LINC00941 knockdown in day 3 ( D3 ) organotypic epidermal tissue cultures ( D ) ( n = 3 - 4 tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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LINC00941 knockdown in day 3 (D3) organotypic epidermal tissue cultures (D) (n = 3-4 tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


LINC00941 knockdown in day 3 ( D3 ) organotypic epidermal tissue cultures results in increased expression of key differentiation genes ( E ) ( n = 3 - 4 tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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LINC00941 knockdown in day 3 (D3) organotypic epidermal tissue cultures results in increased expression of key differentiation genes (E) (n = 3-4 tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


F Immunofluorescence analysis shows increased levels of early and late differentiation proteins . Collagen VII ( col VII , green ) stain indicates the basement membrane , nuclei are shown in blue and the differentiation proteins Keratin 10 and Loricrin are shown in red ( n = 3 - 4 tissue cultures / knockdown group ; one exemplary picture for each group is depicted ) . Scale bar : 50 µm .

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F Immunofluorescence analysis shows increased levels of early and late differentiation proteins. Collagen VII (col VII, green) stain indicates the basement membrane, nuclei are shown in blue and the differentiation proteins Keratin 10 and Loricrin are shown in red (n = 3-4 tissue cultures/knockdown group; one exemplary picture for each group is depicted). Scale bar: 50 µm.


B LINC00941 knockdown efficiency ( siLINC00941 ) in epidermal tissue on day 3 of differentiation as obtained by qRT - PCR measurements ( n = 4 - 5 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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B LINC00941 knockdown efficiency (siLINC00941) in epidermal tissue on day 3 of differentiation as obtained by qRT-PCR measurements (n = 4-5 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


C Heatmap of differentially expressed genes ( padj < 0 . 05 and - 1 > log2FC > 1 ) upon LINC00941 depletion on day 3 in organotypic epidermal tissue with marked keratinocyte differentiation genes ( n = 4 - 5 epidermal tissue cultures / knockdown group ) .

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

C Heatmap of differentially expressed genes (padj < 0.05 and -1 > log2FC > 1) upon LINC00941 depletion on day 3 in organotypic epidermal tissue with marked keratinocyte differentiation genes (n = 4-5 epidermal tissue cultures/knockdown group).


A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex ( EDC ) ( n = 4 - 5 biological replicates / knockdown group ) .

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

A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex (EDC) (n = 4-5 biological replicates/knockdown group).


B LINC00941 knockdown on day 3 in calcium - induced keratinocyte differentiation cultures results in increased expression of SPRR5 ( FC = fold change ) ( n = 3 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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B LINC00941 knockdown on day 3 in calcium-induced keratinocyte differentiation cultures results in increased expression of SPRR5 (FC = fold change) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


C Detected fragments per million ( FPM ) for SPRR5 in control ( siCtrl ) as well as LINC00941 deficient ( siLINC00941 ) epidermal tissue for all biological replicates on day 3 ( D3 ) of differentiation as obtained by DeSeq2 ( FC = fold change ) ( n = 4 ‑ 5 epidermal tissue cultures / knockdown group ) .

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C Detected fragments per million (FPM) for SPRR5 in control (siCtrl) as well as LINC00941 deficient (siLINC00941) epidermal tissue for all biological replicates on day 3 (D3) of differentiation as obtained by DeSeq2 (FC = fold change) (n = 4‑5 epidermal tissue cultures/knockdown group).


SPRR5 induction during the course of keratinocyte ( KC ) differentiation is shown by northern blot ( D ) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes ( n = 8 biological replicates ) . Data is presented as mean ± SD .

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SPRR5 induction during the course of keratinocyte (KC) differentiation is shown by northern blot (D) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes (n = 8 biological replicates). Data is presented as mean ± SD.


SPRR5 induction during the course of keratinocyte ( KC ) differentiation is shown by qRT - PCR analysis ( E ) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes ( n = 8 biological replicates ) . Data is presented as mean ± SD .

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SPRR5 induction during the course of keratinocyte (KC) differentiation is shown by qRT-PCR analysis (E) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes (n = 8 biological replicates). Data is presented as mean ± SD.


F p63 knockdown on day 3 of keratinocyte differentiation leads to decreased SPRR5 transcript levels ( FC = fold change ) ( n = 3 - 4 biological replicates / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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F p63 knockdown on day 3 of keratinocyte differentiation leads to decreased SPRR5 transcript levels (FC = fold change) (n = 3-4 biological replicates /knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool - mediated SPRR5 knockdown on day 4 to 6 ( D4 - D6 ) of calcium - induced keratinocyte differentiation ( A ) ( n = 3 - 5 cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation (A) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool - mediated SPRR5 knockdown on day 4 to 6 ( D4 - D6 ) of calcium - induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown by qRT - PCR ( B ) ( FC = fold change ) ( n = 3 - 5 cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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

siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown by qRT-PCR (B) (FC = fold change) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


siPool - mediated SPRR5 knockdown on day 4 to 6 ( D4 - D6 ) of calcium - induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown western blot analysis ( C ) ( n = 3 - 5 cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown western blot analysis (C) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


D SPRR5 knockdown efficiency in day 3 ( D3 ) organotypic epidermal tissue ( n = 4 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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D SPRR5 knockdown efficiency in day 3 (D3) organotypic epidermal tissue (n = 4 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


E SPRR5 depletion in regenerated organotypic epidermal tissue results in drastically reduced expression of differentiation proteins as shown by immunofluorescence analysis . Collagen VII ( Col VII , green ) stain indicates the basement membrane , nuclei are shown in blue and the differentiation proteins filaggrin and loricrin are shown in red ( n = 4 tissue cultures / knockdown group ; one exemplary picture for each group is depicted ) . Scale bar : 50 µm .

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E SPRR5 depletion in regenerated organotypic epidermal tissue results in drastically reduced expression of differentiation proteins as shown by immunofluorescence analysis. Collagen VII (Col VII, green) stain indicates the basement membrane, nuclei are shown in blue and the differentiation proteins filaggrin and loricrin are shown in red (n = 4 tissue cultures/knockdown group; one exemplary picture for each group is depicted). Scale bar: 50 µm.


