drug vehicle alone , 1 µM of myriocin and 10 μM of PHS ( 3 biological replicates , 2 technical replicates ; n = 5 ) . Arrowhead indicates growth phenotype of tsc3 ∆ cells ; open circle indicates growth phenotype of dfm1 ∆ tsc3 ∆ cells .

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

(A) Indicated strains were spotted 5-fold dilutions on synthetic complete (SC) plates , and plates were incubated at room temperature, 30oC, and 37oC (3 biological replicates, 2 technical replicates; n=5). WT, dfm1∆, tsc3∆, and dfm1∆tsc3∆ were compared for growth in the dilution assay. Arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of dfm1∆tsc3∆ cells. (B) dfm1∆tsc3∆ confers resistance to myrocin. WT, dfm1∆, tsc3∆, and dfm1∆tsc3∆ strains were grown to log-phase in YPD medium, and 5-fold serial dilutions of cultures were spotted on (SC) plates containing either drug vehicle alone, 1 µM of myriocin and 10 μM of PHS (3 biological replicates, 2 technical replicates; n=5). Arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of dfm1∆tsc3∆ cells.


( D ) Indicated strains were spotted 5 - fold dilutions on SC plates in 3 biological replicates and 2 technical replicates ( n = 5 ) , and plates were incubated at room temperature , 30oC , and 37oC ( n = 3 ) . Upper panel : WT , orm1 ∆ , tsc3 ∆ , and orm1 ∆ tsc3 ∆ were compared for growth by dilution assay . Middle panel : WT , orm2 ∆ , tsc3 ∆ , and orm2 ∆ tsc3 ∆ were compared for growth by dilution assay . Bottom panel : WT , orm1 ∆ , orm2 ∆ , tsc3 ∆ , and orm1 ∆ orm2 ∆ tsc3 ∆ , were compared for growth by dilution assay . Upper panel : arrowhead indicates growth phenotype of tsc3 ∆ cells ; open circle indicates growth phenotype of orm1 ∆ tsc3 ∆ cells . Lower panel : arrowhead indicates growth phenotype of orm2 ∆ cells ; open circle indicates growth phenotype of orm2 ∆ tsc3 ∆ cells .

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

(D) Indicated strains were spotted 5-fold dilutions on SC plates in 3 biological replicates and 2 technical replicates (n=5), and plates were incubated at room temperature, 30oC, and 37oC (n=3). Upper panel: WT, orm1∆, tsc3∆, and orm1∆tsc3∆ were compared for growth by dilution assay. Middle panel: WT, orm2∆, tsc3∆, and orm2∆tsc3∆ were compared for growth by dilution assay. Bottom panel: WT, orm1∆, orm2∆, tsc3∆, and orm1∆orm2∆tsc3∆, were compared for growth by dilution assay. Upper panel: arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of orm1∆tsc3∆ cells. Lower panel: arrowhead indicates growth phenotype of orm2∆ cells; open circle indicates growth phenotype of orm2∆tsc3∆ cells.


( A ) Indicated strains were spotted 5 - fold dilutions on SC plates in 3 biological replicates and 2 technical replicates ( n = 5 ) , and plates were incubated at room temperature , 30oC , and 37oC . Upper panel : WT , dfm1 ∆ , orm1 ∆ , and dfm1 ∆ orm1 ∆ were compared for growth by dilution assay . Middle panel : WT , dfm1 ∆ , orm2 ∆ , and orm2 ∆ tsc3 ∆ were compared for growth by dilution assay . Bottom panel : WT , dfm1 ∆ , orm1 ∆ , orm2 ∆ , and orm2 ∆ , and orm1 ∆ orm2 ∆ were compared for growth by dilution assay . Upper , middle , and lower panel : gray arrowhead depicts growth phenotype of dfm1 ∆ orm1 ∆ , dfm1 ∆ orm2 ∆ , and orm1 ∆ orm2 ∆ respectively . ( B ) dfm1 ∆ orm1 ∆ confers resistance to myriocin and sensitivity to PHS . WT , dfm1 ∆ , orm1 ∆ , and dfm1 ∆ orm1 ∆ strains were grown to log - phase in SC medium , and 5 - fold serial dilutions of cultures were spotted on YPD plates containing either drug vehicle alone , 1 mM of myriocin and 10 μM of PHS with each condition performed in 3 biological replicates and 2 technical replicates ( n = 5 ) . Plates were incubated at room temperature and photographed after 3 days . Open circle indicates growth phenotype of dfm1 ∆ orm1 ∆ cells .

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

(A) Indicated strains were spotted 5-fold dilutions on SC plates in 3 biological replicates and 2 technical replicates (n=5), and plates were incubated at room temperature, 30oC, and 37oC. Upper panel: WT, dfm1∆, orm1∆, and dfm1∆orm1∆ were compared for growth by dilution assay. Middle panel: WT, dfm1∆, orm2∆, and orm2∆tsc3∆ were compared for growth by dilution assay. Bottom panel: WT, dfm1∆, orm1∆, orm2∆, and orm2∆, and orm1∆orm2∆ were compared for growth by dilution assay. Upper, middle, and lower panel: gray arrowhead depicts growth phenotype of dfm1∆orm1∆, dfm1∆orm2∆, and orm1∆orm2∆ respectively. (B) dfm1∆orm1∆ confers resistance to myriocin and sensitivity to PHS. WT, dfm1∆, orm1∆, and dfm1∆orm1∆ strains were grown to log-phase in SC medium, and 5-fold serial dilutions of cultures were spotted on YPD plates containing either drug vehicle alone, 1 mM of myriocin and 10 μM of PHS with each condition performed in 3 biological replicates and 2 technical replicates (n=5). Plates were incubated at room temperature and photographed after 3 days. Open circle indicates growth phenotype of dfm1∆orm1∆ cells.


