J - M . Percentage of stem cells ( OLFM4 ) goblet cell ( FGCBP ) , mature enterocytes ( APOA4 ) and cells of the enterocyte lineage ( FGCBP ) from the total organoid cells population ( black bar ) in mock - treated organoids and after HAstV1 infection . The percentage of infected cells ( red bars ) and bystander cells ( blue bars ) within each cell type is shown . The data was obtained from multiplex in situ RNA hybridization in 2D organoids . n = 10 , mean ± SEM . Statistics show comparison of infected cells between mock - treated , 4h pi and 16h pi organoids . An Ordinary one - way ANOVA and Tukey ' s multiple comparisons test was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

J-M. Percentage of stem cells (OLFM4) goblet cell (FGCBP), mature enterocytes (APOA4) and cells of the enterocyte lineage (FGCBP) from the total organoid cells population (black bar) in mock-treated organoids and after HAstV1 infection. The percentage of infected cells (red bars) and bystander cells (blue bars) within each cell type is shown. The data was obtained from multiplex in situ RNA hybridization in 2D organoids. n=10, mean ± SEM. Statistics show comparison of infected cells between mock-treated, 4h pi and 16h pi organoids. An Ordinary one-way ANOVA and Tukey's multiple comparisons test was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001.


A . One image per condition was used to show the multiplex in situ RNA FISH . A representative region of the images was chosen and cropped in order to have a zoom - in of a specific area for better visualization . Shown are the enterocyte lineage marker FABP6 ( green ) , HAstV1 infection ( white ) , and CCL2 gene expression ( red ) of mock - treated and infected organoids . DAPI is in blue . White arrows show co - localization of FABP6 and CCL2 with HAstV1 signal . Scale bar 100µm .

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

A. One image per condition was used to show the multiplex in situ RNA FISH. A representative region of the images was chosen and cropped in order to have a zoom-in of a specific area for better visualization. Shown are the enterocyte lineage marker FABP6 (green), HAstV1 infection (white), and CCL2 gene expression (red) of mock-treated and infected organoids. DAPI is in blue. White arrows show co-localization of FABP6 and CCL2 with HAstV1 signal. Scale bar 100µm.


B - D . Fluorescence intensity ( arbitrary units ) of innate immune marker expression in stem cells ( OLFM4 positive ) , goblet cells ( FCGBP positive ) , enterocyte lineage cells ( FABP6 positive ) , and mature enterocytes ( APOA4 positive ) . Bystander cells are in blue and infected cells in red . 10 - 90 percentile box plots , each dot represents one cell ( mock N = 2274 , 4h N = 3682 , 16h N = 1510 . C . mock N = 2693 , 4h N = 3706 , 16h N = 1921 . D . mock N = 2274 , 4h N = 3706 , 16h N = 1514 . ) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages . Ordinary one - way ANOVA and Tukey ' s multiple comparisons test were used . Grey statistics show the comparison between infected and bystander cells within each cell lineage . Unpaired t - test with Welch ' s correction was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O O B-CELL_TYPE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. Fluorescence intensity (arbitrary units) of innate immune marker expression in stem cells (OLFM4 positive), goblet cells (FCGBP positive), enterocyte lineage cells (FABP6 positive), and mature enterocytes (APOA4 positive). Bystander cells are in blue and infected cells in red. 10-90 percentile box plots, each dot represents one cell (mock N=2274, 4h N=3682, 16h N=1510. C. mock N=2693, 4h N=3706, 16h N=1921. D. mock N=2274, 4h N=3706, 16h N=1514.) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages. Ordinary one-way ANOVA and Tukey's multiple comparisons test were used. Grey statistics show the comparison between infected and bystander cells within each cell lineage. Unpaired t-test with Welch's correction was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001


( A ) Overexpression of RNF26 promoted polyubiquitination of MITA . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with a control or RNF26 expression plasmid ( 0 . 5 , 1 , 2 µg ) . Twenty - four hours after transfection , cells were subjected to immunoprecipitation ( IP ) under denatured conditions with anti - Flag and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - HA ( upper panel ) , anti - Flag ( middle panel ) and anti - RNF26 ( bottom panel ) . Ub , ubiquitin .

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

(A) Overexpression of RNF26 promoted polyubiquitination of MITA. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with a control or RNF26 expression plasmid (0.5, 1, 2 µg). Twenty-four hours after transfection, cells were subjected to immunoprecipitation (IP) under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-HA (upper panel), anti-Flag (middle panel) and anti-RNF26 (bottom panel). Ub, ubiquitin.


( B ) RNF26 - mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26 . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with RNF26 or its mutants ( 1 µg each ) . IP under denatured conditions and ubiquitination assay was performed as described in ( A ) .

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

(B) RNF26-mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with RNF26 or its mutants (1 µg each). IP under denatured conditions and ubiquitination assay was performed as described in (A).


( C ) UbcH5 mediated polyubiquitination of MITA by RNF26 . The RNF26 and MITA proteins were obtained by in vitro transcription and translation , then incubated with biotin - Ub , E1 and the indicated E2s . Polyubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) .

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

(C) UbcH5 mediated polyubiquitination of MITA by RNF26. The RNF26 and MITA proteins were obtained by in vitro transcription and translation, then incubated with biotin-Ub, E1 and the indicated E2s. Polyubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels).


( D ) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro . MITA , RNF26 and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and MITA were incubated with RNF26 or its mutants , followed by ubiquitination and immunoblot analysis as described in ( C ) . All experiments were repeated for at least three times with similar results .

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

(D) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro. MITA, RNF26 and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and MITA were incubated with RNF26 or its mutants, followed by ubiquitination and immunoblot analysis as described in (C). All experiments were repeated for at least three times with similar results.


( A ) RNF26 interacted with MITA . The 293 cells ( 5 × 106 ) were transfected with the indicated plasmids ( 5 µg each ) . Twenty - four hours later , cells were lysed and cell lysates were immunoprecipitated with anti - Flag or control IgG . The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti - HA or anti - Flag .

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

(A) RNF26 interacted with MITA. The 293 cells (5×106) were transfected with the indicated plasmids (5 µg each). Twenty-four hours later, cells were lysed and cell lysates were immunoprecipitated with anti-Flag or control IgG. The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti-HA or anti-Flag.


( B ) RNF26 was physiologically associated with MITA . THP - 1 cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins . NC , negative control .

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

(B) RNF26 was physiologically associated with MITA. THP-1 cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins. NC, negative control.


( C ) RNF26 is localized to the ER and mitochondria . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Twenty - four hours after transfection , the cells were stained with Mito - Tracker Red for 15 minutes or left untreated . The cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis .

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

(C) RNF26 is localized to the ER and mitochondria. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Twenty-four hours after transfection, the cells were stained with Mito-Tracker Red for 15 minutes or left untreated. The cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis.


D ) Subcellular distribution of RNF26 . The 293 cells ( 5 × 107 ) were infected with SeV for the indicated time points or left uninfected . Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins .

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

D) Subcellular distribution of RNF26. The 293 cells (5×107) were infected with SeV for the indicated time points or left uninfected. Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins.


( E ) RNF26 and MITA were colocalized to the ER . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Eighteen hours after transfection , cells were infected with SeV or HSV - 1 for 6 hours or left uninfected , followed by ER - Tracker Blue / White staining for 30 minutes . Cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis . All experiments were repeated for at least three times with similar results .

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

(E) RNF26 and MITA were colocalized to the ER. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Eighteen hours after transfection, cells were infected with SeV or HSV-1 for 6 hours or left uninfected, followed by ER-Tracker Blue/White staining for 30 minutes. Cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis. All experiments were repeated for at least three times with similar results.


( A ) RNF26 mediated polyubiquitination of MITA at K150 . The 293 cells ( 5 × 106 ) were transfected with HA - Ub ( 1 µg ) and RNF26 ( 1 µg ) together with Flag - MITA or the indicated mutants ( 5 µg each ) . Twenty - four hours after transfection , cells were subjected IP under denatured conditions with anti - Flag and immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated .

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

(A) RNF26 mediated polyubiquitination of MITA at K150. The 293 cells (5×106) were transfected with HA-Ub (1 µg) and RNF26 (1 µg) together with Flag-MITA or the indicated mutants (5 µg each). Twenty-four hours after transfection, cells were subjected IP under denatured conditions with anti-Flag and immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.


B ) RNF26 targeted MITA for polyubiquitination at K150 in vitro . RNF26 , MITA and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and RNF26 were incubated with MITA or its mutants . The ubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) . All experiments were repeated for at least three times with similar results .

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

B) RNF26 targeted MITA for polyubiquitination at K150 in vitro. RNF26, MITA and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and RNF26 were incubated with MITA or its mutants. The ubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels). All experiments were repeated for at least three times with similar results.


( A and B ) RNF26 targeted MITA for K11 - linked polyubiquitination . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Twenty - four hours after transfection , cells were subjected for IP under denatured conditions with limited amount of anti - Flag ( 0 . 5 µg ) so that equal amount of Flag - MITA is pulled down . The immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated . Ub - AKR , all lysine residues of ubiquitin were mutated to arginine .

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

(A and B) RNF26 targeted MITA for K11-linked polyubiquitination. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Twenty-four hours after transfection, cells were subjected for IP under denatured conditions with limited amount of anti-Flag (0.5 µg) so that equal amount of Flag-MITA is pulled down. The immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated. Ub-AKR, all lysine residues of ubiquitin were mutated to arginine.


( C ) Effects of RNF26 - RNAi plasmids on the expression of RNF26 . In the upper panels , the 293 cells ( 1 × 106 ) were transfected with the expression plasmids for RNF26 - Flag ( 0 . 5 µg ) and HA - β - actin ( 0 . 1 µg ) together with the indicated RNAi plasmids ( 1 µg each ) . Twenty - four hours after transfection , whole cell lysates were analyzed by immunoblots with anti - Flag or anti - HA . In the lower panels , 293 cells were transduced with a control or RNF26 - RNAi by retrovirus mediated gene transfer . Cells ( 1 × 106 ) were lysed and whole cell lysates were analyzed by immunoblots with anti - RNF26 or anti - β - actin .

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

(C) Effects of RNF26-RNAi plasmids on the expression of RNF26. In the upper panels, the 293 cells (1×106) were transfected with the expression plasmids for RNF26-Flag (0.5 µg) and HA-β-actin (0.1 µg) together with the indicated RNAi plasmids (1 µg each). Twenty-four hours after transfection, whole cell lysates were analyzed by immunoblots with anti-Flag or anti-HA. In the lower panels, 293 cells were transduced with a control or RNF26-RNAi by retrovirus mediated gene transfer. Cells (1×106) were lysed and whole cell lysates were analyzed by immunoblots with anti-RNF26 or anti-β-actin.


( D ) Immunoblot analysis of Flag - tagged ubiquitin expression in THP - 1 cells stably transfected with Flag - Ub - K11O plasmid . Whole cell lysates of THP - 1 - Flag - Ub - K11O - RNF26 - RNAi and control cells ( 1 × 106 ) were analyzed by immunoblots with antibodies against the indicated proteins . RNF26 - RNAi # 1 was used here and in the following experiments if not noted .

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

(D) Immunoblot analysis of Flag-tagged ubiquitin expression in THP-1 cells stably transfected with Flag-Ub-K11O plasmid. Whole cell lysates of THP-1-Flag-Ub-K11O-RNF26-RNAi and control cells (1×106) were analyzed by immunoblots with antibodies against the indicated proteins. RNF26-RNAi #1 was used here and in the following experiments if not noted.


( E and F ) Effects of RNF26 knockdown on virus - induced K11 - linked polyubiquitination of endogenous MITA . In ( E ) , THP - 1 - Flag - Ub - K11O - RNF26 - RNAi or control cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti - MITA . The immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panels ) or anti - MITA ( lower panels ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins . In ( F ) , 293 - HA - Ub - K11O cells ( 2 × 107 ) were transfected with a control or RNF26 - RNAi plasmid ( 10 µg each ) . Twelve hours after transfection , puromycin ( 1 µg / mL ) was added into the culture medium . The cells were selected for twenty - four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in ( E ) . All experiments were repeated for at least three times with similar results .

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

(E and F) Effects of RNF26 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. In (E), THP-1-Flag-Ub-K11O-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti-MITA. The immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panels) or anti-MITA (lower panels). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins. In (F), 293-HA-Ub-K11O cells (2×107) were transfected with a control or RNF26-RNAi plasmid (10 µg each). Twelve hours after transfection, puromycin (1 µg/mL) was added into the culture medium. The cells were selected for twenty-four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in (E). All experiments were repeated for at least three times with similar results.


A ) Effects of RNF26 knockdown on virus - induced polyubiquitination of endogenous MITA . The THP - 1 - RNF26 - RNAi or control cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - Ub ( K48 ) ( upper panel ) , anti - Ub ( K63 ) ( middle panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins .

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

A) Effects of RNF26 knockdown on virus-induced polyubiquitination of endogenous MITA. The THP-1-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Ub(K48) (upper panel), anti-Ub(K63) (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.


( B ) Effects of RNF5 knockdown on virus - induced K11 - linked polyubiquitination of endogenous MITA . The 293 - HA - Ub - K11O cells ( 2 × 107 ) were transfected with the indicated RNAi plasmid ( 10 µg each ) . Twelve hours after transfection , the cells were selected with puromycin ( 1 µg / mL ) for twenty - four hours and infected with SeV for the indicated time points or left uninfected . Cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies .

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

(B) Effects of RNF5 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. The 293-HA-Ub-K11O cells (2×107) were transfected with the indicated RNAi plasmid (10 µg each). Twelve hours after transfection, the cells were selected with puromycin (1 µg/mL) for twenty-four hours and infected with SeV for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies.


( C ) RNF26 and RNF5 competed with each other on MITA polyubiquitination . The 293 cells ( 5 × 106 ) were transfected with MITA ( 5 µg ) , Flag - Ub - K48O and HA - Ub - K11O ( 1 µg each ) together with indicated amount of RNF26 and RNF5 . Twenty - four hours after transfection , cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panel ) , anti - HA ( middle panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins .

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

(C) RNF26 and RNF5 competed with each other on MITA polyubiquitination. The 293 cells (5×106) were transfected with MITA (5 µg), Flag-Ub-K48O and HA-Ub-K11O (1 µg each) together with indicated amount of RNF26 and RNF5. Twenty-four hours after transfection, cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panel), anti-HA (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.


( D ) Effects of RNF26 knockdown on virus - triggered MITA degradation . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins ( upper immunoblots ) . The relative protein levels of MITA in reference to β - actin were analyzed by Quantity One program and data shown are mean ± S . D . of three independent experiments ( lower histographs ) .

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

(D) Effects of RNF26 knockdown on virus-triggered MITA degradation. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins (upper immunoblots). The relative protein levels of MITA in reference to β-actin were analyzed by Quantity One program and data shown are mean ± S.D. of three independent experiments (lower histographs).


( A ) Effects of RNF26 knockdown on SeV - triggered activation of the IFN - β promoter . The 293 cells ( 2 × 105 ) were transfected with the IFN - β promoter reporter ( 0 . 1 µg ) and the indicated RNAi plasmid ( 0 . 5 µg each ) . Thirty hours after transfection , cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed .

O O O O O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O

(A) Effects of RNF26 knockdown on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and the indicated RNAi plasmid (0.5 µg each). Thirty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.


( B and C ) Effects of RNF26 knockdown on virus - triggered induction of IFN - β in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( B )

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

(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B)


( B and C ) Effects of RNF26 knockdown on virus - triggered induction of IFN - β in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( B ) or ELISA analysis (

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

(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) or ELISA analysis (C


( D ) Effects of RNF26 knockdown on virus - triggered induction of IFNB1 gene in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with VSV , EMCV or ECTV for the indicated time points or left uninfected before quantitative real - time PCR analysis was performed as in ( B ) .

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

(D) Effects of RNF26 knockdown on virus-triggered induction of IFNB1 gene in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with VSV, EMCV or ECTV for the indicated time points or left uninfected before quantitative real-time PCR analysis was performed as in (B).


( E and F ) Effects of RNF26 knockdown on virus - triggered induction of TNFα in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( E ) and ELISA analysis ( F ) .

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

(E and F) Effects of RNF26 knockdown on virus-triggered induction of TNFα in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (E) and ELISA analysis (F).


( G ) Effects of RNF26 knockdown on virus - triggered phosphorylation of TBK1 , IRF3 and IκBα . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected , whole cell lysates were analyzed by immunoblots with anti - p - TBK1 , anti - TBK1 , anti - p - IRF3 , anti - IRF3 , anti - p - IκBα , anti - IκBα , anti - RNF26 or anti - β - actin as indicated . All experiments were repeated for at least three times with similar results . The bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

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

(G) Effects of RNF26 knockdown on virus-triggered phosphorylation of TBK1, IRF3 and IκBα. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected, whole cell lysates were analyzed by immunoblots with anti-p-TBK1, anti-TBK1, anti-p-IRF3, anti-IRF3, anti-p-IκBα, anti-IκBα, anti-RNF26 or anti-β-actin as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


A ) Effects of RNF26 knockdown on IRF3 level . In the left panel , whole cell lysates of THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were analyzed by immunoblots with the indicated antibodies . In the right panel , cells ( 1 × 106 ) were subjected to quantitative real - time PCR analysis .

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

A) Effects of RNF26 knockdown on IRF3 level. In the left panel, whole cell lysates of THP-1-RNF26-RNAi or control cells (1×106) were analyzed by immunoblots with the indicated antibodies. In the right panel, cells (1×106) were subjected to quantitative real-time PCR analysis.


B ) Effects of RNF26 on IRF3 stability . The 293 cells ( 1 × 106 ) were transfected with HA - IRF3 ( 0 . 5 µg ) and HA - β - actin ( 0 . 1 µg ) together with RNF26 or its mutant ( 0 . 3 µg each ) . Twenty - four hours later , whole cell lysates were analyzed by immunoblots with anti - HA or anti - RNF26 .

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

B) Effects of RNF26 on IRF3 stability. The 293 cells (1×106) were transfected with HA-IRF3 (0.5 µg) and HA-β-actin (0.1 µg) together with RNF26 or its mutant (0.3 µg each). Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.


( C ) Effects of RNF26 and its mutant on SeV - triggered activation of the IFN - β promoter . The 293 cells ( 2 × 105 ) were transfected with the IFN - β promoter reporter ( 0 . 1 µg ) and RNF26 or its mutant ( 0 . 1 µg each ) . Twenty hours after transfection , cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed .

O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-ORGANISM O O O O O O O O O O O O

(C) Effects of RNF26 and its mutant on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and RNF26 or its mutant (0.1 µg each). Twenty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.


( D ) Effects of RNF26 on IRF3 ubiquitination . The 293 cells ( 5 × 106 ) were transfected with Flag - IRF3 ( 5 µg ) and RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment . The cell lysates were subjected to IP under denatured conditions with anti - Flag and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panels ) or anti - Flag ( lower panels ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated .

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

(D) Effects of RNF26 on IRF3 ubiquitination. The 293 cells (5×106) were transfected with Flag-IRF3 (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Eighteen hours after transfection, 3-MA (500 ng/mL) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment. The cell lysates were subjected to IP under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panels) or anti-Flag (lower panels). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.