F SPRR5 knockdown in regenerated epidermal tissue results in reduced transcript levels of key differentiation genes as obtained by qRT - PCR ( n = 4 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

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F SPRR5 knockdown in regenerated epidermal tissue results in reduced transcript levels of key differentiation genes as obtained by qRT-PCR (n = 4 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


B Overview of SPRR5 levels in control ( siCtrl ) and SPRR5 deficient ( siSPRR5 ) epidermal tissue on day 3 and day 4 as obtained by qRT - PCR analysis ( n = 3 - 5 epidermal tissue cultures / knockdown group ) . Data is presented as mean ± SD . Statistical significance was tested by an unpaired t - test and corrected for multiple testing after Bonferroni ( * = adj . p - value < 0 . 05 , * * = adj . p - value < 0 . 01 , * * * = adj . p ‑ value < 0 . 001 , and n . s . = not significant ) .

O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Overview of SPRR5 levels in control (siCtrl) and SPRR5 deficient (siSPRR5) epidermal tissue on day 3 and day 4 as obtained by qRT-PCR analysis (n = 3-5 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).


Heatmaps of differentially expressed genes ( padj < 0 . 05 and - 0 . 5 > log2FC > 0 . 5 ) upon SPRR5 depletion on day 3 ( C ) in organotypic epidermal tissue ( n = 3 - 5 epidermal tissue cultures / knockdown group ) .

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Heatmaps of differentially expressed genes (padj < 0.05 and -0.5 > log2FC > 0.5) upon SPRR5 depletion on day 3 (C) in organotypic epidermal tissue (n = 3-5 epidermal tissue cultures/knockdown group).


Heatmaps of differentially expressed genes ( padj < 0 . 05 and - 0 . 5 > log2FC > 0 . 5 ) upon SPRR5 depletion on day 4 ( D ) in organotypic epidermal tissue ( n = 3 - 5 epidermal tissue cultures / knockdown group ) .

Heatmaps of differentially expressed genes (padj < 0.05 and -0.5 > log2FC > 0.5) upon SPRR5 depletion on day 4 (D) in organotypic epidermal tissue (n = 3-5 epidermal tissue cultures/knockdown group).


B . Western blot analysis of hnRNP R in three independent HNRNPR wildtype ( + / + ) , heterozygous ( + / - ) and homozygous ( - / - ) HeLa cell lines .

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B. Western blot analysis of hnRNP R in three independent HNRNPR wildtype (+/+), heterozygous (+/-) and homozygous (-/-) HeLa cell lines.


C . Confocal microscopy images of nuclei stained with DAPI . Scale bar : 10 µm . D . Quantification of the DAPI area in ( C ) . Data are mean with standard deviation ( SD ) ; * * * P ≤ 0 . 001 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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C. Confocal microscopy images of nuclei stained with DAPI. Scale bar: 10 µm. D. Quantification of the DAPI area in (C). Data are mean with standard deviation (SD); ***P ≤ 0.001; unpaired two-tailed t-test (n = 3 biological replicates).


E . RNA FISH with a Cy3 - labeled oligo - dT probe . DAPI was used for nuclear staining . Scale bar : 10 µm . F . Quantification of mean fluorescence intensity of the Cy3 oligo - dT signal in ( E ) . Data are mean with SD ; * * * * P ≤ 0 . 0001 ; Mann - Whitney test ( n = 129 cells for HNRNPR + / + and n = 176 cells for HNRNPR - / - ) .

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E. RNA FISH with a Cy3-labeled oligo-dT probe. DAPI was used for nuclear staining. Scale bar: 10 µm. F. Quantification of mean fluorescence intensity of the Cy3 oligo-dT signal in (E). Data are mean with SD; ****P ≤ 0.0001; Mann-Whitney test (n = 129 cells for HNRNPR+/+ and n = 176 cells for HNRNPR-/-).


A . Western blot analysis of Ser2 - phosphorylated and total RNA pol II , Cyclin T1 , CDK9 and GAPDH protein levels .

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A. Western blot analysis of Ser2-phosphorylated and total RNA pol II, Cyclin T1, CDK9 and GAPDH protein levels.


B . Quantification of relative expression of Ser2 - phosphorylated and total RNA pol II , Cyclin T1 and CDK9 in ( A ) . Data are mean with SD ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; unpaired two - tailed t - tests ( n = 3 biological replicates ) .

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B. Quantification of relative expression of Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9 in (A). Data are mean with SD; **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates).


F . Western blot analysis of subcellular fractions for Calnexin , GAPDH , histone H3 , Ser2 - phosphorylated and total RNA pol II , Cyclin T1 and CDK9 . Cyt , cytosol ; nuc + org , nuclear soluble proteins and organelles ; chr , chromatin - associated proteins . G . Quantification of relative expression of Ser2 - phosphorylated and total RNA pol II , Cyclin T1 and CDK9 in subcellular fractions in ( F ) . Data are mean with SD ; * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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

F. Western blot analysis of subcellular fractions for Calnexin, GAPDH, histone H3, Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9. Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins. G. Quantification of relative expression of Ser2-phosphorylated and total RNA pol II, Cyclin T1 and CDK9 in subcellular fractions in (F). Data are mean with SD; *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


A . Western blot analysis of the indicated proteins in the input ( In ) lysates used for co - immunoprecipitation . B . Western blot analysis of Cyclin T1 , HEXIM1 , CDK9 and hnRNP A1 co - immunoprecipitated by an anti - LARP7 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . C . Western blot analysis of Cyclin T1 , HEXIM1 , CDK9 , hnRNP A1 and LARP7 co - immunoprecipitated by an anti - MePCE antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . D . Quantification of CDK9 , Cyclin T1 and HEXIM1 co - immunoprecipitating with LARP7 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 , n . s . not significant ; unpaired two - tailed t - tests ( n = 3 biological replicates ) . E . Quantification of CDK9 , Cyclin T1 and HEXIM1 co - immunoprecipitating with MePCE in ( C ) . Data are mean with SD ; * P ≤ 0 . 05 , n . s . not significant ; unpaired two - tailed t - tests ( n = 3 biological replicates ) . F . Quantification of hnRNP A1 co - immunoprecipitating with LARP7 and MePCE in ( B ) and ( C ) . Data are mean with SD ; * P ≤ 0 . 05 ; unpaired two - tailed t - tests ( n = 3 biological replicates ) .