( A ) Degradation of Orm2 depends on Dfm1 and not Der1 . The indicated strains expressing Orm2 - RFP were grown into log phase and degradation was measured by cycloheximide chase ( CHX ) . After CHX addition , cells were lysed at the indicated times , and analyzed by SDS - PAGE and immunoblotted for Orm2 - RFP with α - RFP ( 3 biological replicates ( n = 3 ) . ( B ) Same as ( A ) except degradation of Orm2 - RFP was measured in WT and der1 ∆ cells ( 3 biological replicates ; n = 3 ) .

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

(A) Degradation of Orm2 depends on Dfm1 and not Der1. The indicated strains expressing Orm2-RFP were grown into log phase and degradation was measured by cycloheximide chase (CHX). After CHX addition, cells were lysed at the indicated times, and analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates (n=3). (B) Same as (A) except degradation of Orm2-RFP was measured in WT and der1∆ cells (3 biological replicates; n=3).


( D ) Dfm1 ' s SHP box is not required for degradation of Orm2 - RFP . In the indicated strains , degradation of Orm2 - RFP was measured by CHX - chase assay . Cells were analyzed by SDS - PAGE and immunoblotted for Orm2 - RFP with α - RFP ( 3 biological replicates ; n = 3 ) .

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

(D) Dfm1's SHP box is not required for degradation of Orm2-RFP. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3).


( G ) Dfm1 ' s WR motif , GxxxG motif , substrate binding and lipid thinning function are required for degradation of Orm2 - RFP . In the indicated strains , degradation of Orm2 - RFP was measured by CHX - chase assay . Cells were analyzed by SDS - PAGE and immunoblotted for Orm2 - RFP with α - RFP ( 3 biological replicates ; n = 3 ) .

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

(G) Dfm1's WR motif, GxxxG motif, substrate binding and lipid thinning function are required for degradation of Orm2-RFP. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3).


( H ) Dfm1 L1 residues are required for binding to Orm2 . Orm2 - RFP and binding to retrotranslocation - deficient Dfm1 L1 mutants was analyzed by co - IP . The IP was analyzed for presence of Dfm1 - HA . As a negative control , cells not expressing Orm2 - RFP were used ( 3 biological replicates ; n = 3 ) . Band intensities for all western blots were normalized to PGK1 loading control and quantified by ImageJ .

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

(H) Dfm1 L1 residues are required for binding to Orm2. Orm2-RFP and binding to retrotranslocation-deficient Dfm1 L1 mutants was analyzed by co-IP. The IP was analyzed for presence of Dfm1-HA. As a negative control, cells not expressing Orm2-RFP were used (3 biological replicates; n=3). Band intensities for all western blots were normalized to PGK1 loading control and quantified by ImageJ.


( A ) E3 ligase Tul1 is required for Orm2 degradation . In the indicated strains , degradation of Orm2 - RFP was measured by CHX - chase assay . Cells were analyzed by SDS - PAGE and immunoblotted for Orm2 - RFP with α - RFP ( 3 biological replicates ; n = 3 ) . t = 0 was taken as 100 % and data is represented as mean ± SEM .

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

(A) E3 ligase Tul1 is required for Orm2 degradation. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.


( B ) Cdc48 and the proteasome are required for Orm2 degradation . Same as ( A ) , except cdc48 - 2 and hrd2 - 1 were analyzed for Orm2 - RFP degradation ( 3 biological replicates ; n = 3 ) . t = 0 was taken as 100 % and data is represented as mean ± SEM .

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

(B) Cdc48 and the proteasome are required for Orm2 degradation. Same as (A), except cdc48-2 and hrd2-1 were analyzed for Orm2-RFP degradation (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.


( C ) Dfm1 does not function in the post - ubiquitination step of Orm2 degradation pathway . Indicated strains expressing Orm2 - RFP were grown into log phase . Cells were lysed , and microsomes were collected and immunoprecipitated with α - RFP conjugated to agarose beads . Samples were then subjected to SDS - PAGE and immunoblot by α - Ubiquitin and α - RFP ( 3 biological replicates ; n = 3 ) .

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(C) Dfm1 does not function in the post-ubiquitination step of Orm2 degradation pathway. Indicated strains expressing Orm2-RFP were grown into log phase. Cells were lysed, and microsomes were collected and immunoprecipitated with α-RFP conjugated to agarose beads. Samples were then subjected to SDS-PAGE and immunoblot by α-Ubiquitin and α-RFP (3 biological replicates; n=3).


( D ) ERAD mutants do not rescue temperature - sensitive lethality of tsc3 ∆ . Indicated strains were grown to log - phase in SC and serially diluted cultures were plated on SC plates and incubated at 37 ° C and imaged at Day 2 ( 3 biological replicates , 2 technical replicates ; n = 5 ) .

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(D) ERAD mutants do not rescue temperature-sensitive lethality of tsc3∆. Indicated strains were grown to log-phase in SC and serially diluted cultures were plated on SC plates and incubated at 37°C and imaged at Day 2 (3 biological replicates, 2 technical replicates; n=5).


( A ) Phosphorylated Orm2 accumulates in the absence of Dfm1 . Phos - tag western blot analysis shows that there is an accumulation of phosphorylated Orm2 in dfm1 ∆ cells . The indicated strains were grown to log phase , treated with vehicle or 1 . 5 μM myriocin for 1 hour and subjected to SDS - PAGE or Phos - tag western blot analysis via blotting for Orm2 with α - RFP and PGK1 with α - PGK1 antibodies ( 2 biological replicates ; n = 2 ) .

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

(A) Phosphorylated Orm2 accumulates in the absence of Dfm1. Phos-tag western blot analysis shows that there is an accumulation of phosphorylated Orm2 in dfm1∆ cells. The indicated strains were grown to log phase, treated with vehicle or 1.5 μM myriocin for 1 hour and subjected to SDS-PAGE or Phos-tag western blot analysis via blotting for Orm2 with α-RFP and PGK1 with α-PGK1 antibodies (2 biological replicates; n=2).


( C ) dfm1 ∆ cells block the degradation of phosphorylated mimic of Orm2 ( Orm2 - 3D ) . In the indicated strains , degradation of Orm2 - 3A - GFP and Orm2 - 3D - GFP was measured by CHX - chase assay . Cells were analyzed by SDS - PAGE and immunoblotted α - GFP ( 3 biological replicates ; n = 3 ) . t = 0 was taken as 100 % and data is represented as mean ± SEM .

