( I - J ) RAB21 knockout reduces endosomal F - actin . ( I ) Immunofluorescence of endogenous F - actin using Alexa - 488 conjugated phalloidin EEA1 in RAB21 - depleted . Boxed region is magnified single channel are depicted . ( J ) Per Pearson Correlation between internal F - actin EEA1 ; , n = 3 independent experiments . Data information scale bars represent 10µm or 2 . 5µm in enlarged views . Statistical used : Mann - Whitney . error bars are . Number of presented on each graph represents total number of analyzed for 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 TGN46 or GLUT1 with LAMP1 in RAB21 - knockout . Boxed region is magnified . ( B ) Per Pearson Correlation between CI - MPR TGN46 or ( C ) GLUT1 LAMP1 ; , 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 used : ( B ) - Unpaired t - ( C Mann - Whitney . error bars are . Number of presented on each graph represents total number of analyzed for

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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 . ( E ) Per integrated MCT1 ( in Relative Unit ) ; , n = 2 independent experiments . Data information : scale bars represent 20µm or 5µm in enlarged views . error bars are . Number of presented on each graph represents total number of analyzed for

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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 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 CD44 trafficking . ( G ) Antibody - uptake assays in parental or knockout RAB21 . 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 CD44 , explaining plasma labeling . ( H ) of with tubules in parental or RAB21 knockout ; , n = 3 independent experiments . Data information scale bars represent 20µm or 5µm in enlarged views . Statistical used Mann - Whitney . error bars are . Number of presented on each graph represents total number of analyzed for

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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 retromer knockouts SLC3A2 . Immunoblots of parental or FAM21 , VARP VPS29 knockout 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 VPS29 . were stained with an Alexa488 conjugated anti - mouse antibody . Arrowheads point to endosomal tubules . Data information : ( B ) scale bars represent

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

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

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(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 - VPS29 - deleted . Boxed region is magnified underneath . Data information scale bars represent 10µm or 2 . 5µm in enlarged views .

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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 - VPS29 - deleted . Boxed region is magnified underneath . Data information : scale bars represent 10µm or 2 . 5µm in enlarged views .

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

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(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 , 72 h incubation bortezomib inhibited growth in a dependent manner . Untreated were measured as controls ( n = 12 , mean ± S . D . ) .

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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 SW - 1353 in presence of 0 ( red ) , 2 . 5 nM ( green ) , 5 nM ( blue ) , 10 nM ( purple ) bortezomib . Data shown are representatives from three independent experiments ( n = 3 , measured in biological quadruplicates ) .

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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 showed a significantly higher level of caspase 3 / 7 activity than untreated . Untreated control served as reference value ( = 1 ) .

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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 . y - axis denotes x - axis represents of APC antibody . black filled histogram represents untreated control , striated histogram represents 5 nM , checkered histogram shows 10 nM bortezomib treated .

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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 of pro - anti - apoptotic markers Bax , Bak , Bcl - 2 , Bcl - xl in Cal - 78 SW - 1353 treated with respective IC50 values of bortezomib for 24 h ( n = 10 ) .

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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 ) human apoptosis antibody array revealed downregulation of heat shock HSP27 , HSP60 , HSP70 as well as HTRA , Livin , p27 , p53 in both lines .

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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 showed an upregulation of cytochrome C in fraction after bortezomib treatment .

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


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

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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 ) human apoptosis antibody array supported important role of IGF binding ( IGFBP - 1 to IGFBP - 6 ) , TNF receptor family , TRAIL receptors in both lines .

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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 showed downregulation of Fas TNF - R1 .

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


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

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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 of whole lysates for expression of autophagy markers Atg 5 / 12 Beclin in Cal - 78 SW - 1353 treated with respective IC50 values of bortezomib for 24 h .

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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 for expression of LC3BI - II . lysosomal protease inhibitors E64d pepstatin A ( Inh ) blocked autophagic flux inhibited degradation of LC3B - II .

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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.


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

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

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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 1 % Triton X - 100 - soluble - insoluble fraction of autophagy - deficient brain homogenates captured with P2UBA resin . ( A ) - matched control Atg7 ( 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 .

