( E ) Northern blot analysis of pre - rRNAs from HT1080PARN WT and HT1080PARN KO cells , transduced with an empty vector . ( F ) Ectopic expression of PARN was induced in HT1080PARN KO cells and compared to cells transduced with an empty vector . Western blot relative to actin assessed PARN levels . ( G ) Quantitative analyses of 18S - E / 21S and 18S - E FL / 21S ratios were as described in ( D ) for HT1080PARN KO cells relative to HT1080PARN WT ( left panel ; see ( E ) ) , and for HT1080PARN KO cells rescued by ectopic expression of PARN relative to HT1080PARN KO cells transduced with an empty expression vector ( right panel ; see ( F ) ) , with Log averages + standard deviation for four independent experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O 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) Northern blot analysis of pre-rRNAs from HT1080PARN WT and HT1080PARN KO cells, transduced with an empty vector. (F) Ectopic expression of PARN was induced in HT1080PARN KO cells and compared to cells transduced with an empty vector. Western blot relative to actin assessed PARN levels. (G) Quantitative analyses of 18S-E/21S and 18S-E FL/21S ratios were as described in (D) for HT1080PARN KO cells relative to HT1080PARN WT (left panel; see (E)), and for HT1080PARN KO cells rescued by ectopic expression of PARN relative to HT1080PARN KO cells transduced with an empty expression vector (right panel; see (F)), with Log averages + standard deviation for four independent experiments.


( A ) Western blot analysis of Parn expression in control and Parn + / - MEFs .

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(A) Western blot analysis of Parn expression in control and Parn+/- MEFs.


( B ) Northern blot analysis of total RNAs from WT and Parn + / - MEFs .

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(B) Northern blot analysis of total RNAs from WT and Parn+/- MEFs.


( D ) Frequencies of expected and observed newborns of the indicated genotype obtained from Parn + / - intercrosses . n indicates the number of animal analyzed . Statistical significance ( P = 1 . 5417E - 08 ) of the differences between the observed and expected genotype distributions was assessed by χ2 - test . ( E ) Frequencies of expected and observed E11 . 5 embryos of the indicated genotype obtained from Parn + / - intercrosses . n indicates the number of animal analysed . Statistical significance ( P = 0 . 02535 ) of the differences between the observed and expected genotype distributions was assessed by χ2 - test .

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(D) Frequencies of expected and observed newborns of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analyzed. Statistical significance (P = 1.5417E-08) of the differences between the observed and expected genotype distributions was assessed by χ2-test. (E) Frequencies of expected and observed E11.5 embryos of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analysed. Statistical significance (P = 0.02535) of the differences between the observed and expected genotype distributions was assessed by χ2-test.


( F ) , ( G ) p53 knock - out does not rescue Parn - / - . No Parn - / - p53 - / - mouse was born from various combinations of Parn + / - and either p53 + / - or p53 - / - crosses ( 49 and 56 pups , respectively ) . Statistical significance ( P = 0 . 000886 ) of the differences between the observed and expected genotype distributions was assessed by χ2 - test .

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(F), (G) p53 knock-out does not rescue Parn-/-. No Parn-/- p53-/- mouse was born from various combinations of Parn+/- and either p53+/- or p53-/- crosses (49 and 56 pups, respectively). Statistical significance (P = 0.000886) of the differences between the observed and expected genotype distributions was assessed by χ2-test.


( C ) Streptavidin Western blotting of total biotinylated proteins in APEX2 : RAB or APEX2 only Flp - In / T - REx HeLa cells .

(C) Streptavidin Western blotting of total biotinylated proteins in APEX2:RAB or APEX2 only Flp-In/T-REx HeLa cells.


( D ) APEX2 : RAB biotinylated proteins ( streptavidin ) are partially colocalized with EEA1 in HeLa cells , n = 2 independent experiments . Scale bars : 10µm or 5µm in enlarged views .

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(D) APEX2:RAB biotinylated proteins (streptavidin) are partially colocalized with EEA1 in HeLa cells, n=2 independent experiments. Scale bars: 10µm or 5µm in enlarged views.


( C ) ProHits - viz generated dotblots of RAB effectors , GEFs , GAPs and sorting complexes . Green , blue and beige shadings along with RAB names refer to previously identified interactions between RABs and the depicted proteins .

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(C) ProHits-viz generated dotblots of RAB effectors, GEFs, GAPs and sorting complexes. Green, blue and beige shadings along with RAB names refer to previously identified interactions between RABs and the depicted proteins.


Strumpellin and VPS35 are in close proximity to RAB5 and RAB21 . ( A ) Anti - biotin immunoprecipitation and immunoblot of endogenous Strumpellin and VPS35 . Lysates correspond to 1 % of input , n = 3 independent experiments . RAB21 can be seen interacting with various WASH and retromer complexes subunits .

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Strumpellin and VPS35 are in close proximity to RAB5 and RAB21. (A) Anti-biotin immunoprecipitation and immunoblot of endogenous Strumpellin and VPS35. Lysates correspond to 1% of input, n=3 independent experiments. RAB21 can be seen interacting with various WASH and retromer complexes subunits.


GFP trap IP of WT , Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and endogenous ( B ) VPS26 immunoblot Lysates correspond to 5 % of input , n ≥ 3 independent experiments .

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GFP trap IP of WT, Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and endogenous (B) VPS26 immunoblot Lysates correspond to 5% of input, n≥ 3 independent experiments.


GFP trap IP of WT , Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and ( C ) FAM21 , Strumpellin and VPS35 immunoblots . Lysates correspond to 5 % of input , n ≥ 3 independent experiments .

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GFP trap IP of WT, Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and (C) FAM21, Strumpellin and VPS35 immunoblots. Lysates correspond to 5% of input, n≥ 3 independent experiments.


RAB21 actively pull - downs WASH and retromer complex subunits ( D ) Bacterially - purified and GTP - loaded GST : RAB21 pull - down of HeLa cell lysates followed by Strumpellin , FAM21 , CAPZα and VPS35 immunoblots . Ponceau staining reveals the purity of the GST and GST : RAB21 used for the pull - downs . n = 3 independent experiments .

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RAB21 actively pull-downs WASH and retromer complex subunits (D) Bacterially-purified and GTP-loaded GST:RAB21 pull-down of HeLa cell lysates followed by Strumpellin, FAM21, CAPZα and VPS35 immunoblots. Ponceau staining reveals the purity of the GST and GST:RAB21 used for the pull-downs. n=3 independent experiments.


( E ) RAB21 colocalizes with the WASH and retromer complexes . Transiently expressing GFP : RAB21 HeLa cells were fixed and stained for endogenous EEA1 , FAM21 , WASH and VPS26 . Boxed region is magnified and single channels are depicted . Scale bars : 10µm or 5µm in enlarged view .

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(E) RAB21 colocalizes with the WASH and retromer complexes. Transiently expressing GFP:RAB21 HeLa cells were fixed and stained for endogenous EEA1, FAM21, WASH and VPS26. Boxed region is magnified and single channels are depicted. Scale bars: 10µm or 5µm in enlarged view.


( F ) Per cell Pearson Correlation between RAB21 and the various markers . Error bars are SEM , n = 2 independent experiments .

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(F) Per cell Pearson Correlation between RAB21 and the various markers. Error bars are SEM, n=2 independent experiments.


( A ) Low RAB21 expression levels in two independent RAB21 KO cell populations . Immunoblot analysis of endogenous RAB21 , RAB5 , RAB7 and GAPDH .

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(A) Low RAB21 expression levels in two independent RAB21 KO cell populations. Immunoblot analysis of endogenous RAB21, RAB5, RAB7 and GAPDH.


( B ) Endo - lysosomal compartments are unaffected by the loss of RAB21 . Immunofluorescences of APPL1 , TfR , RAB7 and LAMP1 in parental HeLa cells and in the two RAB21 knockout cell populations . Scale bars : 10µm , n = 3 independent experiments .