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(C) dfm1∆ cells block the degradation of phosphorylated mimic of Orm2 (Orm2-3D). In the indicated strains, degradation of Orm2-3A-GFP and Orm2-3D-GFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted α-GFP (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.


( A ) Dfm1 - HA binding to Orm2 - GFP , Orm2 - 3A - GFP , Orm2 - 3D - GFP , Orm1 , Lcb1 , and Lcb2 were analyzed by co - IP . As negative control , cells not expressing Dfm1 - HA were used . Also , Sec61 was also included to test for non - specific binding ( 3 biological replicates ; n = 3 ) . ( B ) Same as ( A ) , except co - IP was performed on WT and orm2 ∆ cells and Dfm1 - HA binding to Orm1 , Lcb1 , and Lcb2 was analyzed ( 3 biological replicates ; n = 3 ) . ( C ) Same as ( A ) , except Orm2 - GFP binding to Dfm1 - HA , Orm1 , Lcb1 , and Lcb2 was analyzed by co - IP ( 3 biological replicates ; n = 3 ) .

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(A) Dfm1-HA binding to Orm2-GFP, Orm2-3A-GFP, Orm2-3D-GFP, Orm1, Lcb1, and Lcb2 were analyzed by co-IP. As negative control, cells not expressing Dfm1-HA were used. Also, Sec61 was also included to test for non-specific binding (3 biological replicates; n=3). (B) Same as (A), except co-IP was performed on WT and orm2∆ cells and Dfm1-HA binding to Orm1, Lcb1, and Lcb2 was analyzed (3 biological replicates; n=3). (C) Same as (A), except Orm2-GFP binding to Dfm1-HA, Orm1, Lcb1, and Lcb2 was analyzed by co-IP (3 biological replicates; n=3).


( A ) Cellulase functional screening by Congo Red assay . Halos on the plate indicate cellulase activity . Pc : Pseudomonas cellulosa , Bs : Bacillus subtilis . Numbered strains are from the C . elegans native gut

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(A) Cellulase functional screening by Congo Red assay. Halos on the plate indicate cellulase activity. Pc: Pseudomonas cellulosa, Bs: Bacillus subtilis. Numbered strains are from the C. elegans native gut microbiome


( B ) Production of reducing sugars by cellulose - degrading bacterial strains . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Error bars represent 95 % confidence intervals of the mean .

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(B) Production of reducing sugars by cellulose-degrading bacterial strains. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.


( D ) Colony - forming units ( CFU ) of various bacterial species in the worm gut . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Error bars represent 95 % confidence intervals of the mean .

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(D) Colony-forming units (CFU) of various bacterial species in the worm gut. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.


( E ) C . elegans colonized with cellulolytic P . cellulosa and incubated with different carbon sources . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Error bars represent 95 % confidence intervals of the mean .

(E) C. elegans colonized with cellulolytic P. cellulosa and incubated with different carbon sources. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.


( A ) Incorporation of 14C - labelled cellulose . Worms were first colonized with cellulolytic bacteria and then incubated with 14C - cellulose to allow carbon utilization . Antibiotic treatment was applied before 14C measurement of worms to eliminate bacterial interference in isotopic reading . 14C - cellulose incorporation measurement reflected the isotopic carbon incorporation in C . elegans . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Student ' s t test was used for statistical analysis . In A , experiments were performed in biological triplicates using 200 worms per data point . In A error bars represent 95 % confidence intervals for the mean .

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(A) Incorporation of 14C-labelled cellulose. Worms were first colonized with cellulolytic bacteria and then incubated with 14C-cellulose to allow carbon utilization. Antibiotic treatment was applied before 14C measurement of worms to eliminate bacterial interference in isotopic reading. 14C-cellulose incorporation measurement reflected the isotopic carbon incorporation in C. elegans. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In A, experiments were performed in biological triplicates using 200 worms per data point. In A error bars represent 95% confidence intervals for the mean.


( B ) Induction of the worm fat - 7p : : GFP metabolic reporter . Adult worms pre - colonized with P . cellulosa were incubated in liquid media for 24 h with the indicated carbon sources , and GFP fluorescence was read in individual worms on a BioSorter large object sorter . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Student ' s t test was used for statistical analysis . In B , error bars represent 95 % confidence intervals for the mean . In B , each point represents a different worm in biological replicates , n = 96 , n = 53 , and n = 80 from left to right .

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(B) Induction of the worm fat-7p::GFP metabolic reporter. Adult worms pre-colonized with P. cellulosa were incubated in liquid media for 24 h with the indicated carbon sources, and GFP fluorescence was read in individual worms on a BioSorter large object sorter. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In B, error bars represent 95% confidence intervals for the mean. In B, each point represents a different worm in biological replicates, n=96, n=53, and n=80 from left to right.


( C ) Effect of nutrition on larval yield . Pre - colonized worms were separated into the individual wells of a 384 - well plate , and larvae counts in each well were read after 48 h . Larval yield comparison indicates the nutritional benefit of cellulolytic activity . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Student ' s t test was used for statistical analysis . In C , the central band represents the median , notches represent 95 % confidence intervals for the median , the box edges represent the inter quartile range ( IQR ) and the whiskers extend an additional 1 . 5 IQR beyond the boxes . In C , each point represents a different worm in biological replicates , n = 74 , n = 79 , n = 79 , n = 89 , n = 88 , n = 86 from left to right .

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(C) Effect of nutrition on larval yield. Pre-colonized worms were separated into the individual wells of a 384-well plate, and larvae counts in each well were read after 48 h. Larval yield comparison indicates the nutritional benefit of cellulolytic activity. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In C, the central band represents the median, notches represent 95% confidence intervals for the median, the box edges represent the inter quartile range (IQR) and the whiskers extend an additional 1.5 IQR beyond the boxes. In C, each point represents a different worm in biological replicates, n=74, n=79, n=79, n=89, n=88, n=86 from left to right.