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

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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 B ) Quantitative mass spectrometry of P2UBA captured total ( A ) isopeptide - linked ( B ) Ub from soluble insoluble fractions of autophagy - deficient brains . - matched control Atg7 ( 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 . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

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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 B ) Quantitative mass spectrometry of P2UBA captured p62 ( A ) , total ( A ) , isopeptide - linked ( B ) Ub from soluble insoluble fraction of autophagy - deficient livers ( 28 d after pIpC injection ) . Control ( 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 . * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

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

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

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

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

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(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 ) of accumulation of htt ( Q ) - GFP after autophagy shutoff . At steady - state ( t = 0 h ) , of both htt ( Q25 ) - GFP htt ( Q47 ) - GFP were virtually undetectable . After autophagy shutoff , there was a robust increase in 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 O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O 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 measuring selective autophagy in live . ASI is of relative increase in green red , as assessed by two - color flow cytometry , in response to dox . In control experiments , ASI of two soluble , nonaggregating , htt ( Q25 ) - GFP chFP , was 1 . In contrast , ASI of aggregation - prone , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 of at different time points after autophagy shutoff ( + dox ) . degree of correlated directly with increased htt ( Q47 ) - GFP after autophagy shutoff was significantly enhanced in expressing htt ( Q47 ) - GFP compared with htt ( Q25 ) - GFP ( * * * , P ≤ 0 . 005 ) . Data represent three independent experiments performed on different days .

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

(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 routine Ub standards after P2UBA pull - down of soluble insoluble lysates from htt ( Q ) - GFP - IRES - chFP bicistronic expressing before ( − dox ) after autophagy shutoff ( + dox ) . experimental GFP peptide was only detected in two of four experiments , data shown are representative of these . In other two experiments , experimental GFP peptide was below threshold of detection .

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

(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 ) relative of ( A ) canalicular ( BA ) transporter Bsep , Mrp2 , Mdr1b , Mdr2 , Abcg5 Abcg8 mRNA transcripts , ( B ) basolateral BA transporter Mrp3 , Mrp4 , Ostβ , Ntcp Oatp1a1 mRNA transcripts , ( C ) BA synthetic enzyme Cyp7a1 , Cyp7b1 , Cyp8b1 , Cyp27a1 , 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 WT mice , # P < 0 . 05 versus heterozygous mutant mice . data were analyzed by independent - samples Student ' s t - Mann - Whitney U .

O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of relative of Bsep , Mrp2 , Mdr2 , Mrp3 , Mrp4 , Ostα / β , Ntcp , Cyp7a1 Cyp8b1 expression in mouse livers . Contrary to mRNA alterations , of canalicular Bsep Mrp2 were dramatically reduced in homozygous mouse livers , suggesting that decreased these were might be mediated by post - translational regulation in 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 WT mice , # P < 0 . 05 versus heterozygous mutant mice . data were analyzed by independent - samples Student ' s t - Mann - Whitney U .

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

(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 Mrp2 expression in livers ( E ) hepatocytes in sandwich cultures ( F ) from WT homozygous mice . Scale bar , 50 µm .

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(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 SEMA7A _ WT or SEMA7A _ R148W construct decreased of canalicular Bsep Mrp2 expression in mouse 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 , Sema7aR145W mutation can reduce hepatic Bsep Mrp2 expression lead to intrahepatic BA accumulation cholestasis ( Appendix Table S16 ) . Subsequently , cholestasis triggered an adaptive response in liver by down - regulating expression of BA synthetic enzymes of Cyp7a1 Cyp8b1 up - regulating expression of BA efflux transporters Mrp3 , Mrp4 Ostα / β .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 ) viability ( E ) of H358 A549 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 P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons .

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

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

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

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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 transfected with ATG5 - specific or control siRNAs for 48h before treated for 24h with vehicle ( DMSO ) or combined AZD 4547 ( 5 µM ) / BI2536 ( 5 nM ) .

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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 ) BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * * P < 0 . 001 P ＞ 0 . 05 ( ns ) by one - way ANOVA with Tukey ' s multiple comparisons . Data are mean of volume of each group ( 5 mice / group ) ; error bar : SD

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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 ) BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * * P < 0 . 001 P ＞ 0 . 05 ( ns ) by one - way ANOVA with Tukey ' s multiple comparisons . Data are mean of volume of each group ( 5 mice / group ) ; error bar :

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

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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 ) 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 . Data are mean of volume of each group ( 4 mice / group ) ; error bar : SD Relative volume ( G , J ) weights ( H , K ) of PDX tumors after treatment . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 P ＞ 0 . 05 ( ns ) by unpaired two - t - . Data are mean ± SD ( error bar ) of weights of each group ( 4 mice / group ) .