( E ) Effects of inhibitors on RNF26 - mediated destabilization of IRF3 . The 293 cells ( 1 × 106 ) were transfected with the indicated plasmids as described in ( B ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) , NH4Cl ( 25 mM ) or MG132 ( 100 µM ) was added into culture medium for four hours . Whole cell lysates were analyzed by immunoblots with anti - HA or anti - RNF26 .

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

(E) Effects of inhibitors on RNF26-mediated destabilization of IRF3. The 293 cells (1×106) were transfected with the indicated plasmids as described in (B). Eighteen hours after transfection, 3-MA (500 ng/mL), NH4Cl (25 mM) or MG132 (100 µM) was added into culture medium for four hours. Whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.


( F ) Effects of ATG12 knockdown on RNF26 - mediated destabilization of IRF3 . The 293 - ATG12 - RNAi or control cells ( 1 × 106 ) were transfected with the indicated plasmids . Twenty - four hours later , whole cell lysates were analyzed by immunoblots with anti - HA , anti - RNF26 or anti - ATG12 as indicated . All experiments were repeated for at least three times with similar results . The bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

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

(F) Effects of ATG12 knockdown on RNF26-mediated destabilization of IRF3. The 293-ATG12-RNAi or control cells (1×106) were transfected with the indicated plasmids. Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA, anti-RNF26 or anti-ATG12 as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


( A , B ) Expression of UVRAG under cell stress . HL60 and HCT116 cells were treated with doxorubicin ( 1 μg / ml ) , cisplatin ( 10 μm ) and UV irradiation ( 5 min after 50 mJ / cm2 ) for the indicated time , and then apoptosis , autophagy and fluorescence intensity of UVRAG was assayed . Scale bar , 10 μm .

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

(A,B) Expression of UVRAG under cell stress. HL60 and HCT116 cells were treated with doxorubicin (1 μg/ml), cisplatin (10 μm) and UV irradiation (5 min after 50 mJ/cm2) for the indicated time, and then apoptosis, autophagy and fluorescence intensity of UVRAG was assayed. Scale bar, 10 μm.


( C ) In parallel , the total protein extracts were used for western blot analysis .

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

(C) In parallel, the total protein extracts were used for western blot analysis.


( D , E ) Pharmacological inhibition ( for example , 3 ‐ methyladenine ( 3 ‐ MA ) , 10 mM ) or genetic deletion ( for example , ATG5 − / − ) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin ( Doxo ) and UV irradiation ( n = 3 , * P0 . 05 ) . AU , arbitrary units ; C ‐ PARP , cleaved poly ‐ ADP ribose polymerase ; LC3 , light chain 3 ; mRNA , messenger RNA ; PI , propidium iodide ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance ‐ associated gene .

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

(D,E) Pharmacological inhibition (for example, 3‐methyladenine (3‐MA), 10 mM) or genetic deletion (for example, ATG5−/−) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin (Doxo) and UV irradiation (n=3, *P0.05). AU, arbitrary units; C‐PARP, cleaved poly‐ADP ribose polymerase; LC3, light chain 3; mRNA, messenger RNA; PI, propidium iodide; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance‐associated gene.


( A - C ) After transfection with UVRAG shRNA or control shRNA for 48 h , HL60 ( A ) , HCT116 ( B ) and HeLa ( C ) cells were treated with doxorubicin ( Doxo , 1 μg / ml ) , cisplatin ( Cis , 10 μm ) and UV irradiation ( 5 min after 50 mJ / cm2 ) for 12 h or starvation ( HBSS ) for 3 h , and then apoptosis and autophagy were assayed ( n = 3 , * P0 . 05 ) .

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

(A-C) After transfection with UVRAG shRNA or control shRNA for 48 h, HL60 (A), HCT116 (B) and HeLa (C) cells were treated with doxorubicin (Doxo, 1 μg/ml), cisplatin (Cis, 10 μm) and UV irradiation (5 min after 50 mJ/cm2) for 12 h or starvation (HBSS) for 3 h, and then apoptosis and autophagy were assayed (n=3, *P0.05).


( D ) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A ( 10 μg / ml ) and E64D ( 10 μg / ml ) after doxorubicin ( 1 μg / ml ) treatment for 12 h . * P0 . 05 compared with control shRNA group .

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

(D) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (10 μg/ml) and E64D (10 μg/ml) after doxorubicin (1 μg/ml) treatment for 12 h. *P0.05 compared with control shRNA group.


( E ) After transfection with UVRAG shRNA or control shRNA for 48 h , ATG5 + / + and ATG5 − / − murine embryonic fibroblasts ( MEFs ) were treated with doxorubicin ( 1 μg / ml ) , with or without Z ‐ VAD ‐ FMK ( 20 μM ) for 12 h , and then apoptosis was assayed ( n = 3 , * P0 . 05 ) .

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

(E) After transfection with UVRAG shRNA or control shRNA for 48 h, ATG5+/+ and ATG5−/− murine embryonic fibroblasts (MEFs) were treated with doxorubicin (1 μg/ml), with or without Z‐VAD‐FMK (20 μM) for 12 h, and then apoptosis was assayed (n=3, *P0.05).


( F ) Nude mice were inoculated with 4 × 106 HL60 tumour cells following transfection of control ( shControl ) or UVRAG ( shUVRAG ) ‐ specific shRNA and treated with doxorubicin ( 10 mg / kg ) beginning at day 7 . Tumours were measured twice weekly , and volumes were calculated for 21 days ( n = 10 , * P0 . 05 ; shControl + Doxo compared with shUVRAG + Doxo ) .

O O O O B-ORGANISM O O O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Nude mice were inoculated with 4 × 106 HL60 tumour cells following transfection of control (shControl) or UVRAG (shUVRAG)‐specific shRNA and treated with doxorubicin (10 mg/kg) beginning at day 7. Tumours were measured twice weekly, and volumes were calculated for 21 days (n=10, *P0.05; shControl+Doxo compared with shUVRAG+Doxo).


( G ) On day 21 in the experiments described in ( F ) , apoptosis and UVRAG expression in tumour samples were assayed by TUNEL or western blotting , respectively ( n = 10 , * P0 . 05 ) . BAX , BCL2 ‐ associated X protein ; HBSS , Hank ' s Buffered Salt Solution ; LC3 , light chain 3 ; shRNA , short ‐ hairpin RNA ; TUNEL , terminal deoxytransferase uridine triphosphate nick end labelling ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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

(G) On day 21 in the experiments described in (F), apoptosis and UVRAG expression in tumour samples were assayed by TUNEL or western blotting, respectively (n=10, *P0.05). BAX, BCL2‐associated X protein; HBSS, Hank's Buffered Salt Solution; LC3, light chain 3; shRNA, short‐hairpin RNA; TUNEL, terminal deoxytransferase uridine triphosphate nick end labelling; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


( A ) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid , and apoptosis was assayed ( n = 3 , * P0 . 05 ) .