Synchronized adult worms were colonized for two days on plates containing E . coli OP50 + P . cellulosa ( Pc ) , E . coli OP50 + Lp , or E . coli OP50 + Pc + Lp . ( B ) After 36 h outgrowth of the pathogen , worms pre - colonized with P . cellulosa in the presence of L . plantarum ( E . coli OP50 + Pc + Lp ) showed lower pathogen burden than P . cellulosa monocolonized worms when CMC was used as the carbon source . Data shown are fold change Salmonella infection ( CFU / worm ) relative to the P . cellulosa - only condition . Error bars represent 95 % confidence interval of the mean . CMC = 0 . 01 % carboxymethylcellulose ( w / v ) . All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis .

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Synchronized adult worms were colonized for two days on plates containing E. coli OP50 + P. cellulosa (Pc), E. coli OP50 + Lp, or E. coli OP50 + Pc + Lp. (B) After 36 h outgrowth of the pathogen, worms pre-colonized with P. cellulosa in the presence of L. plantarum (E. coli OP50 + Pc + Lp) showed lower pathogen burden than P. cellulosa monocolonized worms when CMC was used as the carbon source. Data shown are fold change Salmonella infection (CFU/worm) relative to the P. cellulosa-only condition. Error bars represent 95% confidence interval of the mean. CMC = 0.01% carboxymethylcellulose (w/v). All experiments were performed in biological triplicates. Student's t test was used for statistical analysis.


B . Total quantified proteome depth in tissues ( n = 18 ) , primary cells ( n = 12 ) , immortalized cell lines ( n = 4 ) and all samples ( n = 34 ) . In all cases , n means biological replicates unless otherwise indicated . The upper - and lower panel shows the number of quantified protein groups and peptides , respectively . C . Dynamic range of the different proteomes based on median LFQ intensity ordered by abundance rank ( Liver : bulk liver biopsy , HepA : hepatic artery , PorV : portal vein , Cell lines : mixture of human liver - derived immortalized cell lines ) . Number of biological replicates is same as Panel ( A ) .

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B. Total quantified proteome depth in tissues (n=18), primary cells (n=12), immortalized cell lines (n=4) and all samples (n=34). In all cases, n means biological replicates unless otherwise indicated. The upper- and lower panel shows the number of quantified protein groups and peptides, respectively. C. Dynamic range of the different proteomes based on median LFQ intensity ordered by abundance rank (Liver: bulk liver biopsy, HepA: hepatic artery, PorV: portal vein, Cell lines: mixture of human liver-derived immortalized cell lines). Number of biological replicates is same as Panel (A).


B . Cumulative protein abundance of liver biopsy , haptic artery and portal vein as a function of protein rank , with the total of the top ten abundant proteins and number of proteins that comprise four quartiles indicated .

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B. Cumulative protein abundance of liver biopsy, haptic artery and portal vein as a function of protein rank, with the total of the top ten abundant proteins and number of proteins that comprise four quartiles indicated.


D . Cumulative protein abundance of liver cell types as a function of protein rank , indicating the total of the top ten abundant proteins and number of proteins that comprise the four quartiles .

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D. Cumulative protein abundance of liver cell types as a function of protein rank, indicating the total of the top ten abundant proteins and number of proteins that comprise the four quartiles.


F . The bar plot shows the contribution of each organelle to total cellular protein mass , also accounting for unassigned proteins . Percentage of cytosol , mitochondria , nucleoli , plasma membrane and actin filaments is indicated .

F. The bar plot shows the contribution of each organelle to total cellular protein mass, also accounting for unassigned proteins. Percentage of cytosol, mitochondria, nucleoli, plasma membrane and actin filaments is indicated.


A . Total protein copy number of the oxidative phosphorylation complex I - V in bulk liver tissue ( n = 6 independent biological replicates ) and primary cell types ( isolated primary cells from n = 3 individuals for each cell type ) . Values are presented as mean ± s . d . B . Protein copy number for members of the oxidative phosphorylation complex I - V in hepatocytes and Kupffer cells with Pearson correlation coefficient indicated .

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A. Total protein copy number of the oxidative phosphorylation complex I-V in bulk liver tissue (n=6 independent biological replicates) and primary cell types (isolated primary cells from n=3 individuals for each cell type). Values are presented as mean ± s.d. B. Protein copy number for members of the oxidative phosphorylation complex I-V in hepatocytes and Kupffer cells with Pearson correlation coefficient indicated.


D . Top ten uniquely quantified proteins per cell type with LFQ intensities [ Log10 ] and copy numbers [ Log10 ] .

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D. Top ten uniquely quantified proteins per cell type with LFQ intensities [Log10] and copy numbers [Log10].


A . Unsupervised hierarchical clustering of proteins significantly differentially abundant across cell types , with columns showing three biological replicates ( n = 3 ) of four cell types and rows significant proteins . Significance was calculated by one - way ANOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) . Frames and numbers indicate four clusters of proteins highly enriched in each cell type .

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A. Unsupervised hierarchical clustering of proteins significantly differentially abundant across cell types, with columns showing three biological replicates (n=3) of four cell types and rows significant proteins. Significance was calculated by one-way ANOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05). Frames and numbers indicate four clusters of proteins highly enriched in each cell type.


A . Total quantified proteins at day one , three and seven upon primary cell culture in hepatocytes ( hHEP ) , hepatic stellate cells ( hHSC ) and liver sinusoidal endothelial cells ( LSEC ) . In all cases , number of biological replicates is 3 . Values are presented as mean ± s . d . No additional replications of the experiment was done in laboratory .

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A. Total quantified proteins at day one, three and seven upon primary cell culture in hepatocytes (hHEP), hepatic stellate cells (hHSC) and liver sinusoidal endothelial cells (LSEC). In all cases, number of biological replicates is 3. Values are presented as mean ± s.d. No additional replications of the experiment was done in laboratory.


F . Protein expression patterns over the course of primary hHSC culture ( n = 3 ) . Values are presented as mean ± s . d . Significance was calculated by ANOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) with a significance level of * * P < 0 . 01 , and * * * P < 0 . 001 .