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

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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 Pank2 .

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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 after infection differentiated NPC were positive for neuronal markers βIII - tubulin ( Tuj1 ) , Map2 , NeuN humannuclei ( hNu ) synaptic markers , voltage - gated Na + channels ( PanNav ) vesicular glutamate transporter 1 ( VGlut1 ) .

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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 homogenates from humanneurons probed with PANK2 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 total dendritic branching points . Data presented as mean + from at least three independent experiments . A total of 38 neurons were counted for each sample . Statistics were determined by t - resulted not significant .

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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 , E18 cortical miceneurons . Control PKAN NPCs were infected with GFP - LV tdT - LV expressing vectors , respectively , differentiated for 8 . Scale bars 20 μm .

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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 ( top ) ; PKAN ( bottom ) . Traces on left represent trains of action potentials induced by injection of a suprathreshold current step through patch electrode in current - clamp mode . Middle traces show Na + K + currents ( down - 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 right display enlarged portions of traces to magnify fast Na + currents . G Histogram with percentages of recorded showing repetitively firing ( z - ) .

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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 of humanneurons stained with mitochondrial potential sensible probe TMRM , neuronal specific anti - NCAM antibody Hoechst . Left panel , basal conditions . Right panel , after addition of mitochondrial uncoupler FCCP . Scale bar 20 μm . Plot showing of TMRM signal from NCAM + neurons . Data presented as means + of three independent experiments ( unpaired , two - t - ) .

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

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

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

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

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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 of in - gel enzymatic activity of mitochondrial aconitase ( mAco cAco , respectively ) . lower part of gel was cut , stained with Coomassie blue , a band was used as loading control ( Loading ) . Lower panel : of mAco cAco enzymatic activity by densitometry .

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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 of mitochondrial aconitases . Lower panel : of mAco cAco by densitometry .

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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 soluble lysates .

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


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

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

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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 ) relatively large Na + K + currents ( right ) , were restored by CoA . histogram on right shows fractions of repetitively firing recorded in untreated vs . CoA - treated neurons from control PKAN ( z - ) .

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

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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 without CoA on controls PKAN . Basal uncoupled ( FCCP ) respiration increased after CoA supplementation . Data presented as means + 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 by absorbance at 400 nm of soluble NPC lysates . data are presented as means + 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 of changes in intracellular calcium , as measured in ( F340 / 380 ) ( A ) Examples show a TRPM8 - expressing DRG neuron ( left in A responding to both TRPM8 agonist menthol ( 300 μM ) 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 TRPM8 agonist menthol ( 300 μM ) Calcium increase is due to influx from extracellular

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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 TRPA1 agonist AITC ( left in E 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 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 ) , 4 ° C ( blue ) 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).


( C ) Mean cold - sensitive increase in calcium - dependent F340 / 380 in SCG neurons is significantly smaller in neurons recorded in voltage - clamp at - 60mV ( 0 . 56 ± 0 . 08 , mean ± , n = 21 neurons ) than in neurons recorded in current - clamp mode ( 1 . 16 ± 0 . 29 , n = 11 neurons , p = 0 . 0165 , unpaired t - ) .

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(C) Mean cold-sensitive increase in calcium-dependent fluorescence ratio F340/380 in SCG neurons is significantly smaller in neurons recorded in voltage-clamp at -60mV (0.56 ±0.08, mean ± SEM, n=21 neurons) than in neurons recorded in current-clamp mode (1.16 ±0.29, n=11 neurons, p=0.0165, unpaired t-test).


( F ) Summary of results of experiments on SCG neurons ( mean ± , n = 115 neurons , 4 separate cultures ) . YM58483 caused a significant decrease in cold - response amplitude ( p < 0 . 0001 , RM one - way ANOVA + Dunnett ' s ) , with only partial recovery . trace below . ( G ) In neurons with a small response to cold ( F340 / 380 increase < 0 . 2 ; n = 24 neurons ) , many responses are potentiated by verapamil ( 31 % of cold - insensitive neurons ) are fully blocked by ORAI channel antagonist YM58483 ( p < 0 . 0001 , RM one - way ANOVA + Dunnett ' s ) .