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

(A) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid, and apoptosis was assayed (n=3, *P0.05).


( B ) In parallel , the mitochondrial membrane potential , cytochrome c release from mitochondria , and caspase ‐ 3 and ‐ 9 activation in HL60 cells were analysed ( n = 3 , * P0 . 05 ) .

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

(B) In parallel, the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase‐3 and ‐9 activation in HL60 cells were analysed (n=3, *P0.05).


( C ) After transfection with UVRAG vector or control vector for 48 h , cells were transected or treated with BAX ( 1 μg ) , BAD ( 1 μg ) , BID ( 1 μg ) , FAS antibody ( 10 μg / ml ) or TRAIL ( 100 ng / ml ) for 24 h , then cell apoptosis was analysed ( n = 3 , * P0 . 05 ) .

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

(C) After transfection with UVRAG vector or control vector for 48 h, cells were transected or treated with BAX (1 μg), BAD (1 μg), BID (1 μg), FAS antibody (10 μg/ml) or TRAIL (100 ng/ml) for 24 h, then cell apoptosis was analysed (n=3, *P0.05).


( D ) Knockdown of UVRAG in BAX − / − HCT116 cells restored the sensitivity to UV treatment ‐ induced apoptosis ( n = 3 , * P0 . 05 ) . AU , arbitrary units ; BAX , BCL2 ‐ associated X protein ; shRNA , short ‐ hairpin RNA ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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

(D) Knockdown of UVRAG in BAX−/− HCT116 cells restored the sensitivity to UV treatment‐induced apoptosis (n=3, *P0.05). AU, arbitrary units; BAX, BCL2‐associated X protein; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


( A , B ) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co ‐ immunoprecipitation ( RIPA or CHAPS buffer ) or western blotting .

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

(A,B) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co‐immunoprecipitation (RIPA or CHAPS buffer) or western blotting.


( C ) Proteins in the pellet after GST pull ‐ down assays in HCT116 cell lysate were analysed by SDS - PAGE and immunodetected with UVRAG or GST antibodies .

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

(C) Proteins in the pellet after GST pull‐down assays in HCT116 cell lysate were analysed by SDS-PAGE and immunodetected with UVRAG or GST antibodies.


( D ) UVRAG C2 domain deletion terminates BAX binding . HCT116 cells were transfected with Flag ‐ UVRAG or its mutants ( ΔC2 , ΔCCD ) , and then assayed for protein expression levels as indicated by IP or western blot .

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

(D) UVRAG C2 domain deletion terminates BAX binding. HCT116 cells were transfected with Flag‐UVRAG or its mutants (ΔC2, ΔCCD), and then assayed for protein expression levels as indicated by IP or western blot.


( E ) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin ( Doxo , 1 μg / ml ) , UV irradiation ( 5 min after 50 mJ / cm2 ) or transfected with BAX ( 1 μg ) for 24 h . BAX , BCL2 ‐ associated X protein ; CCD , coiled ‐ coil domain ; CHAPS , 3 ‐ [ ( 3 ‐ cholamidopropyl ) ‐ dimethylammonio ] ‐ 1 ‐ propane sulphonate ; GST , glutathione ‐ S ‐ transferase ; IB , immunoblotting ; IgG , immunoglobulin G ; IP , immunoprecipitation ; RIPA , radioimmunoprecipitation assay ; SDS - PAGE , SDS - polyacrylamide gel electrophoresis ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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

(E) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin (Doxo, 1 μg/ml), UV irradiation (5 min after 50 mJ/cm2) or transfected with BAX (1 μg) for 24 h. BAX, BCL2‐associated X protein; CCD, coiled‐coil domain; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate; GST, glutathione‐S‐transferase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; RIPA, radioimmunoprecipitation assay; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


UVRAG inhibits mitochondrial translocation of BAX . After transfection with indicated shRNA or cDNA for 48 h , HL60 cells were treated with doxorubicin ( Doxo ) and UV irradiation for 12 h , and then BAX in cytosol ( Cyt ) and mitochondria ( Mit ) was assayed by western blotting ( A - C ) and activation of BAX ( D - F ) was assayed by western blotting after IP using BAX monoclonal antibody ( clone 6A7 ) * P0 . 05 . AU , arbitrary units ; BAX , BCL2 ‐ associated X protein ; CHAPS , 3 ‐ [ ( 3 ‐ cholamidopropyl ) ‐ dimethylammonio ] ‐ 1 ‐ propane sulphonate buffer ; Cyt c , cytochrome c ; IP , immunoprecipitation ; shRNA , short ‐ hairpin RNA ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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

UVRAG inhibits mitochondrial translocation of BAX. After transfection with indicated shRNA or cDNA for 48 h, HL60 cells were treated with doxorubicin (Doxo) and UV irradiation for 12 h, and then BAX in cytosol (Cyt) and mitochondria (Mit) was assayed by western blotting (A-C) and activation of BAX (D-F) was assayed by western blotting after IP using BAX monoclonal antibody (clone 6A7) *P0.05. AU, arbitrary units; BAX, BCL2‐associated X protein; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate buffer; Cyt c, cytochrome c; IP, immunoprecipitation; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


A . Average ribosome occupancy and their respective codon bias - derived occupancy scores across the CDS of transcript ( in 25 bins ) . Ribosome occupancy for 16 , 423 transcripts and their respective codon bias - derived occupancy were firstly binned into 25 bins and the mean occupancy was calculated for each bin . B . Cumulative distribution plots showing the distributions of correlations between ribosome occupancy and codon bias - derived occupancy . Correlations obtained from the ribosome occupancy and scrambled codon bias - derived occupancy served as the control . A Kolmogorov - Smirnov test was performed between the codon bias - derived occupancy and the control group . Data information : error bars represent the 95 % confidence intervals .

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

A. Average ribosome occupancy and their respective codon bias-derived occupancy scores across the CDS of transcript (in 25 bins). Ribosome occupancy for 16,423 transcripts and their respective codon bias-derived occupancy were firstly binned into 25 bins and the mean occupancy was calculated for each bin. B. Cumulative distribution plots showing the distributions of correlations between ribosome occupancy and codon bias-derived occupancy. Correlations obtained from the ribosome occupancy and scrambled codon bias-derived occupancy served as the control. A Kolmogorov-Smirnov test was performed between the codon bias-derived occupancy and the control group. Data information: error bars represent the 95% confidence intervals.