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F. Protein expression patterns over the course of primary hHSC culture (n=3). Values are presented as mean ± s.d. Significance was calculated by ANOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05) with a significance level of **P < 0.01, and ***P < 0.001.


A . Hierarchical clustering of proteins significantly differentially abundant between NASH ( n = 20 ) , cirrhosis ( n = 10 ) and control groups ( n = 15 ) . Significance was calculated by ANCOVA , followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) . Two major clusters of proteins were identified with Cluster 1 mainly upregulated in cirrhosis compared to NASH and controls and Cluster 2 downregulated . No additional replications of the experiment was done in laboratory .

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A. Hierarchical clustering of proteins significantly differentially abundant between NASH (n=20), cirrhosis (n=10) and control groups (n=15). Significance was calculated by ANCOVA, followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05). Two major clusters of proteins were identified with Cluster 1 mainly upregulated in cirrhosis compared to NASH and controls and Cluster 2 downregulated. No additional replications of the experiment was done in laboratory.


D . Box ‐ and ‐ whisker plot showing the distribution of log2 ‐ intensity values of statistical significantly regulated proteins across three groups . Number of independent biological replicates is n = 15 , 20 and 10 for the control , NASH and cirrhosis group , respectively . The black line in the middle of the box is the median , the top and the bottom of the box represent the upper and lower quartile values of the data and the whiskers represent the upper and lower limits for considering outliers ( Q3 + 1 . 5 * IQR , Q1 ‐ 1 . 5 * IQR ) where IQR is the interquartile range ( Q3 - Q1 ) . Significance was defined by ANCOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) with a significance level of * * * P < 0 . 001 . E . Heatmap of CYP 450 family members that are statistically significantly abundant between three groups . Data was presented as mean protein abundance followed by Z - score normalization across . Number of replicates is same as Panel ( C ) . F . Extracellular matrix ( ECM ) remodeling in liver cirrhosis . Upper and lower panel shows the abundance rank of proteins involved in ECM organization in the control ( upper ) and cirrhosis ( lower ) groups , highlighting top shifted ECM components in the cirrhosis group as compared with the control group .

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D. Box‐and‐whisker plot showing the distribution of log2‐intensity values of statistical significantly regulated proteins across three groups. Number of independent biological replicates is n=15, 20 and 10 for the control, NASH and cirrhosis group, respectively. The black line in the middle of the box is the median, the top and the bottom of the box represent the upper and lower quartile values of the data and the whiskers represent the upper and lower limits for considering outliers (Q3+1.5*IQR, Q1‐1.5*IQR) where IQR is the interquartile range (Q3-Q1). Significance was defined by ANCOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05) with a significance level of ***P < 0.001. E. Heatmap of CYP 450 family members that are statistically significantly abundant between three groups. Data was presented as mean protein abundance followed by Z-score normalization across. Number of replicates is same as Panel (C). F. Extracellular matrix (ECM) remodeling in liver cirrhosis. Upper and lower panel shows the abundance rank of proteins involved in ECM organization in the control (upper) and cirrhosis (lower) groups, highlighting top shifted ECM components in the cirrhosis group as compared with the control group.


A . Lysates of HK2 cells cultured in FM or STV ( 2h ) were immunoprecipitated with anti - OFD1 ( left ) and anti - ATG13 ( right ) antibodies or IgG and analyzed by WB ; ACTIN was used as loading control . n = 3 independent experiments . IP = immunoprecipitation , FM = full medium , STV = HBSS starvation .

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A. Lysates of HK2 cells cultured in FM or STV(2h) were immunoprecipitated with anti-OFD1 (left) and anti-ATG13 (right) antibodies or IgG and analyzed by WB; ACTIN was used as loading control. n=3 independent experiments. IP=immunoprecipitation, FM=full medium, STV=HBSS starvation.


B . Airyscan confocal analysis of endogenous FIP200 / γTUBULIN and ATG13 / γTUBULIN colocalization with lentiviral delivered HA - OFD1 in HK2 cells . Cells were cultured in HBSS ( 90min ) . Green , FIP200 and ATG13 ; red , HA - OFD1 ; Grey , γTUBULIN ; blue , Hoechst for nuclei . Insets show magnifications and single - color channels of selected areas . Scale bar = 3 , 5μm . C . Quantification of HA - OFD1 / ATG13 and HA - OFD1 / FIP200 colocalization , expressed as % of total OFD1 fluorescence ( mean ± SEM ) . n = 3 independent experiments ; n = 53 cells for ATG13 and n = 39 for FIP200 were analyzed .

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B. Airyscan confocal analysis of endogenous FIP200/γTUBULIN and ATG13/γTUBULIN colocalization with lentiviral delivered HA-OFD1 in HK2 cells. Cells were cultured in HBSS (90min). Green, FIP200 and ATG13; red, HA-OFD1; Grey, γTUBULIN; blue, Hoechst for nuclei. Insets show magnifications and single-color channels of selected areas. Scale bar=3,5μm. C. Quantification of HA-OFD1/ATG13 and HA-OFD1/FIP200 colocalization, expressed as % of total OFD1 fluorescence (mean ± SEM). n=3 independent experiments; n=53 cells for ATG13 and n=39 for FIP200 were analyzed.


A . Top , WB of ULK1 - complex components in wt and KO - OFD1 cells assessed in FM ( - ) or STV ( 2 , 4h ) . Bands at same exposure , noncontiguous on the same gel . Bottom , ACTIN - normalized protein levels are expressed as fold increase compared with wt cells , represented by the dashed line ( mean ± SEM ) ; n = 5 independent experiments . FM = full medium , STV = HBSS starvation .

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A. Top, WB of ULK1-complex components in wt and KO-OFD1 cells assessed in FM (-) or STV (2,4h). Bands at same exposure, noncontiguous on the same gel. Bottom, ACTIN-normalized protein levels are expressed as fold increase compared with wt cells, represented by the dashed line (mean ± SEM); n=5 independent experiments. FM=full medium, STV=HBSS starvation.