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(F) Summary of results of experiments on SCG neurons (mean ±SEM, n=115 neurons, 4 separate cultures). YM58483 caused a significant decrease in cold-response amplitude (p<0.0001, RM one-way ANOVA + Dunnett's test), with only partial recovery. Temperature trace below. (G) In neurons with a small response to cold (F340/380 increase <0.2; n=24 neurons), many responses are potentiated by verapamil (31% of cold-insensitive neurons) and are fully blocked by ORAI channel antagonist YM58483 (p<0.0001, RM one-way ANOVA + Dunnett's test).


( D ) Representative of SCG neurons from neonatal mice ( P5 - 7 ) electroporated with plasmids coding for mCherry for siRNA targeting STIM1 . Neurons were cooled from 35 ° C to 7 ° C in presence of 100μM verapamil . Electroporation was indicated by expression of mCherry ( # in left - panel ) ; in these neurons a cold response was absent ( # in right - panel ) while in neurons not expressing mCherry ( * ) cold responses were still observed . Scale bar 20μm .

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(D) Representative fluorescence images of SCG neurons from neonatal mice (age P5-7) electroporated with plasmids coding for mCherry and for siRNA targeting STIM1. Neurons were cooled from 35°C to 7°C in the presence of 100μM verapamil. Electroporation was indicated by expression of mCherry (# in left-hand panel); in these neurons a cold response was absent (# in right-hand panel) while in neurons not expressing mCherry (*) cold responses were still observed. Scale bar 20μm.


( F ) Cold response amplitudes ( F340 / 380 , mean ± ) . Successfully electroporated neurons ( mCherry positive , red bar , n = 111 neurons from four separate cultures ) had a significantly lower cold response amplitude than neurons not electroporated ( mCherry negative , black bar , n = 104 neurons from same cultures , p = 0 . 0082 , unpaired t - ) . ( G ) of neurons that responded to cold ramp with F340 / 380 increase > 0 . 2 . Only 5 % of mCherry - positive neurons ( successfully electroporated , red bar ) responded to cold , compared to 19 % of mCherry - negative neurons in same cultures ( non - electroporated , black bar , p < 0 . 001 , chi - square ) .

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(F) Cold response amplitudes (F340/380, mean ±SEM). Successfully electroporated neurons (mCherry positive, red bar, n=111 neurons from four separate cultures) had a significantly lower cold response amplitude than neurons not electroporated (mCherry negative, black bar, n=104 neurons from same cultures, p=0.0082, unpaired t-test). (G) Percentage of neurons that responded to cold ramp with F340/380 increase >0.2. Only 5% of mCherry-positive neurons (successfully electroporated, red bar) responded to cold, compared to 19% of mCherry-negative neurons in same cultures (non-electroporated, black bar, p<0.001, chi-square test).


( A ) Representative TIRF microscopy ( imaging depth 100nm ) showing that cooling from 35 ° C to 13 ° C increases ORAI1 formation colocalization of ORAI1 with STIM1 in HEK293 transfected with ORAI1 - CFP STIM1 - YFP . Cooling to 13 ° C causes appearance of new ORAI1 ( examples shown by * ) that are not present in 35 ° C image are colocalised with STIM1 . Cooling also causes STIM1 to move towards surface in edge - on views ( arrows ) . To reduce motility , experiment was conducted in nominal 0 [ Ca ] e , which potentially caused partial formation of STIM1 before shown . Scale bar = 5μm . trace shown below . Mean adjusted to compensate for increase in intrinsic of CFP YFP caused by cooling ( see Fig . S12 ) .