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(B) Endo-lysosomal compartments are unaffected by the loss of RAB21. Immunofluorescences of APPL1, TfR, RAB7 and LAMP1 in parental HeLa cells and in the two RAB21 knockout cell populations. Scale bars :10µm, n=3 independent experiments.


The stability of the retromer cargo sorting complex is affected in RAB21 KO cells . Immunoblot analysis of VPS35 , VPS26 , VPS29 , GAPDH and RAB21 in parental and the two RAB21 KO cell populations . Ratio of VPS35 , VPS26 and VPS29 integrated densities to GAPDH for four independent experiments ; SEM . * represents p < 0 . 05 Statistical tests used : One sample t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The stability of the retromer cargo sorting complex is affected in RAB21 KO cells. Immunoblot analysis of VPS35, VPS26, VPS29, GAPDH and RAB21 in parental and the two RAB21 KO cell populations. Ratio of VPS35, VPS26 and VPS29 integrated densities to GAPDH for four independent experiments; SEM. * represents p <0.05 Statistical tests used: One sample t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( B ) WASH complex proteins are not affected by RAB21 deletion . Immunoblot of Strumpellin , WASH , FAM21 , CAPZα , GAPDH and RAB21 . Ratio of WASH , FAM21 , Strumpellin and CAPZα integrated densities to GAPDH for four independent experiments ; SEM . Data information Statistical tests used : One sample t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

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(B) WASH complex proteins are not affected by RAB21 deletion. Immunoblot of Strumpellin, WASH, FAM21, CAPZα, GAPDH and RAB21. Ratio of WASH, FAM21, Strumpellin and CAPZα integrated densities to GAPDH for four independent experiments; SEM. Data information Statistical tests used: One sample t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


RAB21 knockout decreases VPS35 localization at endosomes . ( C ) Immunofluorescence of endogenous VPS35 and EEA1 in RAB21 - depleted cells . Boxed region is magnified and single channel images are depicted . ( D ) Per cell Pearson Correlation between VPS35 and EEA1 ; SEM , n = 3 independent experiments . Data information scale bars represent 10µm or 2 . 5µm in enlarged views . Statistical tests used : ( D ) - Unpaired t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

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RAB21 knockout decreases VPS35 localization at endosomes. (C) Immunofluorescence of endogenous VPS35 and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (D) Per cell Pearson Correlation between VPS35 and EEA1; SEM, n=3 independent experiments. Data information scale bars represent 10µm or 2.5µm in enlarged views. Statistical tests used: (D) - Unpaired t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


RAB21 knockout decreases VPS35 localization at endosomes . ( E ) Average number of VPS35 puncta per cell ; SEM , n = 3 independent experiments . Data information Statistical tests used : Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

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RAB21 knockout decreases VPS35 localization at endosomes. (E) Average number of VPS35 puncta per cell; SEM, n=3 independent experiments. Data information Statistical tests used: Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( F - H ) RAB21 loss impairs endosomal recruitment of the WASH complex . ( F ) Immunofluorescence of endogenous WASH and EEA1 in RAB21 - depleted cells . Boxed region is magnified and single channel images are depicted . ( G ) Per cell Pearson Correlation between VPS35 and EEA1 and ( H ) FAM21 and EEA1 ; SEM , n = 2 independent experiments . Data information : scale bars represent 10µm or 2 . 5µm in enlarged views . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

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(F-H) RAB21 loss impairs endosomal recruitment of the WASH complex. (F) Immunofluorescence of endogenous WASH and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (G) Per cell Pearson Correlation between VPS35 and EEA1 and (H) FAM21 and EEA1; SEM, n=2 independent experiments. Data information: scale bars represent 10µm or 2.5µm in enlarged views. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( I - J ) RAB21 knockout reduces endosomal F - actin levels . ( I ) Immunofluorescence of endogenous F - actin using Alexa - 488 conjugated phalloidin and EEA1 in RAB21 - depleted cells . Boxed region is magnified and single channel images are depicted . ( J ) Per cell Pearson Correlation between internal F - actin and EEA1 ; SEM , n = 3 independent experiments . Data information scale bars represent 10µm or 2 . 5µm in enlarged views . Statistical tests used : Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

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(I-J) RAB21 knockout reduces endosomal F-actin levels. (I) Immunofluorescence of endogenous F-actin using Alexa-488 conjugated phalloidin and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (J) Per cell Pearson Correlation between internal F-actin and EEA1; SEM, n=3 independent experiments. Data information scale bars represent 10µm or 2.5µm in enlarged views. Statistical tests used: Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( A - C ) RAB21 is not required for CI - MPR or GLUT1 trafficking . ( A ) Immunofluorescence of endogenous CI - MPR and TGN46 or GLUT1 with LAMP1 in RAB21 - knockout cells . Boxed region is magnified . ( B ) Per cell Pearson Correlation between CI - MPR and TGN46 or ( C ) GLUT1 and LAMP1 ; SEM , n = 3 independent experiments . Data information : In ( A ) , scale bars represent 20µm or 5µm in enlarged views for CI - MPR , while they represent 10µm in enlarged views for GLUT1 . Statistical tests used : ( B ) - Unpaired t - tests ( C Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all

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(A-C) RAB21 is not required for CI-MPR or GLUT1 trafficking. (A) Immunofluorescence of endogenous CI-MPR and TGN46 or GLUT1 with LAMP1 in RAB21-knockout cells. Boxed region is magnified. (B) Per cell Pearson Correlation between CI-MPR and TGN46 or (C) GLUT1 and LAMP1; SEM, n=3 independent experiments. Data information: In (A), scale bars represent 20µm or 5µm in enlarged views for CI-MPR, while they represent 10µm in enlarged views for GLUT1. Statistical tests used: (B) - Unpaired t-tests (C Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats


MCT1 trafficking requires RAB21 . ( D ) Immunofluorescence of endogenous MCT1 in wild type or RAB21 - depleted cells . ( E ) Per cell integrated MCT1 intensity ( in Relative Fluorescence Unit ) ; SEM , n = 2 independent experiments . Data information : scale bars represent 20µm or 5µm in enlarged views . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all

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MCT1 trafficking requires RAB21. (D) Immunofluorescence of endogenous MCT1 in wild type or RAB21-depleted cells. (E) Per cell integrated MCT1 intensity (in Relative Fluorescence Unit); SEM, n=2 independent experiments. Data information: scale bars represent 20µm or 5µm in enlarged views. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats


( F ) RAB21 interacts with SLC3A2 . FLAG immunoprecipitation of FLAG : RAB21 and immunoblot of co - expressed SLC3A2 : HA . Lysates correspond to 5 % input , n = 3 independent experiments .

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(F) RAB21 interacts with SLC3A2. FLAG immunoprecipitation of FLAG:RAB21 and immunoblot of co-expressed SLC3A2:HA. Lysates correspond to 5% input, n=3 independent experiments.


( G - H ) RAB21 is required for appropriate SLC3A2 , Basigin and CD44 trafficking . ( G ) Antibody - uptake assays in parental or knockout RAB21 cells . Cells were stained with an Alexa488 conjugated anti - mouse antibody . Arrowheads point to endosomal tubules . Note that non - internalized antibodies were removed by an acid wash , which was not fully efficient for CD147 and CD44 , explaining plasma membrane labeling . ( H ) Percentage of cells with tubules in parental or RAB21 knockout cells ; SEM , n = 3 independent experiments . Data information scale bars represent 20µm or 5µm in enlarged views . Statistical tests used Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all

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(G-H) RAB21 is required for appropriate SLC3A2, Basigin and CD44 trafficking. (G) Antibody-uptake assays in parental or knockout RAB21 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Note that non-internalized antibodies were removed by an acid wash, which was not fully efficient for CD147 and CD44, explaining plasma membrane labeling. (H) Percentage of cells with tubules in parental or RAB21 knockout cells; SEM, n=3 independent experiments. Data information scale bars represent 20µm or 5µm in enlarged views. Statistical tests used Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats


( A ) Validation of WASH and retromer knockouts and SLC3A2 protein levels . Immunoblots of parental or FAM21 , VARP and VPS29 knockout cell populations .