C . Comparison of average transcript translation efficiencies ( TEs ) across their respective GC3 - content ranges . Number of transcripts within each gene optimality range is indicated above their respective points . Data information : error bars represent the 95 % confidence intervals .

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

C. Comparison of average transcript translation efficiencies (TEs) across their respective GC3-content ranges. Number of transcripts within each gene optimality range is indicated above their respective points. Data information: error bars represent the 95% confidence intervals.


A . HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts ( left ) as well as IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( right ) , post - doxycycline addition . Data information : data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . The half - lives of the respective transcripts are indicated in brackets .

O O B-CELL_LINE B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (left) as well as IL6-OPT, IL6-WT and IL6-DE transcripts (right), post-doxycycline addition. Data information: data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.


B . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT in HEK293T cells transfected with either empty plasmids , plasmids bearing REL - OPT or REL - WT . The immunoblot is representative of 3 independent experiments . ACTB is shown as the loading controls .

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

B. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells transfected with either empty plasmids, plasmids bearing REL-OPT or REL-WT. The immunoblot is representative of 3 independent experiments. ACTB is shown as the loading controls.


C . ELISA of secreted IL6 concentrations of IL6 - OPT , IL6 - WT and IL6 - DE from HEK293T cells transfected with plasmids bearing IL6 - OPT , IL6 - WT and IL6 - DE . Data information : In ( C ) , the data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A one - way ANOVA with Tukey ' s multiple comparisons was performed between samples where , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ELISA of secreted IL6 concentrations of IL6-OPT, IL6-WT and IL6-DE from HEK293T cells transfected with plasmids bearing IL6-OPT, IL6-WT and IL6-DE. Data information: In (C), the data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A one-way ANOVA with Tukey's multiple comparisons was performed between samples where, p<0.01 (**) and p<0.001 (***).


D . Fold changes of REL - OPT and REL - WT transcript levels ( top ) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions ( below ) . Data represents the mean ± SD for 3 biological replicates . Data information data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . The half - lives of the respective transcripts are indicated in brackets .

O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 O O O O O O O O O O O O O O O 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. Fold changes of REL-OPT and REL-WT transcript levels (top) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions (below). Data represents the mean ± SD for 3 biological replicates. Data information data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.


HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts under vehicle ( DMSO ) - and cycloheximide ( CHX ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( B ) , under vehicle ( DMSO ) - and cycloheximide ( CHX ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (B), under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts ( C ) under vehicle ( PBS ) - and anisomycin ( ANI ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (C) under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( D ) , under vehicle ( PBS ) - and anisomycin ( ANI ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (D), under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing the degradation of REL - OPT , REL - OPT ( + 1 Frameshift ) and REL - WT transcripts ( E ) post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of REL-OPT, REL-OPT (+1 Frameshift) and REL-WT transcripts (E) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT , IL6 - DE and IL6 - OPT ( + 1 Frameshift ) transcripts ( F ) post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT, IL6-DE and IL6-OPT (+1 Frameshift) transcripts (F) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


Volcano plots showing the enrichment of RBPs which bind to REL - WT relative to REL - OPT transcripts ( A ) Data information : vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 .

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

Volcano plots showing the enrichment of RBPs which bind to REL-WT relative to REL-OPT transcripts (A) Data information: vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.


Volcano plots showing the enrichment of RBPs which bind to , IL6 - DE relative to IL6 - WT transcripts ( B ) Data information : vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 .

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

Volcano plots showing the enrichment of RBPs which bind to , IL6-DE relative to IL6-WT transcripts (B) Data information: vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.


Volcano plots showing the enrichment of RBPs which bind to IL6 - WT relative to IL6 - OPT transcripts ( C ) . Data information : , vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 .

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

Volcano plots showing the enrichment of RBPs which bind to IL6-WT relative to IL6-OPT transcripts (C). Data information: , vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.


A . Cumulative distribution plots showing the difference in distribution of transcript optimality between upregulated and downregulated transcripts in K562 cells subject to ILF2 CRISPR interference targeting ILF2 . Transcript quantities are indicated in the figure legend . Data information : Wilcoxon signed rank tests were performed on the upregulated and downregulated groups against the control group . P - values are denoted ( right ) .

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

A. Cumulative distribution plots showing the difference in distribution of transcript optimality between upregulated and downregulated transcripts in K562 cells subject to ILF2 CRISPR interference targeting ILF2. Transcript quantities are indicated in the figure legend. Data information: Wilcoxon signed rank tests were performed on the upregulated and downregulated groups against the control group. P-values are denoted (right).


B , C . HEK293 Tet - off experiments showing the degradation of REL - OPT ( B ) and REL - WT ( C ) transcripts with ILF2 and ILF3 siRNA and Control ( CTR ) siRNA treatment , post - doxycycline addition . Data information : data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

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

B, C. HEK293 Tet-off experiments showing the degradation of REL-OPT (B) and REL-WT (C) transcripts with ILF2 and ILF3 siRNA and Control (CTR) siRNA treatment, post-doxycycline addition. Data information: data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**).


D . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT expressed in HEK293T cells under ILF2 and ILF3 siRNA treatment . The immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls .

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

D. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT expressed in HEK293T cells under ILF2 and ILF3 siRNA treatment. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.


E . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT in HEK293T cells co - expressed with two different isoforms of ILF2 . The immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls .

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

E. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells co-expressed with two different isoforms of ILF2. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.


A . KD efficiency of three different Syt11 shRNAs in DRG neurons . Cultured DRG neurons were infected with control or shRNA - expressing lentiviruses at DIV 1 , and Western blotting for Syt11 , Syt1 , Syt4 , synaptobrevin 2 ( Syb2 ) , SNAP25 , complexin 1 / 2 ( Cpx1 , Cpx2 ) , clathrin , adaptor protein 2 ( AP - 2 ) , and β - actin were performed at DIV 6 - 7 . N = 4 independent experiments .

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

A. KD efficiency of three different Syt11 shRNAs in DRG neurons. Cultured DRG neurons were infected with control or shRNA-expressing lentiviruses at DIV 1, and Western blotting for Syt11, Syt1, Syt4, synaptobrevin 2 (Syb2), SNAP25, complexin 1/2 (Cpx1, Cpx2), clathrin, adaptor protein 2 (AP-2), and β-actin were performed at DIV 6-7. N = 4 independent experiments.