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(A) Representative TIRF microscopy images (imaging depth 100nm) showing that cooling from 35°C to 13°C increases ORAI1 puncta formation and colocalization of ORAI1 with STIM1 in HEK293 cells transfected with ORAI1-CFP and STIM1-YFP. Cooling to 13°C causes the appearance of new ORAI1 puncta (examples shown by *) that are not present in the 35°C image and are colocalised with STIM1. Cooling also causes STIM1 to move towards the cell surface in edge-on views (arrows). To reduce cell motility, the experiment was conducted in nominal 0[Ca]e, which potentially caused partial formation of STIM1 puncta before the images shown. Scale bar = 5μm. Temperature trace shown below. Mean fluorescence intensity adjusted to compensate for increase in intrinsic fluorescence of CFP and YFP caused by cooling (see Fig. S12).


( A ) Representative immunofluorescence showing adult mouse stellate ganglion ( SG ) sections stained for nNOS ( red ) , to label nitrergic neurons , βIII Tubulin ( green ) to label neurons , DAPI to label nuclei . nNOS is widely expressed in neurons of stellate ganglion , but is absent from glial ( glial nuclei identifiable in merged image , similar results in n = 5 ) . Antibody against nNOS from Abcam ; similar results obtained with antibodies from ECM Millipore . three antibodies show nNOS widely expressed in SG bodies neurites .

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(A) Representative immunofluorescence images showing adult mouse stellate ganglion (SG) sections stained for nNOS (red), to label nitrergic neurons, βIII Tubulin (green) to label all neurons, and DAPI to label nuclei. nNOS is widely expressed in neurons of the stellate ganglion, but is absent from glial cells (glial nuclei identifiable in merged image, similar results in n=5 animals). Antibody against nNOS from Abcam; similar results obtained with antibodies from ECM and Millipore. All three antibodies show nNOS widely expressed in SG cell bodies and neurites.


( B ) Adult mouse SCG ( n = 2 , upper ) SG ( n = 5 , lower ) sections stained for nNOS ( red ) , tyrosine hydroxylase ( TH , green ) DAPI ( blue ) to label nitrergic neurons , noradrenergic neurons nuclei respectively . Many noradrenergic also express nNOS .

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(B) Adult mouse SCG (n=2 animals, upper) and SG (n=5 animals, lower) sections stained for nNOS (red), tyrosine hydroxylase (TH, green) and DAPI (blue) to label nitrergic neurons, noradrenergic neurons and nuclei respectively. Many noradrenergic cells also express nNOS.


( B ) of unique histone iCLIP - seq read population to unique histone - seq read population was calculated shown , with unique histone - seq read population set as " 1 " . Error bars represent standard deviations from three replicates of ALYREF iCLIP data . Statistical was performed using Student ' s t - . * * * P < 0 . 001 .

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(B) The ratio of unique histone iCLIP-seq read population to unique histone RNA-seq read population was calculated and shown, with the unique histone RNA-seq read population set as "1". Error bars represent standard deviations from three replicates of ALYREF iCLIP data. Statistical analysis was performed using Student's t-test. ***P< 0.001.


( C ) ALYREF RIP - qPCRs to examine ALYREF binding on histone mRNAs . Relative RIP efficiencies are shown . A tRNA was used as a negative control for ALYREF binding . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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(C) ALYREF RIP-qPCRs to examine ALYREF binding on histone mRNAs. Relative RIP efficiencies are shown. A tRNA was used as a negative control for ALYREF binding. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01, ***P< 0.001, n.s., not significant.


( D ) distribution of histone reads in 5 ' UTR , CDS 3 ' UTR in rRNA - depleted - seq ALYREF - iCLIP - seq .

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(D) The distribution of histone reads in 5' UTR, CDS and 3' UTR in rRNA-depleted nuclear RNA-seq and ALYREF-iCLIP-seq.


( E ) Metagene of normalized ALYREF signals on histone mRNA on iCLIP - seq data . iCLIP - seq signal at each position of an mRNA is divided by sum of signal of mRNA to normalize each mRNA ' s contribution to plot .

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(E) Metagene analysis of normalized ALYREF signals on histone mRNA based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot.


( F ) Screenshots of two histone mRNAs showing clustered ALYREF binding sites . 3 ' UTR regions are highlighted .

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(F) Screenshots of two histone mRNAs showing clustered ALYREF binding sites. The 3' UTR regions are highlighted.


( G ) Distribution profile of clustered ALYREF binding sites on histone mRNA 3 ' UTRs relative to cleavage site ( CS ) . CS is set as " 0 " in x axis , relative position to CS is marked . y axis shows total occurrence of binding sites in three repeats at each nucleotide .