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(A) Validation of WASH and retromer knockouts and SLC3A2 protein levels. Immunoblots of parental or FAM21, VARP and VPS29 knockout cell populations.


( B ) Antibody - uptake assays in parental or knockout FAM21 , VARP and VPS29 cells . Cells were stained with an Alexa488 conjugated anti - mouse antibody . Arrowheads point to endosomal tubules . Data information : ( B ) scale bars represent

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O

(B) Antibody-uptake assays in parental or knockout FAM21, VARP and VPS29 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Data information: (B) scale bars represent 20µm


( C ) Percentage of cells with tubules in parental or in various knockout cells ; SEM , n = 3 independent experiments . Statistical tests used : ( C ) - Unpaired t - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of cells with tubules in parental or in various knockout cells; SEM, n=3 independent experiments. Statistical tests used: (C) - Unpaired t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( D ) Ratio of SLC3A2 integrated densities to GAPDH ; SEM , n = 3 independent experiments . Statistical tests used : ( D ) One sample T - tests All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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) Ratio of SLC3A2 integrated densities to GAPDH; SEM, n=3 independent experiments. Statistical tests used: (D) One sample T-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


Retromer is required for endosomal localization of RAB21 . Immunofluorescence of transiently expressed GFP : RAB21 with endogenous ( E ) EEA1 in parental or VARP - and VPS29 - deleted cells . Boxed region is magnified underneath . Data information scale bars represent 10µm or 2 . 5µm in enlarged views .

B-SUBCELLULAR O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (E) EEA1 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. Data information scale bars represent 10µm or 2.5µm in enlarged views.


Retromer is required for endosomal localization of RAB21 . Immunofluorescence of transiently expressed GFP : RAB21 with endogenous ( F ) VPS26 in parental or VARP - and VPS29 - deleted cells . Boxed region is magnified underneath . Data information : scale bars represent 10µm or 2 . 5µm in enlarged views .

B-SUBCELLULAR O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (F) VPS26 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. Data information: scale bars represent 10µm or 2.5µm in enlarged views.


( G ) Per cell Pearson Correlation between GFP : RAB21 and EEA1 or VPS26 ; SEM , n = 3 independent experiments . Statistical tests used : ( G ) - Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all repeats .

O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Per cell Pearson Correlation between GFP:RAB21 and EEA1 or VPS26; SEM, n=3 independent experiments. Statistical tests used: (G) - Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


A , C ) After 24 , 48 , and 72 h incubation bortezomib inhibited cell growth in a concentration dependent manner . Untreated cells were measured as controls ( n = 12 , mean ± S . D . ) .

O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O 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,C) After 24, 48, and 72 h incubation bortezomib inhibited cell growth in a concentration dependent manner. Untreated cells were measured as controls (n = 12, mean ± S.D.).


B , D ) Dynamic proliferation curves for Cal - 78 and SW - 1353 cells in the presence of 0 ( red ) , 2 . 5 nM ( green ) , 5 nM ( blue ) , and 10 nM ( purple ) bortezomib . Data shown are representatives from three independent experiments ( n = 3 , measured in biological quadruplicates ) .

O O O O O B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

B,D) Dynamic proliferation curves for Cal-78 and SW-1353 cells in the presence of 0 (red), 2.5 nM (green), 5 nM (blue), and 10 nM (purple) bortezomib. Data shown are representatives from three independent experiments (n = 3, measured in biological quadruplicates).


A , C ) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3 / 7 activity than untreated cells . Untreated control cells served as reference value ( ratio = 1 ) .

O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

A,C) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3/7 activity than untreated cells. Untreated control cells served as reference value (ratio = 1).


B , D ) Cleavage of caspase - 3 was detected after 24 h of bortezomib treatment by flow cytometry . The y - axis denotes cell counts and the x - axis represents fluorescence intensity of the APC antibody . The black filled histogram represents untreated control cells , the striated histogram represents 5 nM , and the checkered histogram shows 10 nM bortezomib treated cells .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O 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-SMALL_MOLECULE O O O

B,D) Cleavage of caspase-3 was detected after 24 h of bortezomib treatment by flow cytometry. The y-axis denotes cell counts and the x-axis represents fluorescence intensity of the APC antibody. The black filled histogram represents untreated control cells, the striated histogram represents 5 nM, and the checkered histogram shows 10 nM bortezomib treated cells.


A , B ) Relative gene expression analysis of the pro - and anti - apoptotic markers Bax , Bak , Bcl - 2 , and Bcl - xl in Cal - 78 and SW - 1353 cells treated with the respective IC50 values of bortezomib for 24 h ( n = 10 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

A,B) Relative gene expression analysis of the pro- and anti-apoptotic markers Bax, Bak, Bcl-2, and Bcl-xl in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h (n = 10).


C ) The human apoptosis antibody protein array revealed the downregulation of the heat shock proteins HSP27 , HSP60 , and HSP70 as well as HTRA , Livin , p27 , and p53 in both cell lines .

O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O

C) The human apoptosis antibody protein array revealed the downregulation of the heat shock proteins HSP27, HSP60, and HSP70 as well as HTRA, Livin, p27, and p53 in both cell lines.


D ) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O

D) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment.


A , B ) Relative gene expression analysis of IGF1R , Fas , and the death receptors TRAILR - 1 and TRAILR - 2 after bortezomib treatment with the respective IC50 concentrations for 48 h . Untreated control cells served as reference value ( ratio = 1 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

A,B) Relative gene expression analysis of IGF1R, Fas, and the death receptors TRAILR-1 and TRAILR-2 after bortezomib treatment with the respective IC50 concentrations for 48 h. Untreated control cells served as reference value (ratio = 1).


C ) The human apoptosis antibody protein array supported the important role of the IGF binding proteins ( IGFBP - 1 to IGFBP - 6 ) , the TNF receptor family , and the TRAIL receptors in both cell lines .

O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O

C) The human apoptosis antibody protein array supported the important role of the IGF binding proteins (IGFBP-1 to IGFBP-6), the TNF receptor family, and the TRAIL receptors in both cell lines.


D ) Western blot analysis showed the downregulation of Fas and TNF - R1 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

D) Western blot analysis showed the downregulation of Fas and TNF-R1.


A ) Relative gene expression and B ) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5 / 12 and Beclin in Cal - 78 and SW - 1353 cells treated with the respective IC50 values of bortezomib for 24 h .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O

A) Relative gene expression and B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h.


B ) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5 / 12 and Beclin in Cal - 78 and SW - 1353 cells treated with the respective IC50 values of bortezomib for 24 h .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O

B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h.


C ) Western blot analysis for the expression of LC3BI - II . The lysosomal protease inhibitors E64d and pepstatin A ( Inh ) blocked the autophagic flux and inhibited the degradation of LC3B - II .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O

C) Western blot analysis for the expression of LC3BI-II. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II.


D ) Quantification of the relative LC3B protein expression .

D) Quantification of the relative LC3B protein expression.


A ) Effect of bortezomib treatment on LC3B immunostaining of Cal - 78 and SW - 1353 cells . Double immunolabeling with DAPI ( blue ) and anti - LC3B ( green ) antibodies was performed as described in the methods section . The lysosomal protease inhibitors E64d and pepstatin A ( Inh ) blocked the autophagic flux and inhibited the degradation of LC3B - II ( bar : 20 μm ) .

O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

A) Effect of bortezomib treatment on LC3B immunostaining of Cal-78 and SW-1353 cells. Double immunolabeling with DAPI (blue) and anti-LC3B (green) antibodies was performed as described in the methods section. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II (bar: 20 μm).