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

A. Western blot analysis of the indicated proteins in the input (In) lysates used for co-immunoprecipitation. B. Western blot analysis of Cyclin T1, HEXIM1, CDK9 and hnRNP A1 co-immunoprecipitated by an anti-LARP7 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. C. Western blot analysis of Cyclin T1, HEXIM1, CDK9, hnRNP A1 and LARP7 co-immunoprecipitated by an anti-MePCE antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. D. Quantification of CDK9, Cyclin T1 and HEXIM1 co-immunoprecipitating with LARP7 in (B). Data are mean with SD; **P ≤ 0.01, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates). E. Quantification of CDK9, Cyclin T1 and HEXIM1 co-immunoprecipitating with MePCE in (C). Data are mean with SD; *P ≤ 0.05, n.s. not significant; unpaired two-tailed t-tests (n = 3 biological replicates). F. Quantification of hnRNP A1 co-immunoprecipitating with LARP7 and MePCE in (B) and (C). Data are mean with SD; *P ≤ 0.05; unpaired two-tailed t-tests (n = 3 biological replicates).


G . Western blot analysis of subcellular fractions Calnexin , GAPDH , histone H3 , hnRNP R and hnRNP A1 . Cyt , cytosol ; nuc + org , nuclear soluble proteins and organelles ; chr , chromatin - associated proteins .

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G. Western blot analysis of subcellular fractions Calnexin, GAPDH, histone H3, hnRNP R and hnRNP A1. Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins.


H . Western blot analysis of hnRNP R co - immunoprecipitated by an anti - hnRNP A1 antibody from subcellular fractions . Immunoprecipitation with mouse - IgG antibody was used as control .

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H. Western blot analysis of hnRNP R co-immunoprecipitated by an anti-hnRNP A1 antibody from subcellular fractions. Immunoprecipitation with mouse-IgG antibody was used as control.


I . Quantification of 7SK RNA in subcellular fractions by qPCR . Data are mean with SD ; * * P ≤ 0 . 01 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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

I. Quantification of 7SK RNA in subcellular fractions by qPCR. Data are mean with SD; **P ≤ 0.01, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


J . Quantification of 7SK RNA co - immunoprecipitating with hnRNP A1 from subcellular fractions . Data are mean with SD ; * P ≤ 0 . 05 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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J. Quantification of 7SK RNA co-immunoprecipitating with hnRNP A1 from subcellular fractions. Data are mean with SD; *P ≤ 0.05, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


A . Western blot analysis of BRD4 , hnRNP R , Cyclin T1 , CDK9 , α - Tubulin and histone H3 in the input lysates used for co - immunoprecipitation .

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

A. Western blot analysis of BRD4, hnRNP R, Cyclin T1, CDK9, α-Tubulin and histone H3 in the input lysates used for co-immunoprecipitation.


B . Western blot analysis of hnRNP R , Cyclin T1 , CDK9 and histone H3 co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control .

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B. Western blot analysis of hnRNP R, Cyclin T1, CDK9 and histone H3 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.


C . Quantification of CDK9 co - immunoprecipitating with BRD4 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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C. Quantification of CDK9 co-immunoprecipitating with BRD4 in (B). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


D . Western blot analysis of BRD4 ( long and short exposure ) co - immunoprecipitated by an anti - CDK9 antibody . Immunoprecipitation with mouse - IgG antibody was used as control .

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D. Western blot analysis of BRD4 (long and short exposure) co-immunoprecipitated by an anti-CDK9 antibody. Immunoprecipitation with mouse-IgG antibody was used as control.


E . Quantification of BRD4 co - immunoprecipitating with CDK9 in ( D ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

E. Quantification of BRD4 co-immunoprecipitating with CDK9 in (D). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


F . Quantification of histone H3 co - immunoprecipitating with BRD4 in ( B ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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F. Quantification of histone H3 co-immunoprecipitating with BRD4 in (B). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


G . Western blot analysis of BRD4 , hnRNP R , Cyclin T1 , CDK9 , α - Tubulin and histone H3 in the input lysates used for co - immunoprecipitation . Lysates were pre - treated with RNase as indicated .

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

G. Western blot analysis of BRD4, hnRNP R, Cyclin T1, CDK9, α-Tubulin and histone H3 in the input lysates used for co-immunoprecipitation. Lysates were pre-treated with RNase as indicated.


H . Western blot analysis of hnRNP R , Cyclin T1 , CDK9 and histone H3 co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control .

H. Western blot analysis of hnRNP R, Cyclin T1, CDK9 and histone H3 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.


I . Western blot analysis of BRD4 , hnRNP R , CDK9 and α - Tubulin in the input lysates used for co - immunoprecipitation .

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

I. Western blot analysis of BRD4, hnRNP R, CDK9 and α-Tubulin in the input lysates used for co-immunoprecipitation.


J . Western blot analysis of hnRNP R and CDK9 co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . K . Quantification of CDK9 co - immunoprecipitating with BRD4 in ( J ) . Data are mean with SD ; * * * * P ≤ 0 . 0001 ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

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

J. Western blot analysis of hnRNP R and CDK9 co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. K. Quantification of CDK9 co-immunoprecipitating with BRD4 in (J). Data are mean with SD; ****P ≤ 0.0001; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


L . Western blot analysis of phospho - CDK9 ( Thr186 ) co - immunoprecipitated by an anti - BRD4 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control . M . Quantification of phospho - CDK9 ( Thr186 ) co - immunoprecipitating with BRD4 in ( L ) . Data are mean with SD ; * * P ≤ 0 . 01 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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

L. Western blot analysis of phospho-CDK9(Thr186) co-immunoprecipitated by an anti-BRD4 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control. M. Quantification of phospho-CDK9(Thr186) co-immunoprecipitating with BRD4 in (L). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates).