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(G) Distribution profile of clustered ALYREF binding sites on histone mRNA 3' UTRs relative to the cleavage site (CS). The CS is set as "0" in the x axis, and the relative position to the CS is marked. The y axis shows the total occurrence of binding sites in three repeats at each nucleotide.


IPs from RNase A - treated S - phase HeLa lysate using SLBP antibody or IgG ( A ) Western blotting was performed with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . delineates boundary where irrelevant lanes have been removed from same

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IPs from RNase A-treated S-phase HeLa cell lysate using the SLBP antibody or IgG (A) Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots


IPs from RNase A - treated S - phase HeLa lysate using ALYREF antibody or IgG ( B ) . Western blotting was performed with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . delineates boundary where irrelevant lanes have been removed from same

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IPs from RNase A-treated S-phase HeLa cell lysate using the ALYREF antibody or IgG (B). Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots


( C ) Pull - downs of in vitro translated 35S labelled SLBP luciferase ( Cntl ) using MBP or MBP - ALYREF in presence of RNase A . pulled down were visualized by coomassie ( left ) or phosphoimager ( right ) . 3 % of input was loaded .

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(C) Pull-downs of in vitro translated 35S labelled SLBP and luciferase (Cntl) using MBP or MBP-ALYREF in the presence of RNase A. The proteins pulled down were visualized by coomassie staining (left) or phosphoimager (right). 3% of input was loaded.


( D ) GST - SLBP , GST - UAP56 GST were used for pull - down of purified MBP - ALYREF or MBP in presence of RNase A . pulled down were separated by SDS - PAGE , followed by coomassie . 37 . 5 % of input were loaded .

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(D) GST-SLBP, GST-UAP56 and GST were used for pull-down of purified MBP-ALYREF or MBP in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE, followed by coomassie staining. 37.5% of input proteins were loaded.


( E ) ( Top ) Domain schematic representation of SLBP . ( Bottom ) Flag IPs from RNase A - treated HeLa lysate individually expressing indicated Flag tagged , followed by western blotting using Flag ALYREF antibodies . 3 % of input was loaded . * indicates a band probably resulted from degradation of Flag - SLBP . delineates boundary where irrelevant lanes have been removed from same

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(E) (Top) Domain schematic representation of SLBP. (Bottom) Flag IPs from RNase A-treated HeLa cell lysate individually expressing indicated Flag tagged proteins, followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


( E ) ( Top ) Domain schematic representation of SLBP . followed by western blotting using Flag ALYREF antibodies . 3 % of input was loaded . * indicates a band probably resulted from degradation of Flag - SLBP . delineates boundary where irrelevant lanes have been removed from same blot except that Flag IPs were carried out from HA - SLBP stable expression transfected with plasmids expressing ALYREF fragments .

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(E) (Top) Domain schematic representation of SLBP. followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot except that Flag IPs were carried out from HA-SLBP stable expression cells transfected with plasmids expressing ALYREF fragments.


( G ) Western blotting to examine KD efficiency of SLBP . GAPDH was used as a loading control . delineates boundary where irrelevant lanes have been removed from same

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(G) Western blotting to examine the KD efficiency of SLBP. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


Cntl - or SLBP - siRNA treated HeLa were used for IPs with IgG or ALYREF antibody . immunoprecipitates were subjected to western ( H ) Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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Cntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to western analysis (H) Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant.


Cntl - or SLBP - siRNA treated HeLa were used for IPs with IgG or ALYREF antibody . immunoprecipitates were subjected to RT - qPCRs ( I ) . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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Cntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to RT-qPCRs (I). Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant.


( J ) Metagene of normalized ALYREF signals on histone mRNAs in Cntl SLBP KD on iCLIP - seq data . iCLIP - seq signal at each position of an mRNA is divided by sum of signal of mRNA to normalize each mRNA ' s contribution to plot .

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(J) Metagene analysis of normalized ALYREF signals on histone mRNAs in Cntl and SLBP KD cells based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot.


( B ) List of histone genes with defective 3 ' - end processing detected by polyA + polyA - - seq . PolyA + ALYREF / Cntl shows polyA + - seq RPM of each histone gene in ALYREF KD versus Cntl .