B ) Cal - 78 and SW - 1353 cells were treated either with bortezomib alone or in combination with the lysosomal protease inhibitors E64d and pepstatin A ( Inh ; striated bars ) and cell viability was analysed by the MTS assay . Untreated cells were measured as controls ( light grey ; n = 8 , mean ± S . D . ) .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY 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) Cal-78 and SW-1353 cells were treated either with bortezomib alone or in combination with the lysosomal protease inhibitors E64d and pepstatin A (Inh; striated bars) and cell viability was analysed by the MTS assay. Untreated cells were measured as controls (light grey; n = 8, mean ± S.D.).


Accumulation of Ub in autophagy - deficient brain . Ub profile from the 1 % Triton X - 100 - soluble and - insoluble fraction of autophagy - deficient brain homogenates captured with P2UBA resin . ( A ) Age - matched control Atg7 littermates ( Atg7flox / + ; nestin - Cre : p62 + / − ; denoted + ) at 6 wk ( open circles ; n = 4 ) or Atg7 KO ( Atg7flox / flox ; nestin - Cre : p62 + / − ; denoted − ) at 6 wk ( closed diamonds ; n = 3 ) are shown .

O O B-GENEPROD O O O O B-TISSUE O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (A) Age-matched control Atg7 littermates (Atg7flox/+;nestin-Cre:p62+/−; denoted +) at 6 wk (open circles; n = 4) or Atg7 KO (Atg7flox/flox;nestin-Cre:p62+/−; denoted −) at 6 wk (closed diamonds; n = 3) are shown.


Accumulation of Ub in autophagy - deficient brain . Ub profile from the 1 % Triton X - 100 - soluble and - insoluble fraction of autophagy - deficient brain homogenates captured with P2UBA resin . ( B ) Control Atg5 littermates ( Atg5flox / + ; nestin - Cre ; denoted + ) at 16 ( open circles ; n = 4 ) and 26 wk ( open diamonds ; n = 3 ) or Atg5 KO ( Atg5flox / flox ; nestin - Cre ; denoted − ) at 16 ( closed circles ; n = 4 ) and 26 wk ( closed diamonds ; n = 3 ) are shown . Each symbol represents one animal . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

O O B-GENEPROD O O O O B-TISSUE O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O 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-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (B) Control Atg5 littermates (Atg5flox/+;nestin-Cre; denoted +) at 16 (open circles; n = 4) and 26 wk (open diamonds; n = 3) or Atg5 KO (Atg5flox/flox;nestin-Cre; denoted −) at 16 (closed circles; n = 4) and 26 wk (closed diamonds; n = 3) are shown. Each symbol represents one animal. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.


Suppressed accumulation of Ub in Atg7 - / p62 - deficient brains . ( A and B ) Quantitative mass spectrometry analysis of P2UBA captured total ( A ) and isopeptide - linked ( B ) Ub from the soluble and insoluble fractions of autophagy - deficient brains . Age - matched control Atg7 littermates ( Atg7flox / + : nestin - Cre : p62 + / − ; denoted + ) at 6 wk ( open circles ; n = 4 ) , p62 KO ( Atg7flox / flox : p62 − / − ; denoted − ) at 6 wk ( closed circles ; n = 3 ) , Atg7 KO ( Atg7flox / flox : nestin - Cre : p62 + / − ; denoted − ) at 6 wk ( closed diamonds ; n = 3 ) , or Atg7 - / p62 - DKO ( Atg7flox / flox : nestin - Cre : p62 − / − ) at 6 wk ( closed triangles ; n = 3 ) are shown . Each symbol represents one animal . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Suppressed accumulation of Ub in Atg7-/p62-deficient brains. (A and B) Quantitative mass spectrometry analysis of P2UBA captured total (A) and isopeptide-linked (B) Ub from the soluble and insoluble fractions of autophagy-deficient brains. Age-matched control Atg7 littermates (Atg7flox/+:nestin-Cre:p62+/−; denoted +) at 6 wk (open circles; n = 4), p62 KO (Atg7flox/flox:p62−/−; denoted −) at 6 wk (closed circles; n = 3), Atg7 KO (Atg7flox/flox:nestin-Cre:p62+/−; denoted −) at 6 wk (closed diamonds; n = 3), or Atg7-/p62-DKO (Atg7flox/flox:nestin-Cre:p62−/−) at 6 wk (closed triangles; n = 3) are shown. Each symbol represents one animal. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.


Suppressed accumulation of Ub in Atg7 - / Nrf2 - deficient livers . ( A and B ) Quantitative mass spectrometry analysis of P2UBA captured p62 ( A ) , total ( A ) , and isopeptide - linked ( B ) Ub from the soluble and insoluble fraction of autophagy - deficient livers ( 28 d after pIpC injection ) . Control littermates ( F / F or F / F : Nrf2 + / − ; denoted + ; open circles ; n = 4 ) , Nrf2 KO ( F / F : Nrf2 − / − ; denoted − ; closed circles ; n = 4 ) , Atg7 KO ( F / F : Mx1 or F / F : Mx1 : Nrf2 + / − ; denoted − ; closed diamonds ; n = 6 ) , or Atg7 - / Nrf2 - DKO ( F / F : Mx1 : Nrf2 − / − ; closed triangles ; n = 6 ) are shown . Each symbol represents one animal . * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Suppressed accumulation of Ub in Atg7-/Nrf2-deficient livers. (A and B) Quantitative mass spectrometry analysis of P2UBA captured p62 (A), total (A), and isopeptide-linked (B) Ub from the soluble and insoluble fraction of autophagy-deficient livers (28 d after pIpC injection). Control littermates (F/F or F/F:Nrf2+/−; denoted +; open circles; n = 4), Nrf2 KO (F/F:Nrf2−/−; denoted −; closed circles; n = 4), Atg7 KO (F/F:Mx1 or F/F:Mx1:Nrf2+/−; denoted −; closed diamonds; n = 6), or Atg7-/Nrf2-DKO (F/F:Mx1:Nrf2−/−; closed triangles; n = 6) are shown. Each symbol represents one animal. **, P ≤ 0.01; ***, P ≤ 0.005.


( B ) Elevated Ub mRNA levels from Atg5 - deficient brains . Total RNA was extracted from control Atg5 littermates ( Atg5flox / + ; nestin - Cre ) at 16 wk or Atg5 KO ( Atg5flox / flox ; nestin - Cre ) at 16 wk ( n = 4 for each genotype ; * * , P ≤ 0 . 01 ) and the amount of transcript from Ubc , Ubb , Uba52 , and Uba80 genes was quantified using real - time RT - PCR and normalized to actin . Error bars indicate SEM . au , arbitrary unit .

O O O O O O O O B-GENEPROD O O B-TISSUE O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O

(B) Elevated Ub mRNA levels from Atg5-deficient brains. Total RNA was extracted from control Atg5 littermates (Atg5flox/+;nestin-Cre) at 16 wk or Atg5 KO (Atg5flox/flox;nestin-Cre) at 16 wk (n = 4 for each genotype; **, P ≤ 0.01) and the amount of transcript from Ubc, Ubb, Uba52, and Uba80 genes was quantified using real-time RT-PCR and normalized to actin. Error bars indicate SEM. au, arbitrary unit.


( B ) The addition of dox for 96 h decreased autophagy in the bicistronic stably expressing cell lines similar to the parental line , m5 - 7 , as measured by the decreased levels of phosphatidylethanolamine - modified LC3 ( LC3 - II ) .

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O

(B) The addition of dox for 96 h decreased autophagy in the bicistronic stably expressing cell lines similar to the parental line, m5-7, as measured by the decreased levels of phosphatidylethanolamine-modified LC3 (LC3-II).


( C ) Western blot ( WB ) analysis of the accumulation of htt ( Q ) - GFP after autophagy shutoff . At steady - state ( t = 0 h ) , the levels of both htt ( Q25 ) - GFP and htt ( Q47 ) - GFP were virtually undetectable . After autophagy shutoff , there was a robust increase in the level of htt ( Q47 ) - GFP , as detected by GFP Western blot , whereas there was very little or no detectable increase in htt ( Q25 ) - GFP , GAPDH , LDH , or chFP .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O

(C) Western blot (WB) analysis of the accumulation of htt(Q)-GFP after autophagy shutoff. At steady-state (t = 0 h), the levels of both htt(Q25)-GFP and htt(Q47)-GFP were virtually undetectable. After autophagy shutoff, there was a robust increase in the level of htt(Q47)-GFP, as detected by GFP Western blot, whereas there was very little or no detectable increase in htt(Q25)-GFP, GAPDH, LDH, or chFP.