A , B . Western blot analysis of Ser2 - phosphorylated and total RNA pol II levels in HNRNPR + / + ( A ) and - / - ( B ) cells treated with DMSO or CHX for the indicated durations . GAPDH and α - Tubulin served as loading control .

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

A, B. Western blot analysis of Ser2-phosphorylated and total RNA pol II levels in HNRNPR+/+ (A) and -/- (B) cells treated with DMSO or CHX for the indicated durations. GAPDH and α-Tubulin served as loading control.


C . Quantification of relative expression levels of Ser2 - phosphorylated and total RNA pol II in ( A , B ) . Data are mean with SD ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ; unpaired two - tailed t - tests ( n = 4 biological replicates ) .

O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-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. Quantification of relative expression levels of Ser2-phosphorylated and total RNA pol II in (A, B). Data are mean with SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; unpaired two-tailed t-tests (n = 4 biological replicates).


D , Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Calnexin , GAPDH and histone H3 ( D ) Cyt , cytosol ; nuc + org , nuclear soluble proteins and organelles ; chr , chromatin - associated proteins .

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

D, Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Calnexin, GAPDH and histone H3 (D) Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins.


E . Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Cyclin T1 and CDK9 ( E ) . Cyt , cytosol ; nuc + org , nuclear soluble proteins and organelles ; chr , chromatin - associated proteins . F . Quantification of relative expression of Cyclin T1 and CDK9 protein levels in subcellular fractions in ( E ) . Data are mean with SD ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; one - way ANOVA with Tukey ' s multiple comparisons test ( n = 3 biological replicates ) .

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

E. Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Cyclin T1 and CDK9 (E). Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins. F. Quantification of relative expression of Cyclin T1 and CDK9 protein levels in subcellular fractions in (E). Data are mean with SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; one-way ANOVA with Tukey's multiple comparisons test (n = 3 biological replicates).


G . Western blot analysis of Cyclin T1 levels in HNRNPR + / + and - / - cells treated with DMSO or MG132 . α - Tubulin served as loading control . H . Quantification of relative expression levels of Cyclin T1 in ( G ) . Data are mean with SD ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . not significant ; two - way ANOVA with Sidak ' s multiple comparisons test ( n = 3 biological replicates ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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 O O O O O O O O O O O O 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. Western blot analysis of Cyclin T1 levels in HNRNPR+/+ and -/- cells treated with DMSO or MG132. α-Tubulin served as loading control. H. Quantification of relative expression levels of Cyclin T1 in (G). Data are mean with SD; ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).


B . Western blot analysis of Cyclin K , CDK9 and α - Tubulin in the input lysates used for co - immunoprecipitation .

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B. Western blot analysis of Cyclin K, CDK9 and α-Tubulin in the input lysates used for co-immunoprecipitation.


C . Western blot analysis of Cyclin K co - immunoprecipitated by an anti - CDK9 antibody . Immunoprecipitation with rabbit - IgG antibody was used as control .

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

C. Western blot analysis of Cyclin K co-immunoprecipitated by an anti-CDK9 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.


D . Quantification of Cyclin K co - immunoprecipitating with CDK9 in ( C ) . Data are mean with SD ; * P ≤ 0 . 05 ; unpaired two - tailed t - test ( n = 3 biological replicates ) .

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

D. Quantification of Cyclin K co-immunoprecipitating with CDK9 in (C). Data are mean with SD; *P ≤ 0.05; unpaired two-tailed t-test (n = 3 biological replicates).


Immunoblot analysis of indicated cell lines , starved for 6 hours ( HBSS ) as indicated , and treated with 25 μM MG132 or 200 ng / ml of bafilomycin A1 ( Baf A1 ) for the indicated times .

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

Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times.


In C endogenous CALCOCO1 is analysed and the bars represent the mean ± sd of band intensities relative to the actin loading control , as quantified using ImageJ of three independent experiments . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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

In C endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot analysis of indicated cell lines , starved for 6 hours ( HBSS ) as indicated , and treated with 25 μM MG132 or 200 ng / ml of bafilomycin A1 ( Baf A1 ) for the indicated times . In E , endogenous CALCOCO1 is analysed and the bars represent the mean ± sd of band intensities relative to the actin loading control , as quantified using ImageJ of three independent experiments . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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

Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In E, endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


F A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 and immunostained for endogenous GM130 . Scale bars are 5 μm and 2 μm ( zoomed inset ) .

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F A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 and immunostained for endogenous GM130. Scale bars are 5 μm and 2 μm (zoomed inset).


G Same cells as in E were left untreated or treated with Baf A1 for 6 hours , and then immunostained for endogenous p62 and LC3B . Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans .

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G Same cells as in E were left untreated or treated with Baf A1 for 6 hours, and then immunostained for endogenous p62 and LC3B. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.


H CALCOCO1 , p62 and LC3B puncta in in the indicated conditions , counted using an automated system . The error bars represent mean ± SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test . Significance is displayed as * * * p < 0 . 001 .

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H CALCOCO1, p62 and LC3B puncta in in the indicated conditions, counted using an automated system. The error bars represent mean±SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001.


I Percentage of co - localization of CALCOCO1 puncta with p62 and / or LC3B in cells treated as indicated . The error bars represent mean ± SEM of three independent experiments per condition and over 250 cells per experiment . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test . Significance is displayed as * * * p < 0 . 001 .

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I Percentage of co-localization of CALCOCO1 puncta with p62 and/or LC3B in cells treated as indicated. The error bars represent mean±SEM of three independent experiments per condition and over 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001.


J HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1 . Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans .

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J HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.