B , C . Representative Cm traces induced by 200 - ms pulse depolarization ( arrows ) ib DRG neurons . DRG neurons were transfected with plasmids expressing shSyt11 - 2 ( Syt11 KD , KD ) or scrambled shRNA ( Sc ) and Cm recording was performed 5 days after transfection . Endo - 5s represents the Cm decay 5 s after stimulation . Insets show Ca2 + currents recorded in the same neurons .

O O O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O B-TISSUE B-CELL_TYPE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-CELL_TYPE O

B, C. Representative Cm traces induced by 200-ms pulse depolarization (arrows) ib DRG neurons. DRG neurons were transfected with plasmids expressing shSyt11-2 (Syt11 KD, KD) or scrambled shRNA (Sc) and Cm recording was performed 5 days after transfection. Endo-5s represents the Cm decay 5 s after stimulation. Insets show Ca2+ currents recorded in the same neurons.


D . Representative Cm overshoot recorded from an Syt11 KD neuron . About 45 % of the KD neurons showed excessive membrane retrieval after a 200 - ms depolarizing pulse .

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

D. Representative Cm overshoot recorded from an Syt11 KD neuron. About 45% of the KD neurons showed excessive membrane retrieval after a 200-ms depolarizing pulse.


E . Averaged Cm traces recorded from Control ( Ctrl ) , Sc , KD ( from all three shRNAs , n = 39 for shSyt11 - 2 , n = 7 for shSyt11 - 1 , n = 14 for shSyt11 - 3 ) , and rescued ( with the shSyt11 - 2 - resistant form of Syt11 ) DRG neurons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE B-CELL_TYPE O

E. Averaged Cm traces recorded from Control (Ctrl), Sc, KD (from all three shRNAs, n = 39 for shSyt11-2, n = 7 for shSyt11-1, n = 14 for shSyt11-3), and rescued (with the shSyt11-2-resistant form of Syt11) DRG neurons.


F - H . Statistics of Cm jumps , Endo - 5s , and Ca2 + current recorded from DRGneurons as in ( B - E ) . Data were collected from 4 ( Sc , Rescue ) , 8 ( Ctrl ) , and 16 ( KD ) independent experiments .

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

F-H. Statistics of Cm jumps, Endo-5s, and Ca2+ current recorded from DRGneurons as in (B-E). Data were collected from 4 (Sc, Rescue), 8 (Ctrl), and 16 (KD) independent experiments.


I . Normalized Cm changes induced by two pulses of 200 - ms depolarization ( arrows ) at a 1 - s interval . Data were normalized to the capacitance jump induced by the first depolarization .

O O O O O O O O O O O O O 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. Normalized Cm changes induced by two pulses of 200-ms depolarization (arrows) at a 1-s interval. Data were normalized to the capacitance jump induced by the first depolarization.


J . Paired - pulse ratios of ΔCm with different interstimulus intervals as in ( I ) . DRG neurons were stimulated with two 200 - ms pulses at different intervals as indicated , and the paired - pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one .

O O O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. Paired-pulse ratios of ΔCm with different interstimulus intervals as in (I). DRG neurons were stimulated with two 200-ms pulses at different intervals as indicated, and the paired-pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one.


A , B . FM 1 - 43 uptake into DRG neurons stimulated by 100 mM K + for 30 s . The right - shifted cumulative frequency in ( B ) indicates the increased uptake level in KD neurons . Scale bars , 5 μm .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-TISSUE B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O

A, B. FM 1-43 uptake into DRG neurons stimulated by 100 mM K+ for 30 s. The right-shifted cumulative frequency in (B) indicates the increased uptake level in KD neurons. Scale bars, 5 μm.


C . Quantification of KCl - evoked FM 1 - 43 uptake with different stimulation times .

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

C. Quantification of KCl-evoked FM 1-43 uptake with different stimulation times.


D . Representative micrographs showing the increased constitutive FM 1 - 43 uptake ( for 5 min ) in KD neurons . Scale bars , 5 μm .

O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O

D. Representative micrographs showing the increased constitutive FM 1-43 uptake (for 5 min) in KD neurons. Scale bars, 5 μm.


E . Quantification of constitutive FM 1 - 43 uptake with different loading times .

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

E. Quantification of constitutive FM 1-43 uptake with different loading times.


F . Basal [ Ca2 + ] i in DRG neurons measured with Fura - 2 .

F. Basal [Ca2+]i in DRG neurons measured with Fura-2.


G . FM1 - 43 uptake by Syt11 KD ( RFP - positive ) and control ( RFP - negative ) hippocampal neurons stimulated by 100 mM K + for 2 min . Quantitative data are shown in the lower - right panel . Scale bars , 20 μm .

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

G. FM1-43 uptake by Syt11 KD (RFP-positive) and control (RFP-negative) hippocampal neurons stimulated by 100 mM K+ for 2 min. Quantitative data are shown in the lower-right panel. Scale bars, 20 μm.


H . Representative images showing the preloaded FM4 - 64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz . Scale bars , 5 μm .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O

H. Representative images showing the preloaded FM4-64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz. Scale bars, 5 μm.


I . Time - course of FM4 - 64 unloading from control , Syt11 KD , and rescued nerve terminals within the same field of view ( 6 coverslips from at least 3 biological repeats each ) .

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

I. Time-course of FM4-64 unloading from control, Syt11 KD, and rescued nerve terminals within the same field of view (6 coverslips from at least 3 biological repeats each).


A . Averaged ΔCm traces induced by 200 - ms depolarization in the presence and absence of dynasore . Endocytic inhibition by dynasore ( Dyna ) was estimated by comparison of Cm traces recorded during 1 - 3 min ( KD ) and 5 - 10 min ( KD + Dyna and Ctrl + Dyna ) after whole - cell dialysis .

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

A. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynasore. Endocytic inhibition by dynasore (Dyna) was estimated by comparison of Cm traces recorded during 1-3 min (KD) and 5-10 min (KD + Dyna and Ctrl + Dyna) after whole-cell dialysis.


B , C . Endo - 5s and Cm jumps recorded as in ( A ) .

B, C. Endo-5s and Cm jumps recorded as in (A).


D . Averaged ΔCm traces induced by 200 - ms depolarization in the presence and absence of dynole - 34 - 2TM ( Dynole ) . Cm traces and endocytic rate were measured as in ( A ) except that 10 μM dynole - 34 - 2TM with 0 . 1 % DMSO was dialyzed into patched cells .