( D ) Quantitative flow cytometry time course analysis measuring selective autophagy in live cells . The ASI is the ratio of the relative increase in green and red fluorescence , as assessed by two - color flow cytometry , in response to dox . In control experiments , the ASI of two soluble , nonaggregating proteins , htt ( Q25 ) - GFP and chFP , was 1 . In contrast , the ASI of the aggregation - prone protein , htt ( Q47 ) - GFP , compared with chFP after autophagy shutoff ( + dox ; 100 h ) was 2 . Data represent three independent experiments performed on different days . * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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) Quantitative flow cytometry time course analysis measuring selective autophagy in live cells. The ASI is the ratio of the relative increase in green and red fluorescence, as assessed by two-color flow cytometry, in response to dox. In control experiments, the ASI of two soluble, nonaggregating proteins, htt(Q25)-GFP and chFP, was 1. In contrast, the ASI of the aggregation-prone protein, htt(Q47)-GFP, compared with chFP after autophagy shutoff (+dox; 100 h) was 2. Data represent three independent experiments performed on different days. **, P ≤ 0.01; ***, P ≤ 0.005.


( E ) Flow cytometry time course analysis of cell death at different time points after autophagy shutoff ( + dox ) . The degree of cell death correlated directly with increased htt ( Q47 ) - GFP levels after autophagy shutoff and was significantly enhanced in cells expressing htt ( Q47 ) - GFP compared with htt ( Q25 ) - GFP ( * * * , P ≤ 0 . 005 ) . Data represent three independent experiments performed on different days .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(E) Flow cytometry time course analysis of cell death at different time points after autophagy shutoff (+dox). The degree of cell death correlated directly with increased htt(Q47)-GFP levels after autophagy shutoff and was significantly enhanced in cells expressing htt(Q47)-GFP compared with htt(Q25)-GFP (***, P ≤ 0.005). Data represent three independent experiments performed on different days.


( F ) AQUA using heavy isotope - labeled GFP standard in addition to the routine Ub standards after P2UBA pull - down of soluble and insoluble lysates from htt ( Q ) - GFP - IRES - chFP bicistronic expressing cells before ( − dox ) and after autophagy shutoff ( + dox ) . The experimental GFP peptide was only detected in two of the four experiments , and the data shown are representative of these . In the other two experiments , the experimental GFP peptide was below the threshold of detection .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O 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-GENEPROD O O O O O O O O

(F) AQUA using heavy isotope-labeled GFP standard in addition to the routine Ub standards after P2UBA pull-down of soluble and insoluble lysates from htt(Q)-GFP-IRES-chFP bicistronic expressing cells before (−dox) and after autophagy shutoff (+dox). The experimental GFP peptide was only detected in two of the four experiments, and the data shown are representative of these. In the other two experiments, the experimental GFP peptide was below the threshold of detection.


( A - C ) The relative levels of ( A ) canalicular membrane ( BA ) transporter Bsep , Mrp2 , Mdr1b , Mdr2 , Abcg5 and Abcg8 mRNA transcripts , ( B ) basolateral membrane BA transporter Mrp3 , Mrp4 , Ostβ , Ntcp and Oatp1a1 mRNA transcripts , and ( C ) BA synthetic enzyme Cyp7a1 , Cyp7b1 , Cyp8b1 , Cyp27a1 , and Cyp2c70 mRNA transcripts . Wild type , wild type mice , n = 6 ; Heterozygote , Sema7aR145W heterozygous mice , n = 5 ; Homozygote , Sema7aR145W homozygous mice , n = 7 . Data are shown as means ± SD . * P < 0 . 05 versus the WT mice , # P < 0 . 05 versus the heterozygous mutant mice . The data were analyzed by the independent - samples Student ' s t - test and the Mann - Whitney U test .

O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM 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-C) The relative levels of (A) canalicular membrane (BA) transporter Bsep, Mrp2, Mdr1b, Mdr2, Abcg5 and Abcg8 mRNA transcripts, (B) basolateral membrane BA transporter Mrp3, Mrp4, Ostβ, Ntcp and Oatp1a1 mRNA transcripts, and (C) BA synthetic enzyme Cyp7a1, Cyp7b1, Cyp8b1, Cyp27a1, and Cyp2c70 mRNA transcripts. Wild type, wild type mice, n=6; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=7. Data are shown as means ± SD. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.


( D ) Western blot analysis of the relative levels of Bsep , Mrp2 , Mdr2 , Mrp3 , Mrp4 , Ostα / β , Ntcp , Cyp7a1 and Cyp8b1 protein expression in mouse livers . Contrary to the mRNA alterations , the levels of canalicular membrane Bsep and Mrp2 proteins were dramatically reduced in homozygous mouse livers , suggesting that the decreased these proteins were might be mediated by post - translational regulation in the livers of Sema7aR145W mutant mice . Wild type , wild type mice , n = 4 ; Heterozygote , Sema7aR145W heterozygous mice , n = 5 ; Homozygote , Sema7aR145W homozygous mice , n = 5 . * P < 0 . 05 versus the WT mice , # P < 0 . 05 versus the heterozygous mutant mice . The data were analyzed by the independent - samples Student ' s t - test and the Mann - Whitney U test .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM 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) Western blot analysis of the relative levels of Bsep, Mrp2, Mdr2, Mrp3, Mrp4, Ostα/β, Ntcp, Cyp7a1 and Cyp8b1 protein expression in mouse livers. Contrary to the mRNA alterations, the levels of canalicular membrane Bsep and Mrp2 proteins were dramatically reduced in homozygous mouse livers, suggesting that the decreased these proteins were might be mediated by post-translational regulation in the livers of Sema7aR145W mutant mice. Wild type, wild type mice, n=4; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=5. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.


( E , F ) Multiplex IF analyses of Bsep and Mrp2 expression in the livers ( E ) and primary hepatocytes in sandwich cultures ( F ) from WT and homozygous mice . Scale bar , 50 µm .

O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O B-CELL_TYPE O O O O O O O O O O B-ORGANISM O O O O O O O

(E, F) Multiplex IF analyses of Bsep and Mrp2 expression in the livers (E) and primary hepatocytes in sandwich cultures (F) from WT and homozygous mice. Scale bar, 50 µm.


( G ) Multiplex IF analyses revealed that transfection with the SEMA7A _ WT or SEMA7A _ R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose - dependent manner ( 0 , 0 . 2 , 1 . 0 μg per well of 6 - well plate ; 1 . 5 ml culture medium per well ) . Scale bar , 50 µm . Together , the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis ( Appendix Table S16 ) . Subsequently , cholestasis triggered an adaptive response in the liver by down - regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up - regulating the expression of BA efflux transporters Mrp3 , Mrp4 and Ostα / β .

O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O B-GENEPROD O O B-ORGANISM O B-CELL_TYPE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O 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-GENEPROD O O O B-TISSUE B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O B-DISEASE O O O O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O

(G) Multiplex IF analyses revealed that transfection with the SEMA7A_WT or SEMA7A_R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose-dependent manner (0, 0.2, 1.0 μg per well of 6-well plate; 1.5 ml culture medium per well). Scale bar, 50 µm. Together, the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis (Appendix Table S16). Subsequently, cholestasis triggered an adaptive response in the liver by down-regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up-regulating the expression of BA efflux transporters Mrp3, Mrp4 and Ostα/β.


Treatment with UDCA ( 13 mg / kg / d ) and GSH ( 40 mg / kg / d ) for 2 weeks restored normal liver function in the child . However , when the treatment was ceased the levels of serum ALT , AST and TBA returned to abnormally high levels in the patient . Interestingly , treatment with half of the dose of UDCA and GSH also maintained the normal levels of serum ALT and AST , and reduced serum TBA in the child patient .