A GST pulldown binding assay of in vitro transcribed / translated 35S - Myc - CALCOCO1 with recombinant GST - tagged ATG8 family proteins . GST and GST fusion proteins were visualized by coomassie brilliant blue staining ( bottom panel ) , and the co - precipitated Myc - CALCOCO1 was detected by autoradiography ( upper panel ) . The numbers below the AR represent % binding in the shown AR .

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A GST pulldown binding assay of in vitro transcribed/translated 35S-Myc-CALCOCO1 with recombinant GST-tagged ATG8 family proteins. GST and GST fusion proteins were visualized by coomassie brilliant blue staining (bottom panel), and the co-precipitated Myc-CALCOCO1 was detected by autoradiography (upper panel). The numbers below the AR represent % binding in the shown AR .


B GST pulldown assay of transiently transfected Myc - CALCOCO1 from HEK293 cell extracts with recombinant GST - tagged ATG8 family proteins . GST and GST fusions were visualized by Ponceau S staining ( bottom panel ) , and co - precipitated Myc - CALCOCO1 detected by immunoblotting with anti - Myc antibody ( upper panel ) .

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B GST pulldown assay of transiently transfected Myc-CALCOCO1 from HEK293 cell extracts with recombinant GST-tagged ATG8 family proteins. GST and GST fusions were visualized by Ponceau S staining (bottom panel), and co-precipitated Myc-CALCOCO1 detected by immunoblotting with anti-Myc antibody (upper panel).


GST pulldown assays of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteins . Precipitated GST and GST fusions and co - precipitated Myc - CALCOCO1 constructs were analyzed as in A .

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GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.


I GST pulldown assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST - tagged CALCOCO1 constructs . GST and GST fusions were visualized as in A , and co - precipitated GABARAP with anti - GABARAP antibody ( upper panel ) .

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I GST pulldown assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST-tagged CALCOCO1 constructs. GST and GST fusions were visualized as in A, and co-precipitated GABARAP with anti-GABARAP antibody (upper panel).


GST pulldowns testing binding of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family proteins ( left ) . Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs . The presence of two well separated binding surfaces on ATG8 proteins binding to LIR ( LDS ) and UIR ( UDS ) is indicated ( right ) .

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GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins (left). Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs. The presence of two well separated binding surfaces on ATG8 proteins binding to LIR (LDS) and UIR (UDS) is indicated (right).


B GST pulldowns testing binding of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family

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B GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins


C GST pulldown assays of in vitro transcribed / translated 35S - Myc - CALCOCO1 and 35S - Myc - p62 with recombinant GST - GABARAPL2 ( WT and indicated mutants ) .

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C GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 and 35S-Myc-p62 with recombinant GST-GABARAPL2 (WT and indicated mutants).


D GST pulldown assays of in vitro transcribed / translated 35S - Myc - TAX1BP1 ( WT and indicated mutants ) with recombinant GST - GABARAP ( WT and indicated mutants ) .

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D GST pulldown assays of in vitro transcribed/translated 35S-Myc-TAX1BP1 (WT and indicated mutants) with recombinant GST-GABARAP (WT and indicated mutants).


Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP - CALCOCO1 or EGFP - CALCOCO1 mLIR + ∆ 623 - 691 . Cells were induced with tetracycline for 24 hours and then starved or treated with MG132 or Baf A1 as indicated . The blot panels are from more than one western blot experiment but for clarity , only a single actin / GAPDH loading control is shown . In F , the bars represent the mean ± sd of band intensities relative to the actin or GAPDH loading controls of three independent experiments quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691. Cells were induced with tetracycline for 24 hours and then starved or treated with MG132 or Baf A1 as indicated. The blot panels are from more than one western blot experiment but for clarity, only a single actin/GAPDH loading control is shown. In F, the bars represent the mean±sd of band intensities relative to the actin or GAPDH loading controls of three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, *p ˂ 0.01; ns is not significant.


G HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 or EGFP - CALCOCO1 mLIR + ∆ 623 - 691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B . Scale bars , 5 μm .

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G HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B. Scale bars, 5 μm.


HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 were starved for 4 hours and then immunostained with anti - ATG13 and anti - WIPI2 antibodies . Scale bars in A are 5 μm for the confocal microscopy images and 2 μm for the airyscans .

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HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were starved for 4 hours and then immunostained with anti-ATG13 and anti-WIPI2 antibodies. Scale bars in A are 5 μm for the confocal microscopy images and 2 μm for the airyscans.


In B , the error bars represent mean ± sd of puncta per cell from three independent experiments and100 - 200 cells per each experiment .

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In B, the error bars represent mean±sd of puncta per cell from three independent experiments and100-200 cells per each experiment.


Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH In D the bars represent the mean ± sd of band intensities relative to the GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH In D the bars represent the mean±sd of band intensities relative to the GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH In , the bars represent the mean ± sd of band intensities relative to the GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH In , the bars represent the mean±sd of band intensities relative to the GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin ) . In , the bars represent the mean ± sd of band intensities relative to the actin loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin). In , the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


, I the bars represent the mean ± sd of band intensities relative to the actin loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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, I the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (

B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O

Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH


Immunoblot analysis of indicated cell lines treated as indicated . Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control ( GAPDH The asterisk in K indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP - CALCOCO1 in cells extracts from the rescued cells . In the bars represent the mean ± sd of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH The asterisk in K indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP-CALCOCO1 in cells extracts from the rescued cells. In the bars represent the mean±sd of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


B Co - IP of Myc - VAPA or Myc - VAPB with EGFP - CALCOCO1 from transiently transfected HEK293 cells . The asterisks ( * ) indicate Myc - VAP previously detected on the same

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B Co- IP of Myc-VAPA or Myc-VAPB with EGFP-CALCOCO1 from transiently transfected HEK293 cells. The asterisks (*) indicate Myc-VAP previously detected on the same membrane


C , D GST pulldown assays of in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - VAPA or GST - VAPB constructs . The scheme is a representation of CALCOCO1 domains and motifs .

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C, D GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-VAPA or GST-VAPB constructs.The scheme is a representation of CALCOCO1 domains and motifs.