B . Top , wild - type and KO - OFD1 cells were incubated with 50µg / ml CHX . Immunoblots were probed with indicated antibodies . Bottom , quantification of βTUBULIN and ACTIN - normalized protein levels versus untreated conditions ( - ) , data are expressed as mean ± SEM ; n = 4 independent experiments . CHX = Cycloheximide , ns = not significative .

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B. Top, wild-type and KO-OFD1 cells were incubated with 50µg/ml CHX. Immunoblots were probed with indicated antibodies. Bottom, quantification of βTUBULIN and ACTIN-normalized protein levels versus untreated conditions (-), data are expressed as mean ± SEM; n=4 independent experiments. CHX=Cycloheximide, ns=not significative.


C . Immunofluorescence of ULK1 and ATG13 puncta in wt and KO - OFD1 cells cultured in HBSS ( 90min ) . Green , ATG13 and ULK1 ; blue , Hoechst for nuclei . Scale bar = 10μm . ( Right ) Graphs display quantification of puncta / cell , data are expressed as mean ± SEM ; n = 5 independent experiments , n ≥ 200 cells / antibody .

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C. Immunofluorescence of ULK1 and ATG13 puncta in wt and KO-OFD1 cells cultured in HBSS (90min). Green, ATG13 and ULK1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) Graphs display quantification of puncta/cell, data are expressed as mean ± SEM; n=5 independent experiments, n≥200 cells/antibody.


D . Western Blot of ULK1 complex components in KO - OFD1 cells transfected with empty vector ( Empty ) or 3xFLAG - OFD1 ( OFD1 ) . Histograms show quantification of band intensities normalized versus ACTIN expressed as mean ± SEM . n = 5 independent experiments .

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D. Western Blot of ULK1 complex components in KO-OFD1 cells transfected with empty vector (Empty) or 3xFLAG-OFD1 (OFD1). Histograms show quantification of band intensities normalized versus ACTIN expressed as mean ± SEM. n=5 independent experiments.


E . Western Blot of ULK1 - complex components in KO - OFD1 cells transfected with 3xFLAG - OFD1 ( OFD1 ) or empty vector ( Empty ) and treated ( + ) or not ( - ) with SAR405 ( 10μM , 6h ) . ACTIN is the loading control . On the right , ATG13 levels are expressed as fold increase compared with empty vector ( mean ± SEM ) ; n = 3 independent experiments .

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E. Western Blot of ULK1-complex components in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or empty vector (Empty) and treated (+) or not (-) with SAR405 (10μM, 6h). ACTIN is the loading control. On the right, ATG13 levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.


F . Representative confocal images of co - staining of 3xFLAG - OFD1 with ATG13 in KO - OFD1 cells incubated in HBSS , treated ( + ) or not ( - ) with Baf - A1 ( 100nM , 90min ) . Green , ATG13 ; red , 3xFLAG - OFD1 ; blue , Hoechst for nuclei . Scale bar = 10μm . ( Right ) ATG13 puncta / cell are quantified and expressed as mean ± SEM ; n = 3 independent experiments , n ≥ 100 cells .

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F. Representative confocal images of co-staining of 3xFLAG-OFD1 with ATG13 in KO-OFD1 cells incubated in HBSS, treated (+) or not (-) with Baf-A1(100nM, 90min). Green, ATG13; red, 3xFLAG-OFD1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=3 independent experiments, n≥100 cells.


A . Middle . Lysates from 3xFLAG - OFD1 overexpressing HEK293 cells were added to beads with immobilized GST - fused LC3B , LC3BF52A - V53AdG , dNLC3BdG and GST alone . Right . Lysates from 3xFLAG - OFD1 ( OFD1 ) and empty vector ( Empty ) overexpressing HEK293 cells were added to GST - GABARAP - L1 and GST . Left . WB of total lysates ( input ) . All panels display WB for FLAG , ACTIN is the loading control .

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A. Middle. Lysates from 3xFLAG-OFD1 overexpressing HEK293 cells were added to beads with immobilized GST-fused LC3B, LC3BF52A-V53AdG, dNLC3BdG and GST alone. Right. Lysates from 3xFLAG-OFD1(OFD1) and empty vector (Empty) overexpressing HEK293 cells were added to GST-GABARAP-L1 and GST. Left. WB of total lysates (input). All panels display WB for FLAG, ACTIN is the loading control.


B . Representative confocal images of endogenous OFD1 ( green ) co - staining with LC3B ( red ) , and of 3xFLAG - OFD1 ( red ) with GABARAP ( green ) in HK2 cells ( HBSS , 90min ) . Hoechst labels nuclei ( blue ) . Insets show magnifications and single - color channels of selected areas . Scale bar = 10μm ; n ≥ 50 cells / sample .

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B. Representative confocal images of endogenous OFD1 (green) co-staining with LC3B (red), and of 3xFLAG-OFD1 (red) with GABARAP (green) in HK2 cells (HBSS,90min). Hoechst labels nuclei (blue). Insets show magnifications and single-color channels of selected areas. Scale bar=10μm; n≥50 cells/sample.


D . Lysates from 3xFLAG - OFD1 ( OFD1 ) , 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) and empty vector ( Empty ) overexpressing cells were added to GST - GABARAP - L1 , GST - LC3B and GST .

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D. Lysates from 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1∆LIR(OFD1∆LIR) and empty vector (Empty) overexpressing cells were added to GST-GABARAP-L1, GST-LC3B and GST.


E . Representative confocal images of OFD1 ∆ LIR co - staining with LC3B ( left ) and GABARAP ( right ) in HK2 cells ( HBSS , 90min ) . Green , LC3B and GABARAP ; red , 3xFLAG - OFD1 ∆ LIR ; blue , Hoechst for nuclei . Scale bar = 5μm . n ≥ 50 cells / sample .

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E. Representative confocal images of OFD1∆LIR co-staining with LC3B (left) and GABARAP (right) in HK2 cells (HBSS,90min). Green, LC3B and GABARAP; red, 3xFLAG-OFD1∆LIR; blue, Hoechst for nuclei. Scale bar=5μm. n≥50 cells/sample.