O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-TISSUE B-GENEPROD O B-GENEPROD O O O B-TISSUE B-SMALL_MOLECULE O O O O O

Treatment with UDCA (13 mg/kg/d) and GSH (40 mg/kg/d) for 2 weeks restored normal liver function in the child. However, when the treatment was ceased the levels of serum ALT, AST and TBA returned to abnormally high levels in the patient. Interestingly, treatment with half of the dose of UDCA and GSH also maintained the normal levels of serum ALT and AST, and reduced serum TBA in the child patient.


C , Dose - response curves of BEAS - 2B and BEAS - 2B - KRAS cells treated for 72 hours with inhibitors of FGFR ( AZD4547 , BGJ398 ) and PLK1 ( BI2536 , BI6727 ) , alone or combination . Values of Fa and CI are shown underneath , with CI < 1 . 0 indicating synergistic effect . Data were from three independent experiments ( n = 3 ) , error bar : SD

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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, Dose-response curves of BEAS-2B and BEAS-2B-KRAS cells treated for 72 hours with inhibitors of FGFR (AZD4547, BGJ398) and PLK1 (BI2536, BI6727), alone or combination. Values of Fa and CI are shown underneath, with CI<1.0 indicating synergistic effect. Data were from three independent experiments (n=3), error bar: SD


C , Dose - response curves of BEAS - 2B and BEAS - 2B - KRAS cells treated for 72 hours with inhibitors of FGFR ( AZD4547 , BGJ398 ) and PLK1 ( BI2536 , BI6727 ) , alone or combination . Values of Fa and CI are shown underneath , with CI < 1 . 0 indicating synergistic effect . Data were from three independent experiments ( n = 3 ) , error bar :

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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 , BEAS - 2B and BEAS - 2B - KRAS treated with the indicated drugs , alone or combination for 72 hours , were cultured in drug - free medium for additional 7 - 21 days . Surviving cells after the treatment were fixed and visualized by crystal violet staining .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

D, BEAS-2B and BEAS-2B-KRAS treated with the indicated drugs, alone or combination for 72 hours, were cultured in drug-free medium for additional 7-21 days. Surviving cells after the treatment were fixed and visualized by crystal violet staining.


C , Clongenic assay of KRAS - mutant lung and pancreatic cells ( MIA PaCa - 2 , H2122 , H2009 , H23 , H358 and A549 ) treated with control ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or combination .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE O B-TISSUE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O

C, Clongenic assay of KRAS-mutant lung and pancreatic cells (MIA PaCa-2, H2122, H2009, H23, H358 and A549) treated with control (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination.


D , Apoptotic assay of KRAS - mutant cancer cells ( H358 , H441 , A549 , and SW620 ) and KRAS - WT lung cancer cells ( EBC - 1 ) treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or in combination for 48h . Data are presented as mean ± SD ( n = 3 ) . * * * P < 0 . 001 and * * * * P < 0 . 0001 by two - way ANOVA with Tukey ' s multiple comparisons test .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O B-CELL_LINE O O B-GENEPROD O O B-DISEASE I-DISEASE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Apoptotic assay of KRAS-mutant cancer cells (H358, H441, A549, and SW620) and KRAS-WT lung cancer cells (EBC-1) treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 48h. Data are presented as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.


E , KRAS mutant lung ( A549 , H358 ， H441 ) , pancreatic ( MIAPaCa - 2 ) and colon ( SW620 ) cancer cells were transfected with control siRNAs , FGFR1 - and PLK1 - specific siRNAs , alone or combination . Cell viability was determined 72 h post transfection . Data are presented as mean ± SD ( n = 3 ) . * * p < 0 . 01 , * * * * p < 0 . 0001 , and ns P ＞ 0 . 05 by two - way ANOVA with Tukey ' s multiple comparisons test . ns , no significance .

O O B-GENEPROD O B-TISSUE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O B-TISSUE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-TISSUE O B-CELL_LINE O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, KRAS mutant lung (A549, H358，H441), pancreatic (MIAPaCa-2) and colon (SW620) cancer cells were transfected with control siRNAs, FGFR1- and PLK1-specific siRNAs, alone or combination. Cell viability was determined 72 h post transfection. Data are presented as mean ± SD (n=3). **p<0.01, ****p<0.0001, and ns P＞0.05 by two-way ANOVA with Tukey's multiple comparisons test. ns, no significance.


D , Clonogenic assay of H358 , A549 and MIA PaCa - 2 cells treated with AZD4547 , BI2536 , and NAC , alone or in combination as indicated .

D, Clonogenic assay of H358, A549 and MIA PaCa-2 cells treated with AZD4547, BI2536, and NAC, alone or in combination as indicated.


E , Immunoblots of H358 and A549 cells treated for 24 h with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , in the presence or absence of NAC ( 5 mM ) .

O O B-EXP_ASSAY O B-CELL_LINE O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

E, Immunoblots of H358 and A549 cells treated for 24 h with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of NAC (5 mM).


clonogenic assay of H358 cells treated for 48 h with vehicle , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , in the presence or absence of Q - VD - OPh ( 20 µM ) . Data shown as mean ± SD ( n = 3 ) . * * * P < 0 . 001 and * * * * P < 0 . 0001 by two - way ANOVA with Tukey ' s multiple comparisons test .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

clonogenic assay of H358 cells treated for 48 h with vehicle, AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of Q-VD-OPh (20 µM). Data shown as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.


B , C , Immunoblots ( B ) and viability assay ( C ) of H358 and A549 cells preincubated overnight with SP600125 ( 2 µM ) and SB203580 ( 1 µM ) and subsequently treated with AZD 4547 ( 5 µM ) and BI2536 ( 5 nM ) for 24 hours . Data were shown as mean ± SD ( n = 3 ) ; * * * P < 0 . 001 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test .

O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C, Immunoblots (B) and viability assay (C) of H358 and A549 cells preincubated overnight with SP600125 (2 µM) and SB203580 (1 µM) and subsequently treated with AZD 4547 (5 µM) and BI2536 (5 nM) for 24 hours. Data were shown as mean ± SD (n=3); ***P<0.001, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.


D , E , Immunoblots ( D ) and viability assay ( E ) of H358 and A549 cells transfected with E2F1 - specific or control siRNAs followed by treatment ( 48 h post transfection ) with vehicle ( DMSO ) or combined AZD 4547 ( 5 µM ) / BI2536 ( 5 nM ) for additional 24 hours . Data were shown as mean ± SD ( n = 3 ) . * * P < 0 . 01 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test .

O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E, Immunoblots (D) and viability assay (E) of H358 and A549 cells transfected with E2F1-specific or control siRNAs followed by treatment (48 h post transfection) with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536(5 nM) for additional 24 hours. Data were shown as mean ± SD (n=3). **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.


F , H358 cells transfected with E2F1 - specific or control siRNAs for 24h were subsequently treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or combination for 48h before apoptotic assay . Data were shown as mean ± SD ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test .

O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H358 cells transfected with E2F1-specific or control siRNAs for 24h were subsequently treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination for 48h before apoptotic assay. Data were shown as mean ± SD (n=3). *P<0.05, **P<0.01 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.


B , A549 cells stably expressing mCHerry - GFP - LC3B were exposed to vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or in combination for 24h . Cell were then fixed and processed for immunofluorescence , scale bar = 10 µm .

O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

B, A549 cells stably expressing mCHerry-GFP-LC3B were exposed to vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h. Cell were then fixed and processed for immunofluorescence, scale bar=10 µm.


E , Flow cytometry - based ROS measure in H358 , A549 and MIA PaCa - 2 cells treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or the combination , in the presence or absence of HCQ ( 10 µM ) for 24h . Quantification of relative ROS levels was shown to the right . Data are presented as mean ± SD ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Flow cytometry-based ROS measure in H358, A549 and MIA PaCa-2 cells treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or the combination, in the presence or absence of HCQ (10 µM) for 24h. Quantification of relative ROS levels was shown to the right. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.