E HeLa cells transiently co - transfected with EGFP - CALCOCO1 and Myc - VAPA or - VAPB were immunostained with anti - Myc antibody . Scale bars , 5 μm .

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E HeLa cells transiently co-transfected with EGFP-CALCOCO1 and Myc-VAPA or -VAPB were immunostained with anti-Myc antibody. Scale bars, 5 μm.


A , B Immunoblot analysis of indicated cell lines , starved for 6 hours ( HBSS ) as indicated , and treated with MG132 or Baf A1 as indicated . Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control . In B , the bars represent the mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA and significance displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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A, B Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with MG132 or Baf A1 as indicated. Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In B, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


C Immunoblot analysis of HeLa cells transfected with the indicated siRNAs .

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C Immunoblot analysis of HeLa cells transfected with the indicated siRNAs.


D , E Immunoblot analysis of HeLa cells transfected with the indicated siRNAs and treated with Baf A1 as indicated . In D , the panels are from more than one western blot experiment but only a single GAPDH loading control is shown . In E , the bars represent the mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 ; ns is not significant .

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D, E Immunoblot analysis of HeLa cells transfected with the indicated siRNAs and treated with Baf A1 as indicated. In D, the panels are from more than one western blot experiment but only a single GAPDH loading control is shown. In E, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005; ns is not significant.


F HeLa KO CALCOCO1 cells were transiently co - transfected with EGFP - CALCOCO1 and Myc - VAPA or Myc - VAPB , and then immunostained with anti - Myc and anti - LC3B antibodies . Arrows indicate dots of co - localization of all three proteins . Scale bars , 5 μm for large merged images and 1 μm for zoomed images .

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F HeLa KO CALCOCO1 cells were transiently co-transfected with EGFP-CALCOCO1 and Myc-VAPA or Myc-VAPB, and then immunostained with anti-Myc and anti-LC3B antibodies. Arrows indicate dots of co-localization of all three proteins. Scale bars, 5 μm for large merged images and 1 μm for zoomed images.


A , B Immunoblot analysis of HeLa WT and HeLa CALCOCO1 KO cells . The cells were starved for 6 hours ( HBSS ) as indicated and treated with Baf A1 as indicated . Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control . In A , the panels are collected from more than one western blot experiment but for clarity , only a single GAPDH loading control is shown . In B , the bars represent the mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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

A, B Immunoblot analysis of HeLa WT and HeLa CALCOCO1 KO cells. The cells were starved for 6 hours (HBSS) as indicated and treated with Baf A1 as indicated. Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In A, the panels are collected from more than one western blot experiment but for clarity, only a single GAPDH loading control is shown. In B, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


C , D Immunoblot analysis of HeLa CALCOCO1 KO cell lines reconstituted with EGFP - CALCOCO1 . Expression of EGFP - CALCOCO1 was induced or not with tetracycline and the cells were treated with MG132 or Baf A1 as indicated . Numbers below the blots in C represent relative intensity of the bands in the shown blots normalized against actin loading control . In C , the panels are collected from more than one western blot experiment but only a single actin loading control is shown . In D , the bars represent the mean ± sd of band intensities of three independent experiments as quantified using ImageJ . Statistical comparison was analyzed as in B and significance displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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C, D Immunoblot analysis of HeLa CALCOCO1 KO cell lines reconstituted with EGFP-CALCOCO1. Expression of EGFP-CALCOCO1 was induced or not with tetracycline and the cells were treated with MG132 or Baf A1 as indicated. Numbers below the blots in C represent relative intensity of the bands in the shown blots normalized against actin loading control. In C, the panels are collected from more than one western blot experiment but only a single actin loading control is shown. In D, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed as in B and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


E HeLa CALCOCO1 KO cell lines reconstituted with EGFP - CALCOCO1 were treated with tetracycline or not to induce expression of EGFP - CALCOCO1 . Abundance of the ER was quantified from widefield fluorescence images of endogenous RTN3 staining ( see Materials and methods ) . Data are presented as mean ± sd of three independent experiments . Statistical comparison was analyzed as in B and significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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E HeLa CALCOCO1 KO cell lines reconstituted with EGFP-CALCOCO1 were treated with tetracycline or not to induce expression of EGFP-CALCOCO1. Abundance of the ER was quantified from widefield fluorescence images of endogenous RTN3 staining (see Materials and methods). Data are presented as mean ± sd of three independent experiments. Statistical comparison was analyzed as in B and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


F , G Immunoblot analysis of cells analyzed in C . The bars in G represent the mean ± sd of band intensities relative to the loading control from three independent experiments quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test . Significance is displayed as * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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F, G Immunoblot analysis of cells analyzed in C. The bars in G represent the mean±sd of band intensities relative to the loading control from three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


H , I Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 in fed or starved conditions and transfected with the indicated VAPA / B siRNAs .

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H, I Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 in fed or starved conditions and transfected with the indicated VAPA/B siRNAs.


A Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 in fed or starved conditions and treated as indicated with Baf A1 or PI3KC3 inhibitor SAR405 .

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A Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 in fed or starved conditions and treated as indicated with Baf A1 or PI3KC3 inhibitor SAR405.


B Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP - CALCOCO1 mLIR + ∆ 623 - 691 for 24 hours or not , and then treated as indicated .

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B Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 mLIR+∆623-691 for 24 hours or not, and then treated as indicated.


C , Representative confocal images of HeLa CALCOCO1 KO cells transiently co - transfected with mCherry - CALCOCO1 , Myc - VAPA and EGFP - LAMP1 , and then treated as indicated before immunostaining with anti - Myc anti - body . Arrows indicate co - localization . Scale bars in C , 5 μm for large merged images and 1 μm for zoomed images .

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C, Representative confocal images of HeLa CALCOCO1 KO cells transiently co-transfected with mCherry-CALCOCO1, Myc-VAPA and EGFP-LAMP1, and then treated as indicated before immunostaining with anti-Myc anti-body. Arrows indicate co-localization. Scale bars in C, 5 μm for large merged images and 1 μm for zoomed images.