A . Representative confocal images of 3xFLAG - OFD1 ( OFD1 ) and 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) co - staining with ATG13 in KO - OFD1 cells ( HBSS , 90min ) . Green , ATG13 ; red , FLAG ; blue , Hoechst for nuclei . Scale bar = 10μm . ( Right ) ATG13 puncta / cell are quantified and expressed as mean ± SEM ; n = 4 independent experiments , n ≥ 100 cells .

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A. Representative confocal images of 3xFLAG-OFD1 (OFD1) and 3xFLAG-OFD1∆LIR (OFD1∆LIR) co-staining with ATG13 in KO-OFD1 cells (HBSS,90min). Green, ATG13; red, FLAG; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=4 independent experiments, n≥100 cells.


B . Western blot of ATG13 in KO - OFD1 cells transfected with 3xFLAG - OFD1 ( OFD1 ) or 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) or empty vector ( Empty ) . ( Right ) ACTIN - normalized ATG13 protein levels are expressed as fold increase compared with empty vector ( mean ± SEM ) ; n = 3 independent experiments .

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B. Western blot of ATG13 in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) or empty vector (Empty). (Right) ACTIN-normalized ATG13 protein levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.


C . Top . OFD1 fragments ( a , b and c ) . Bottom left . Lysates from empty vector ( Empty ) , 3xFLAG - OFD1 ( OFD1 ) , 3xFLAG - OFD1a ( OFD1a ) , 3xFLAG - OFD1b ( OFD1b ) , 3xFLAG - OFD1c ( OFD1c ) overexpressing HEK293 cells were added to GST - ATG13 ; lysates from 3xFLAG - OFD1 ( OFD1 ) overexpressing HEK293 cells were added to GST as control . Right . WB of total lysates ( input ) . All panels display WB for FLAG , HSP90 is the loading control . LIR = LC3 interacting region .

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C. Top. OFD1 fragments (a,b and c). Bottom left. Lysates from empty vector (Empty), 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1a (OFD1a), 3xFLAG-OFD1b (OFD1b), 3xFLAG-OFD1c (OFD1c) overexpressing HEK293 cells were added to GST-ATG13; lysates from 3xFLAG-OFD1(OFD1) overexpressing HEK293 cells were added to GST as control. Right. WB of total lysates (input). All panels display WB for FLAG, HSP90 is the loading control. LIR=LC3 interacting region.


D . Scanning confocal microscopy analysis of wt and KO - OFD1 cells incubated in HBSS ( 8h ) , treated ( + ) or not ( - ) with Baf - A1 ( 50nM , 8h ) . Green , LAMP1 ; red , ATG13 ; blue , Hoechst for nuclei . Scale bar = 6μm . Insets show higher magnification and single colour channels of the boxed area . ( Bottom ) Bar graphs show quantification of lysosomes containing ATG13 expressed as % of total amount of LAMP1 / cell ( mean ± SEM ) . n = 30 WT cells , n = 28 KO - OFD1 cells counted ; three independent experiments .

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D. Scanning confocal microscopy analysis of wt and KO-OFD1 cells incubated in HBSS (8h), treated (+) or not (-) with Baf-A1(50nM, 8h). Green, LAMP1; red, ATG13; blue, Hoechst for nuclei. Scale bar=6μm. Insets show higher magnification and single colour channels of the boxed area. (Bottom) Bar graphs show quantification of lysosomes containing ATG13 expressed as % of total amount of LAMP1/cell (mean ± SEM). n=30 WT cells, n=28 KO-OFD1 cells counted; three independent experiments.


E . Time course of OFD1 levels in HBSS - starved wt HK2 cells treated with Baf - A1 ( 100nM ) and collected at different time points . LC3B is the control for Baf - A1 treatment . ( Right ) Graphs show ACTIN - normalized OFD1 levels versus FM condition ( - ) expressed as mean ± SEM ; n = 4 independent experiments . STV = starvation , Baf - A1 = bafilomycin , Ctrl = control .

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E. Time course of OFD1 levels in HBSS-starved wt HK2 cells treated with Baf-A1 (100nM) and collected at different time points. LC3B is the control for Baf-A1 treatment. (Right) Graphs show ACTIN-normalized OFD1 levels versus FM condition (-) expressed as mean ± SEM; n=4 independent experiments. STV=starvation, Baf-A1=bafilomycin, Ctrl=control.


F . Western blot of FLAG in KO - OFD1 cells transfected with 3xFLAG - OFD1 ( OFD1 ) or 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) and treated or not ( - ) with Baf - A1 ( 100nM , 2h ) . ( Right ) Quantification of GAPDH - normalized FLAG levels versus untreated ( - ) is expressed as mean ± SEM ; n = 3 independent experiments . Baf - A1 = bafilomycin .

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F. Western blot of FLAG in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) and treated or not (-) with Baf-A1 (100nM,2h). (Right) Quantification of GAPDH-normalized FLAG levels versus untreated (-) is expressed as mean ± SEM; n=3 independent experiments. Baf-A1=bafilomycin.


A . Representative blots of ATG13 ( left ) and VPS34 ( right ) phosphorylation levels in wt and KO - OFD1 cells transiently expressing GFP - ATG13 and FLAG - VPS34 in FM .

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A. Representative blots of ATG13 (left) and VPS34 (right) phosphorylation levels in wt and KO-OFD1 cells transiently expressing GFP-ATG13 and FLAG-VPS34 in FM.


B . Wild - type and KO - OFD1 cells were starved in HBSS with / without Baf - A1 ( 100nM , 2h ) and chloroquine ( CQ ) ( 50μM , 2h ) and total lysates were analyzed by immunoblotting using anti - OFD1 , - LC3B ( LC3B - I 18 kDa and LC3B - II 16 kDa ) and - ACTIN antibodies . ( Right ) ACTIN - normalized LC3B - II levels are expressed as fold change versus untreated conditions ( - ) of wt cells . n = 5 independent experiments .