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(B) List of histone genes with defective 3'-end processing detected by polyA+ and polyA- RNA-seq. PolyA+ ALYREF/Cntl shows polyA+ RNA-seq RPM ratio of each histone gene in ALYREF KD versus Cntl.


( C ) Screenshots of two exemplified histone mRNAs with 3 ' - end processing defects are shown . Transcription direction is indicated . Black arrowheads corresponding lines mark position of CS .

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(C) Screenshots of two exemplified histone mRNAs with 3'-end processing defects are shown. Transcription direction is indicated. Black arrowheads and the corresponding lines mark the position of CS.


( D ) RPM of each histone mRNA in Cntl RPM of each histone mRNA in KDs to Cntl detected in 3 ' READS + .

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(D) RPM of each histone mRNA in Cntl cells and RPM ratio of each histone mRNA in KDs to Cntl detected in 3'READS+.


( E , F ) RT - qPCRs to detect histone mRNA in polyA + ( E ) or polyA - fraction ( F ) in treated with Cntl , ALYREF or ARS2 siRNA . arrowhead arrows indicate cleavage site primer location , respectively . bars show relative abundance of polyA + forms to actin mRNA ( E ) that of polyA - forms relative to 18S rRNA ( F ) . Data information Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(E, F) RT-qPCRs to detect histone mRNA levels in the polyA+ (E) or the polyA- fraction (F) in cells treated with Cntl, ALYREF or ARS2 siRNA. The arrowhead and arrows indicate the cleavage site and primer location, respectively. The bars show the relative abundance of polyA+ forms to the actin mRNA (E) and that of polyA- forms relative to the 18S rRNA (F). Data information Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


Western blotting to examine ALYREF level ( G ) in Flag - Cntl ( eIF4A3 ) or siRNA - resistant Flag - ALYREF stable expression treated with Cntl or ALYREF siRNA . delineates boundary where irrelevant lanes have been removed from same

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Western blotting to examine ALYREF protein level (G) in Flag-Cntl (eIF4A3) or siRNA-resistant Flag-ALYREF stable expression cells treated with Cntl or ALYREF siRNA. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


RT - qPCRs to detect polyA + histone mRNA ( H ) in Flag - Cntl ( eIF4A3 ) or siRNA - resistant Flag - ALYREF stable expression treated with Cntl or ALYREF siRNA . bars show relative abundance of histone mRNAs to GAPDH mRNA . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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RT-qPCRs to detect polyA+ histone mRNA levels (H) in Flag-Cntl (eIF4A3) or siRNA-resistant Flag-ALYREF stable expression cells treated with Cntl or ALYREF siRNA. The bars show the relative abundance of histone mRNAs to the GAPDH mRNA. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


IPs were carried out from RNase A - treated S - phase HeLa lysate using Lsm11 antibody IgG ( A ) followed by western blotting with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . delineates boundary where irrelevant lanes have been removed from same

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IPs were carried out from RNase A-treated S-phase HeLa cell lysate using the Lsm11 antibody and IgG (A) followed by western blotting with indicated antibodies. 2 % of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


IPs were carried out from RNase A - treated S - phase HeLa lysate using ALYREF antibody IgG ( B ) , followed by western blotting with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . delineates boundary where irrelevant lanes have been removed from same

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IPs were carried out from RNase A-treated S-phase HeLa cell lysate using the ALYREF antibody and IgG (B), followed by western blotting with indicated antibodies. 2 % of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


( C ) Pull - down of purified His - Cntl ( PGK1 ) or His - Lsm11 using equal amount of GST GST - ALYREF in presence of RNase A . pulled down were separated by SDS - PAGE followed by coomassie . * indicates a nonspecific band .

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(C) Pull-down of purified His-Cntl (PGK1) or His-Lsm11 using equal amount of GST and GST-ALYREF proteins in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE followed by coomassie staining. * indicates a nonspecific band.


Cntl or ALYREF siRNA treated Flag - Lsm11 stable expression were used for IPs with IgG or Flag antibody . immunoprecipitates were subjected western ( D ) . * indicates antibody heavy chain .

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

Cntl or ALYREF siRNA treated Flag-Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody. The immunoprecipitates were subjected western analysis (D). * indicates the antibody heavy chain.