D , data are presented as mean ± sd . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison test and significance displayed as * * * p ˂ 0 . 001 ; ns is not significant , n = 3 .

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D, data are presented as mean ± sd. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001; ns is not significant, n=3.


Schematic , western blot , and immunofluorescence analysis of the 3 × FLAG - UBXD8 knockin U2OS cell line . Scale bar , 5 μm . Data information : In C , mitochondria were visualized by anti - Tom20 immunostaining ; ER was labeled by anti - PDI immunostaining .

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Schematic, western blot, and immunofluorescence analysis of the 3×FLAG-UBXD8 knockin U2OS cell line. Scale bar, 5 μm. Data information: In C, mitochondria were visualized by anti-Tom20 immunostaining; ER was labeled by anti-PDI immunostaining.


Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆ UBXD8 HeLa cells .

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Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆UBXD8 HeLa cells.


Immunoprecipitation analysis of UBXD8 interaction with mitochondrial ( MARCH5 and MUL1 ) and ER ( RNF185 ) ubiquitin E3 ligases . HeLa cells were infected with lentivirus to stably express HA - tagged ubiquitin E3 ligases . Whole cell lysates were prepared for anti - HA immunoprecipitation .

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Immunoprecipitation analysis of UBXD8 interaction with mitochondrial (MARCH5 and MUL1) and ER (RNF185) ubiquitin E3 ligases. HeLa cells were infected with lentivirus to stably express HA-tagged ubiquitin E3 ligases. Whole cell lysates were prepared for anti-HA immunoprecipitation.


Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185 . Whole cell lysates from 3 × FLAG - UBXD8 knockin HEK293T cells were prepared for anti - FLAG immunoprecipitation . MARCH5 and RNF185 : short exposure for whole cell lysate blots , long exposure for FLAG - IP blots .

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Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185. Whole cell lysates from 3×FLAG-UBXD8 knockin HEK293T cells were prepared for anti-FLAG immunoprecipitation. MARCH5 and RNF185: short exposure for whole cell lysate blots, long exposure for FLAG-IP blots.


Fractionation analysis of UBXD8 ' s effect on VCP recruitment to mitochondria and the ER . Whole cell lysate , mitochondria , and ER fractions were purified from WT , ∆ UBXD8 , and ∆ UBXD8 HeLa cells rescued with UBXD8 or the UBX * mutant . UBX * - UBXD8 carries four point mutations ( K367A , F407A , P408G , R409A ) in the UBX domain to disrupt the UBXD8 - VCP interaction . For each sample , 5 μg proteins were loaded for western blot analysis .

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Fractionation analysis of UBXD8's effect on VCP recruitment to mitochondria and the ER. Whole cell lysate, mitochondria, and ER fractions were purified from WT, ∆UBXD8, and ∆UBXD8 HeLa cells rescued with UBXD8 or the UBX* mutant. UBX*-UBXD8 carries four point mutations (K367A, F407A, P408G, R409A) in the UBX domain to disrupt the UBXD8-VCP interaction. For each sample, 5 μg proteins were loaded for western blot analysis.


Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells . Data are shown as mean ± SE from three biological repeats . Statistics : one - way ANOVA ; * * P < 0 . 01 , * * * P < 0 . 001 .

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Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells. Data are shown as mean ± SE from three biological repeats. Statistics: one-way ANOVA; **P < 0.01, ***P < 0.001.


A , B . Western blot ( A ) and quantitative ( B ) analyses of MiD49 and Mcl1 degradation in WT and ∆ MARCH5 HeLa cells . Cycloheximide ( CHX : 200 μg / ml ; protein synthesis inhibitor ) ; MG132 : 20 μM , proteasome inhibitor . Data information : Data are shown as mean ± SE from three biological repeats ( B , Statistics : two - tailed unpaired Student ' s t - test ( B , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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A, B. Western blot (A) and quantitative (B) analyses of MiD49 and Mcl1 degradation in WT and ∆MARCH5 HeLa cells. Cycloheximide (CHX: 200 μg/ml; protein synthesis inhibitor); MG132: 20 μM, proteasome inhibitor. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


C , D . Western blot ( C ) and quantitative ( D ) analyses of Mcl1 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats D , Statistics : two - tailed unpaired Student ' s t - test D , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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C, D. Western blot (C) and quantitative (D) analyses of Mcl1 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


E , F . Western blot ( E ) and quantitative ( F ) analyses of MiD49 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats F ) . Statistics : two - tailed unpaired Student ' s t - test F ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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E, F. Western blot (E) and quantitative (F) analyses of MiD49 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats F). Statistics: two-tailed unpaired Student's t-test F); *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.


Fractionation analysis of the subcellular localization of mito - UBXD8 and ER - UBXD8 . Whole cell lysate , mitochondria , and ER fractions were purified from the indicated HeLa cells . Exogenously - expressed mito - UBXD8 has similar expression level with endogenous UBXD8 in WT cells ; exogenously - expressed WT UBXD8 and ER - UBXD8 have similar expression levels .

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Fractionation analysis of the subcellular localization of mito-UBXD8 and ER-UBXD8. Whole cell lysate, mitochondria, and ER fractions were purified from the indicated HeLa cells. Exogenously-expressed mito-UBXD8 has similar expression level with endogenous UBXD8 in WT cells; exogenously-expressed WT UBXD8 and ER-UBXD8 have similar expression levels.


D , E . Western blot ( D ) and quantitative ( E ) analyses of Mcl1 degradation in the indicated HeLa cells . ∆ UBXD8 HeLa cells were rescued with vector , WT - , mito - , and ER - UBXD8 . Data are shown as mean ± SE from three biological repeats ( E ) . Statistics : two - tailed unpaired Student ' s t - test ( E ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 B-CELL_LINE O O O B-GENEPROD B-CELL_LINE O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR 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 O O O

D, E. Western blot (D) and quantitative (E) analyses of Mcl1 degradation in the indicated HeLa cells. ∆UBXD8 HeLa cells were rescued with vector, WT-, mito-, and ER-UBXD8. Data are shown as mean ± SE from three biological repeats (E). Statistics: two-tailed unpaired Student's t-test (E); *P < 0.05, **P < 0.01, ***P < 0.001.