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B. Wild-type and KO-OFD1 cells were starved in HBSS with/without Baf-A1 (100nM, 2h) and chloroquine (CQ) (50μM, 2h) and total lysates were analyzed by immunoblotting using anti-OFD1, -LC3B (LC3B-I 18 kDa and LC3B-II 16 kDa) and -ACTIN antibodies. (Right) ACTIN-normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of wt cells. n=5 independent experiments.


C . Representative images of LC3B and WIPI2 puncta in wt and KO - OFD1 cells ( HBSS , 90min ) . Green , LC3B ; red , WIPI2 ; blue , Hoechst , nuclei . Scale bar = 10μm . ( Right ) Quantification of LC3B and WIPI2 puncta is shown . ≥ 100 cells analyzed / sample , n = 5 independent experiments .

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C. Representative images of LC3B and WIPI2 puncta in wt and KO-OFD1 cells (HBSS, 90min). Green, LC3B; red, WIPI2; blue, Hoechst, nuclei. Scale bar=10μm. (Right) Quantification of LC3B and WIPI2 puncta is shown. ≥100 cells analyzed/sample, n=5 independent experiments.


D . Transmission electron microscopy ( left ) and quantification ( right ) of autophagic vacuoles ( arrows ) in wt and KO - OFD1 cells ( HBSS , 90min ) .

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D. Transmission electron microscopy (left) and quantification (right) of autophagic vacuoles (arrows) in wt and KO-OFD1 cells (HBSS, 90min).


E . Representative images of siOFD1 depleted and control ( Ctrl ) HeLa cells transiently expressing mRFP - eGFP - LC3B ( HBSS , 90min ) . Insets show higher magnification of colocalization in selected areas . Scale bar = 10μm . Bottom , tandem structure of the mRFP - GFP - LC3B construct and quantification of RFP + GFP + and RFP + GFP - puncta in siOFD1 - depleted cells and controls , cultured in full medium ( FM ) or kept in HBSS for 2h ( STV ) are depicted . n ≥ 90 cells / sample analyzed , n = 5 independent experiments .

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E. Representative images of siOFD1 depleted and control (Ctrl) HeLa cells transiently expressing mRFP-eGFP-LC3B (HBSS, 90min). Insets show higher magnification of colocalization in selected areas. Scale bar=10μm. Bottom, tandem structure of the mRFP-GFP-LC3B construct and quantification of RFP+GFP+ and RFP+GFP- puncta in siOFD1-depleted cells and controls, cultured in full medium (FM) or kept in HBSS for 2h (STV) are depicted. n≥90 cells/sample analyzed, n=5 independent experiments.


F . Representative images of WIPI2 staining in wt HK2 cells transiently expressing eGFP - OFD1 ( HBSS , 90min ) . Green , eGFP - OFD1 , red , WIPI2 ; blue , Hoechst labels nuclei . On the right , quantification of WIPI2 puncta in eGFP - OFD1 transfected cells compared with non - transfected cells ( Ctrl ) is shown . Scale bar = 10μm . n ≥ 90 cells analyzed / sample , n = 3 independent experiments .

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F. Representative images of WIPI2 staining in wt HK2 cells transiently expressing eGFP-OFD1 (HBSS,90min). Green, eGFP-OFD1, red, WIPI2; blue, Hoechst labels nuclei. On the right, quantification of WIPI2 puncta in eGFP-OFD1 transfected cells compared with non-transfected cells (Ctrl) is shown. Scale bar=10μm. n≥90 cells analyzed/sample, n=3 independent experiments.


G . Representative images of WIPI2 and OFD1 co - staining in KO - OFD1 cells transfected with empty vector ( Empty ) , 3xFLAG - OFD1 ( OFD1 ) or 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) ( HBSS , 90min ) . Red , WIPI2 ; green , OFD1 ; blue , Hoechst labels nuclei . Scale bars : 10μm . On the right , quantification of WIPI2 puncta . ≥ 100 cells analyzed / sample , n = 4 independent experiments .

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

G. Representative images of WIPI2 and OFD1 co-staining in KO-OFD1 cells transfected with empty vector (Empty), 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) (HBSS,90min). Red, WIPI2; green, OFD1; blue, Hoechst labels nuclei. Scale bars: 10μm. On the right, quantification of WIPI2 puncta. ≥100 cells analyzed/sample, n=4 independent experiments.


A . Western blot of LC3B in lymphoblasts of OFD type I patients and controls , either untreated ( - ) or exposed to Baf - A1 ( 10nM , 2h ) ( + ) . Cells were cultured in complete medium ( FM , top ) or under serum starvation for 4h ( STV , bottom ) . ACTIN and GAPDH were used as loading controls .

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A. Western blot of LC3B in lymphoblasts of OFD type I patients and controls, either untreated (-) or exposed to Baf-A1 (10nM, 2h)(+). Cells were cultured in complete medium (FM, top) or under serum starvation for 4h (STV, bottom). ACTIN and GAPDH were used as loading controls.


B . ( Left ) Graphs show LC3B - II levels normalized on ACTIN in patients lymphoblasts and controls cultured in complete medium ( FM ) . ( Right ) Graphs show LC3B - II levels normalized on GAPDH in patients lymphoblasts and controls in serum starvation for 4h ( STV ) . Normalized LC3B - II levels are expressed as fold change versus untreated conditions ( - ) of control cells , n = 4 patients and 3 controls .

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B. (Left) Graphs show LC3B-II levels normalized on ACTIN in patients lymphoblasts and controls cultured in complete medium (FM). (Right) Graphs show LC3B-II levels normalized on GAPDH in patients lymphoblasts and controls in serum starvation for 4h (STV). Normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of control cells, n=4 patients and 3 controls.


C . Western blot of ATG13 and ATG101 proteins in serum starved lymphoblasts from controls and OFD type I patients . ( Right ) Protein levels relative to ACTIN ( loading control ) are expressed as fold change versus controls , represented by the dashed line , n = 4 patients and 3 controls .

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C. Western blot of ATG13 and ATG101 proteins in serum starved lymphoblasts from controls and OFD type I patients. (Right) Protein levels relative to ACTIN (loading control) are expressed as fold change versus controls, represented by the dashed line, n=4 patients and 3 controls.