F , Immunoblots of H358 cells transfected with ATG5 - specific or control siRNAs for 48h and before treated for 24h with vehicle ( DMSO ) or combined AZD 4547 ( 5 µM ) / BI2536 ( 5 nM ) .

O O B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O

F, Immunoblots of H358 cells transfected with ATG5-specific or control siRNAs for 48h and before treated for 24h with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536 (5 nM).


A , Growth curve of a KRAS - mutant lung cancer PDX ( BE564T ) treated with vehicle , HCQ ( 30 mg / kg / day ) , AZD4547 ( 10 mg / kg / day ) and BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * * P < 0 . 001 and P ＞ 0 . 05 ( ns ) by one - way ANOVA with Tukey ' s multiple comparisons test . Data are the mean of tumor volume of each group ( 5 mice / group ) ; error bar : SD

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-DISEASE I-DISEASE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O

A, Growth curve of a KRAS-mutant lung cancer PDX (BE564T) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, ***P<0.001 and P＞0.05 (ns) by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (5 mice/group); error bar: SD


A , Growth curve of a KRAS - mutant lung cancer PDX ( BE564T ) treated with vehicle , HCQ ( 30 mg / kg / day ) , AZD4547 ( 10 mg / kg / day ) and BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * * P < 0 . 001 and P ＞ 0 . 05 ( ns ) by one - way ANOVA with Tukey ' s multiple comparisons test . Data are the mean of tumor volume of each group ( 5 mice / group ) ; error bar :

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-DISEASE I-DISEASE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O


D , E , H & E and IHC analysis ( Ki67 and Caspase - 3 ) of PDX ( BE 564T ) tumors after the treatment ( D ) . Quantification of the IHC data for the positivity of Ki - 67 and Caspase - 3 are shown in ( E ) . * P < 0 . 05 and * * P < 0 . 01 by two - way ANOVA with Tukey ' s multiple comparisons test . Data are shown as mean ± SD ( error bar ) of three independent experiments ( n = 3 ) .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E, H&E and IHC analysis (Ki67 and Caspase-3) of PDX (BE 564T) tumors after the treatment (D). Quantification of the IHC data for the positivity of Ki-67 and Caspase-3 are shown in (E). *P<0.05 and **P<0.01 by two-way ANOVA with Tukey's multiple comparisons test. Data are shown as mean ± SD (error bar) of three independent experiments (n=3).


F - K , Growth curves ( F , I ) of KRAS - mutant lung cancer PDXs ( PF139 , PF563 ) treated with vehicle , HCQ ( 30 mg / kg / day ) , AZD4547 ( 10 mg / kg / day ) and BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by one - way ANOVA with Tukey ' s multiple comparisons test . Data are the mean of tumor volume of each group ( 4 mice / group ) ; error bar : SD Relative tumor volume ( G , J ) and weights ( H , K ) of the PDX tumors after the treatment . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and P ＞ 0 . 05 ( ns ) by unpaired two - tailed t - test . Data are the mean ± SD ( error bar ) of tumor weights of each group ( 4 mice / group ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-DISEASE I-DISEASE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O

F-K, Growth curves (F, I) of KRAS-mutant lung cancer PDXs (PF139, PF563) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (4 mice/group); error bar: SD Relative tumor volume (G, J) and weights (H, K) of the PDX tumors after the treatment. *P<0.05, **P<0.01, ***P<0.001 and P＞0.05 (ns) by unpaired two-tailed t-test. Data are the mean ± SD (error bar) of tumor weights of each group (4 mice/group).


F , Box plots showing the percentage of Ki67 - positive cells in different treatment groups . Data are based on IHC staining of lung tissue section of LSL - KrasG12D mice . * P < 0 . 05 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test . The boxplots span from the first to third quartile , the whiskers extend to 1 . 5 × the interquartile range ( IQR ) . The horzontal line within the boxes represents the median of each group of 10 replicates ( n = 10 ) .

O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Box plots showing the percentage of Ki67-positive cells in different treatment groups. Data are based on IHC staining of lung tissue section of LSL-KrasG12D mice. *P<0.05 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. The boxplots span from the first to third quartile, the whiskers extend to 1.5× the interquartile range (IQR). The horzontal line within the boxes represents the median of each group of 10 replicates (n=10).


A Representative IF image of NPCs stained for Nestin , FoxG1 and Pank2 .

O O B-EXP_ASSAY O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O

A Representative IF image of NPCs stained for Nestin, FoxG1 and Pank2.


B NPCs differentiated into neurons by overexpressing Ngn2 ( one representative experiment is shown ) . Two weeks after the infection differentiated NPC were positive for neuronal markers βIII - tubulin ( Tuj1 ) , Map2 , NeuN and humannuclei ( hNu ) and synaptic markers , the voltage - gated Na + channels ( PanNav ) and the vesicular glutamate transporter 1 ( VGlut1 ) .

O B-CELL_TYPE O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-ORGANISM 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-GENEPROD O O

B NPCs differentiated into neurons by overexpressing Ngn2 (one representative experiment is shown). Two weeks after the infection differentiated NPC were positive for neuronal markers βIII-tubulin (Tuj1), Map2, NeuN and humannuclei (hNu) and synaptic markers, the voltage-gated Na+ channels (PanNav) and the vesicular glutamate transporter 1 (VGlut1).


C Western blot of soluble cell homogenates from humanneurons probed with PANK2 and β - actin antibodies ( arrows ) . Asterisk indicates nonspecific band . Data are representative of three independent experiments .

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C Western blot of soluble cell homogenates from humanneurons probed with PANK2 and β-actin antibodies (arrows). Asterisk indicates nonspecific band. Data are representative of three independent experiments.


D Plots showing the total dendritic length and branching points . Data presented as mean and + SEM from at least three independent experiments . A total of 38 neurons were counted for each sample . Statistics were determined by the t - test and resulted not significant .

O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

D Plots showing the total dendritic length and branching points. Data presented as mean and + SEM from at least three independent experiments. A total of 38 neurons were counted for each sample. Statistics were determined by the t-test and resulted not significant.


E Representative example of co - culture containing control ( green ) , PKAN ( red ) humanneurons , and E18 cortical miceneurons . Control and PKAN NPCs were infected with GFP - LV and tdT - LV expressing vectors , respectively , and differentiated for 8 weeks . Scale bars 20 μm .

O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-CELL_TYPE O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O

E Representative example of co-culture containing control (green), PKAN (red) humanneurons, and E18 cortical miceneurons. Control and PKAN NPCs were infected with GFP-LV and tdT-LV expressing vectors, respectively, and differentiated for 8 weeks. Scale bars 20 μm.


F Examples of electrophysiological properties of humanneurons : control individual ( top ) ; PKAN patient ( bottom ) . Traces on the left represent trains of action potentials induced by injection of a suprathreshold current step through the patch electrode in current - clamp mode . Middle traces show Na + and K + currents ( down - and upward - deflecting from baseline , respectively ) in response to a 60 mV step from a holding voltage of - 70 mV in voltage - clamp mode . Insets on the right display enlarged portions of the traces to magnify fast Na + currents . G Histogram with percentages of recorded cells showing repetitively firing ( z - test ) .

O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Examples of electrophysiological properties of humanneurons: control individual (top); PKAN patient (bottom). Traces on the left represent trains of action potentials induced by injection of a suprathreshold current step through the patch electrode in current-clamp mode. Middle traces show Na+ and K+ currents (down- and upward-deflecting from baseline, respectively) in response to a 60 mV step from a holding voltage of -70 mV in voltage-clamp mode. Insets on the right display enlarged portions of the traces to magnify fast Na+ currents. G Histogram with percentages of recorded cells showing repetitively firing (z-test).