Cell survival analysis of the indicated HeLa cells treated with Actinomycin D ( ActD , 1 μM ) or Doxorubicin ( Doxo , 10 μM ) . Representative images are shown . Cell viability was measured by Cell Titer - Glo . Scale bar , 50 μm . Data information : Data are shown as mean ± SE from three biological repeats D , Statistics : two - tailed unpaired Student ' s t - test D , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Cell survival analysis of the indicated HeLa cells treated with Actinomycin D (ActD, 1 μM) or Doxorubicin (Doxo, 10 μM). Representative images are shown. Cell viability was measured by Cell Titer-Glo. Scale bar, 50 μm. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001.


Western blot analysis of apoptosis activation ( caspase cleavage and PARP cleavage ) in the indicated HeLa cells treated similarly as in ( D ) .

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Western blot analysis of apoptosis activation (caspase cleavage and PARP cleavage) in the indicated HeLa cells treated similarly as in (D).


Cell survival analysis of the indicated HeLa cells . 7 × 105 cells in one well of 6 - well plate were treated with ActD ( 200 nM ) for 4 hours or Doxo ( 50 nM ) for 2 hours and subsequently cultured in normal medium for 21 days . Surviving colonies were stained by 0 . 1 % ( W / V ) methylene blue . Representative images and quantifications of the surviving colony numbers are shown . Data information : Data are shown as mean ± SE from three biological repeats G ) . Statistics : one - way ANOVA ( G ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Cell survival analysis of the indicated HeLa cells. 7 × 105 cells in one well of 6-well plate were treated with ActD (200 nM) for 4 hours or Doxo (50 nM) for 2 hours and subsequently cultured in normal medium for 21 days. Surviving colonies were stained by 0.1% (W/V) methylene blue. Representative images and quantifications of the surviving colony numbers are shown. Data information: Data are shown as mean ± SE from three biological repeats G). Statistics: one-way ANOVA (G); *P < 0.05, **P < 0.01, ***P < 0.001.


Western blot analysis of BH3 - domain containing proteins in the indicated HeLa cells treated with Doxo ( 10 μM ) . Three proteins with enhanced level in ∆ UBXD8 cells ( Noxa , Bik , and Bnip3 ) are highlighted in red .

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Western blot analysis of BH3-domain containing proteins in the indicated HeLa cells treated with Doxo (10 μM). Three proteins with enhanced level in ∆UBXD8 cells (Noxa, Bik, and Bnip3) are highlighted in red.


Western blot and quantitative analysis of Noxa degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats ( B , Statistics : two - tailed unpaired Student ' s t - test ( B , ; * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 .

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Western blot and quantitative analysis of Noxa degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.001, ***P < 0.001.


Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells . Data information : Data are shown as mean ± SE from three biological repeats C , Statistics : two - tailed unpaired Student ' s t - test C , ; * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 .

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Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells. Data information: Data are shown as mean ± SE from three biological repeats C, Statistics: two-tailed unpaired Student's t-test C, ; *P < 0.05, **P < 0.001, ***P < 0.001.


Western blot ( D , analysis of the indicated HeLa cells .

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Western blot (D, analysis of the indicated HeLa cells.


Western blot F ) analysis of the indicated HeLa cells .

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Western blot F) analysis of the indicated HeLa cells.


Representative images of the mitophagy events in the indicated HeLa cells . mtKeima emission excited by 405 nm laser is shown as green ; mtKeima emission excited by 561 nm laser is shown as red . Mitolysosomes are pointed by white arrows . Scale bar , 5 μm .

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

Representative images of the mitophagy events in the indicated HeLa cells. mtKeima emission excited by 405 nm laser is shown as green; mtKeima emission excited by 561 nm laser is shown as red. Mitolysosomes are pointed by white arrows. Scale bar, 5 μm.


C - F . Quantitative analysis of mitophagy events in the indicated HeLa cells . Only cells with mitophagy were selected to quantify mitolysosome numbers ( D , F ) . Data information : Data are shown as mean ± SE from three biological repeats ( C - F , Statistics : two - tailed unpaired Student ' s t - test ( C - F , ; * * P < 0 . 01 , * * * P < 0 . 001 .

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

C-F. Quantitative analysis of mitophagy events in the indicated HeLa cells. Only cells with mitophagy were selected to quantify mitolysosome numbers (D, F). Data information: Data are shown as mean ± SE from three biological repeats (C-F, Statistics: two-tailed unpaired Student's t-test (C-F, ; **P < 0.01, ***P < 0.001.


I - K , Representative images ( I ) and quantitative analysis of mitophagy events ( J , K ) in the indicated HeLa cells . White arrows point to mitolysosomes . Scale bar , 5 μm . Data information : Data are shown as mean ± SE from three biological repeats J , K , Statistics : two - tailed unpaired Student ' s t - test J , K ) ; * * P < 0 . 01 , * * * P < 0 . 001 .

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

I-K, Representative images (I) and quantitative analysis of mitophagy events (J, K) in the indicated HeLa cells. White arrows point to mitolysosomes. Scale bar, 5 μm. Data information: Data are shown as mean ± SE from three biological repeats J, K, Statistics: two-tailed unpaired Student's t-test J, K); **P < 0.01, ***P < 0.001.


B . Carfilzomib , Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il - 1β - luciferase transgenic mice . BMDMs from IL - 1β - luciferase transgenic mice were treated by IL - 4 ( 20 ng / mL ) for 24 hours to differentiate into mature M2 macrophages , and then used for screening of FDA compounds ( 5 μM ) . The X axis stands for the code of the drug .

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

B. Carfilzomib, Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il-1β-luciferase transgenic mice. BMDMs from IL-1β-luciferase transgenic mice were treated by IL-4 (20 ng/mL) for 24 hours to differentiate into mature M2 macrophages, and then used for screening of FDA compounds (5 μM). The X axis stands for the code of the drug.