A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM , the neuronal specific anti - NCAM antibody and the nuclear staining Hoechst . Left panel , basal conditions . Right panel , after addition of the mitochondrial uncoupler FCCP . Scale bar 20 μm . Plot showing the quantification of TMRM fluorescence signal from NCAM + neurons . Data presented as means + SEM of three independent experiments ( unpaired , two - tailed t - test ) .

O O O O B-ORGANISM O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O

A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM, the neuronal specific anti-NCAM antibody and the nuclear staining Hoechst. Left panel, basal conditions. Right panel, after addition of the mitochondrial uncoupler FCCP. Scale bar 20 μm. Plot showing the quantification of TMRM fluorescence signal from NCAM+neurons. Data presented as means + SEM of three independent experiments (unpaired, two-tailed t-test).


B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope . PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein . Scale bar 500nm . Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in > 30 field ( 200 mitochondria in total ) for each sample ( one - way ANOVA ) .

O O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O

B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope. PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein. Scale bar 500nm. Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in >30 field (200 mitochondria in total) for each sample (one-way ANOVA).


C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls , PKAN patients and PKAN patients overexpressing PANK2 . The plots show OCR normalization to cell number . OCR was measured in basal conditions , and after oligomycin and FCCP addition . Bars indicate means + SEM of three independent experiments ( Two - way ANOVA ) .

O B-EXP_ASSAY O O O O 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-GENEPROD O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls, PKAN patients and PKAN patients overexpressing PANK2. The plots show OCR normalization to cell number. OCR was measured in basal conditions, and after oligomycin and FCCP addition. Bars indicate means + SEM of three independent experiments (Two-way ANOVA).


A Representative images of neurons stained with the neuron specific anti - NCAM antibody , ROS sensible fluorescent probe DCF , and the nuclei dye Hoechst . Scale bar 20 μm . Plots of the DCF fluorescence signal from NCAM positive controls - and PKAN humanneurons , infected or not with Ngn2 - PANK2 - LV . Data presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O

A Representative images of neurons stained with the neuron specific anti-NCAM antibody, ROS sensible fluorescent probe DCF, and the nuclei dye Hoechst. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM positive controls- and PKAN humanneurons, infected or not with Ngn2-PANK2-LV. Data presented as means + SEM of at least three independent experiments (One-way ANOVA).


B Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1 . Scale bar 20 μm . ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots . Data presented as means + SEM of at least three independent experiments ( unpaired , two - tailed t - test ) .

O O O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-CELL_TYPE O O O O O O O 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 Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1. Scale bar 20 μm. ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots. Data presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).


A Upper panel : representative images of in - gel enzymatic activity of mitochondrial and cytosolic aconitase ( mAco and cAco , respectively ) . The lower part of the gel was cut , stained with Coomassie blue , and a protein band was used as loading control ( Loading ) . Lower panel : quantification of mAco and cAco enzymatic activity by densitometry .

O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O

A Upper panel: representative images of in-gel enzymatic activity of mitochondrial and cytosolic aconitase (mAco and cAco, respectively). The lower part of the gel was cut, stained with Coomassie blue, and a protein band was used as loading control (Loading). Lower panel: quantification of mAco and cAco enzymatic activity by densitometry.


B Upper panel : Western blot analysis of mitochondrial and cytosolic aconitases . Lower panel : quantification of mAco and cAco by densitometry .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O

B Upper panel: Western blot analysis of mitochondrial and cytosolic aconitases. Lower panel: quantification of mAco and cAco by densitometry.


C Hemequantification by absorbance at 400 nm of the soluble cell lysates .

O B-SMALL_MOLECULE O O O O O O O O O O O

C Hemequantification by absorbance at 400 nm of the soluble cell lysates.


D Upper panel : western blot analysis of transferrin receptor ( TfR1 ) and ferritin ( FtH ) . Lower panel : quantification of TfR1 or FtH normalized on actin by densitometry . All the data are presented as means + SEM of at least three independent experiments ( unpaired , two - tailed t - test ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O 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 Upper panel: western blot analysis of transferrin receptor (TfR1) and ferritin (FtH). Lower panel: quantification of TfR1 or FtH normalized on actin by densitometry. All the data are presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).


A Representative images of co - cultures of control and PKAN NPCs infected with GFP - LV and tdT - LV expressing vectors , respectively , at the beginning ( Day 1 ) , after 120 ( Day 120 ) and 150 days of differentiation ( Day 150 ) . Scale bar 20 μm . Plots show the number of green and red humanneurons counted at different time points . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O 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 Representative images of co-cultures of control and PKAN NPCs infected with GFP-LV and tdT-LV expressing vectors, respectively, at the beginning (Day 1), after 120 (Day 120) and 150 days of differentiation (Day 150). Scale bar 20 μm. Plots show the number of green and red humanneurons counted at different time points. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days . Repetitive firing activity ( left ) and relatively large Na + and K + currents ( right ) , were restored by CoA . The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs . CoA - treated neurons from control and PKAN patients ( z - test ) .

O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O

B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days. Repetitive firing activity (left) and relatively large Na+ and K+ currents (right), were restored by CoA. The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs. CoA-treated neurons from control and PKAN patients (z-test).


C An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst . Anti - NCAM antibody was used to detect neurons . Scale bar 20 μm . Plots of the DCF fluorescence signal from NCAM + controls - and PKAN humanneurons differentiated or not in the presence of CoA ( 25 μM ) in the medium for 3 weeks . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O 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 An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst. Anti-NCAM antibody was used to detect neurons. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM+ controls- and PKAN humanneurons differentiated or not in the presence of CoA (25 μM) in the medium for 3 weeks. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


D Oxygen consumption rate ( OCR ) with and without CoA on controls and PKAN patients . Basal and uncoupled ( FCCP ) respiration increased after CoA supplementation . Data presented as means + SEM of 24 independent replicates for each condition ( Two - way ANOVA ) .

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D Oxygen consumption rate (OCR) with and without CoA on controls and PKAN patients. Basal and uncoupled (FCCP) respiration increased after CoA supplementation. Data presented as means + SEM of 24 independent replicates for each condition (Two-way ANOVA).


E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

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E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


Representative images of changes in intracellular calcium , as measured in fluorescence ratio images ( F340 / 380 ) ( A ) Examples show a TRPM8 - expressing DRG neuron ( left in A responding to both the TRPM8 agonist menthol ( 300 μM ) and to a cold stimulus from 32 ° C to 4 ° C a neuron that responds to cold ( right in A but does not express any of TRPM8 , TRPA1 or TRPC5 , as shown by lack of response to the TRPM8 agonist menthol ( 300 μM ) Calcium increase is due to influx from extracellular

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE B-CELL_TYPE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O

Representative images of changes in intracellular calcium, as measured in fluorescence ratio images (F340/380) (A) Examples show a TRPM8-expressing DRG neuron (left in A responding to both the TRPM8 agonist menthol (300 μM) and to a cold stimulus from 32°C to 4°C a neuron that responds to cold (right in A but does not express any of TRPM8, TRPA1 or TRPC5, as shown by lack of response to the TRPM8 agonist menthol (300 μM) Calcium increase is due to influx from extracellular solution


experiments showing an SCG neuron responding to the TRPA1 agonist AITC ( left in E and a novel cold - sensitive SCG neuron that responds to cold but does not express any of TRPM8 , TRPA1 or TRPC5 ( right in E Many glial cells are also visibly activated by AITC but not by cold .

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experiments showing an SCG neuron responding to the TRPA1 agonist AITC (left in E and a novel cold-sensitive SCG neuron that responds to cold but does not express any of TRPM8, TRPA1 or TRPC5 (right in E Many glial cells are also visibly activated by AITC but not by cold.


( G ) Mean current voltage relation of 10 SCG neurons at 32 ° C ( dashed line ) , 4 ° C ( blue ) and 32 ° C ( red dots ) . ( H ) Difference trace from ( G ) .

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(G) Mean current voltage relation of 10 SCG neurons at 32°C (dashed line), 4°C (blue) and 32°C (red dots). (H) Difference trace from (G).