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

D. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynole-34-2TM (Dynole). Cm traces and endocytic rate were measured as in (A) except that 10 μM dynole-34-2TM with 0.1% DMSO was dialyzed into patched cells.


E , F . Endo - 5s and Cm jumps as in ( D ) .

E, F. Endo-5s and Cm jumps as in (D).


G , H . Averaged ∆ Cm traces and Endo - 5s recorded from KD neurons under control conditions ( 0 . 1 % DMSO ) .

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

G, H. Averaged ∆Cm traces and Endo-5s recorded from KD neurons under control conditions (0.1 % DMSO).


I . KCl - evoked FM 1 - 43 uptake in DRG neurons after 10 - min pre - incubation with 0 . 1 % DMSO ( control ) or with 100 μM dynasore .

O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O

I. KCl-evoked FM 1-43 uptake in DRG neurons after 10-min pre-incubation with 0.1% DMSO (control) or with 100 μM dynasore.


A . DRG neurons expressing Myc - Syt11 were immunostained for Myc - Syt11 , dynamin ( Dyn ) , AP - 2 , clathrin ( CL ) , TGN46 , synaptobrevin 2 ( Syb2 ) , and LAMP1 . For dynamin , the box along the plasma membrane was straightened , enlarged , and is shown in the upper right panels with arrows indicating co - localized puncta . An enlarged inset of the inside of the cell ( box ) is shown in the lower panel . For localization of the transferrin receptor , EGFP - transferrin receptor ( TfR ) was expressed in DRG neurons . Scale bars , 10 μm .

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

A. DRG neurons expressing Myc-Syt11 were immunostained for Myc-Syt11, dynamin (Dyn), AP-2, clathrin (CL), TGN46, synaptobrevin 2 (Syb2), and LAMP1. For dynamin, the box along the plasma membrane was straightened, enlarged, and is shown in the upper right panels with arrows indicating co-localized puncta. An enlarged inset of the inside of the cell (box) is shown in the lower panel. For localization of the transferrin receptor, EGFP-transferrin receptor (TfR) was expressed in DRG neurons. Scale bars, 10 μm.


B . Analysis of co - localization of Myc - Syt11 with dynamin , AP - 2 , clathrin , TGN46 , synaptobrevin 2 , transferrin receptor , and LAMP1 . N = 3 independent experiments .

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

B. Analysis of co-localization of Myc-Syt11 with dynamin, AP-2, clathrin, TGN46, synaptobrevin 2, transferrin receptor, and LAMP1. N = 3 independent experiments.


A . Transferrin ( Tf , red ) uptake in Syt11 KD ( GFP - positive ) and control neurons . Quantitative data are on the right . Scale bars , 20 μm .

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

A. Transferrin (Tf, red) uptake in Syt11 KD (GFP-positive) and control neurons. Quantitative data are on the right. Scale bars, 20 μm.


B . Tf uptake in Syt11 over - expressing and control neurons . Quantitative data are on the right . Scale bars , 20 μm .

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

B. Tf uptake in Syt11 over-expressing and control neurons. Quantitative data are on the right. Scale bars, 20 μm.


C . Normalized ∆ Cm induced by 200 - ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double - exponential decay function ( solid black and red , fitted curves ) .

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

C. Normalized ∆Cm induced by 200-ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double-exponential decay function (solid black and red, fitted curves).


D . Fast and slow time - constants of endocytosis in Syt11 KD neurons recorded as in ( C ) .

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

D. Fast and slow time-constants of endocytosis in Syt11 KD neurons recorded as in (C).


E . KCl - evoked FM 1 - 43 uptake in DRG neurons after 1 - h pre - incubation with 0 . 1 % DMSO or 100 μM MDC in the bath solution .

O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O

E. KCl-evoked FM 1-43 uptake in DRG neurons after 1-h pre-incubation with 0.1% DMSO or 100 μM MDC in the bath solution.


A , B . Cm traces with bulk endocytosis ( arrowheads ) in control ( A ) and KD ( B ) neurons induced by 200 - ms depolarization . Expanded bulk events are shown on the right .

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

A, B. Cm traces with bulk endocytosis (arrowheads) in control (A) and KD (B) neurons induced by 200-ms depolarization. Expanded bulk events are shown on the right.


C , D . Frequency distribution and bulk endocytic events / cell within 16 s after stimulation .

C, D. Frequency distribution and bulk endocytic events/cell within 16 s after stimulation.


E , F . Size and duration of Cm decay during bulk endocytosis .

E, F. Size and duration of Cm decay during bulk endocytosis.


G , H . Representative z - projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons . Neurons were loaded with 50 μM tetramethylrhodamine - dextran ( 40 kD ) by 2 - min exposure to 100 mM KCl . Scale bars , 10 μm .

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

G, H. Representative z-projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons. Neurons were loaded with 50 μM tetramethylrhodamine-dextran (40 kD) by 2-min exposure to 100 mM KCl. Scale bars, 10 μm.


A . Representative electron micrographs showing a small / clear vesicle ( SV , arrow ) , dense - core vesicles ( DCVs , arrowheads ) and a clathrin - coated pit ( hatched arrow ) in DRG neurons .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-TISSUE B-CELL_TYPE O

A. Representative electron micrographs showing a small/clear vesicle (SV, arrow), dense-core vesicles (DCVs, arrowheads) and a clathrin-coated pit (hatched arrow) in DRG neurons.


B , C . Diameter distributions of SVs and DCVs ( statistics from 6 control cells and 9 KD cells ) in DRG neurons . The red and green lines are curves fitted to a Gaussian function .

O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O

B, C. Diameter distributions of SVs and DCVs (statistics from 6 control cells and 9 KD cells) in DRG neurons. The red and green lines are curves fitted to a Gaussian function.


D , E . Diameters of SVs and DCVs in Syt11 KD and control neurons with ( 100K , statistics from 6 control cells and 9 KD cells ) or without 100 mM K + stimulation ( SE , statistics from 6 cells for each group ) .

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

D, E. Diameters of SVs and DCVs in Syt11 KD and control neurons with (100K, statistics from 6 control cells and 9 KD cells) or without 100 mM K+ stimulation (SE, statistics from 6 cells for each group).


A . Representative electron micrographs of HRP uptake from standard ( SE ) or 100 mM K + ( 100K ) bath solution . Scale bar , 2 μm .

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

A. Representative electron micrographs of HRP uptake from standard (SE) or 100 mM K+ (100K) bath solution. Scale bar, 2 μm.


B - D . HRP - labeled small vesicles ( filled arrows ) and endosomes ( arrowheads ) in DRG neurons . Quantitative data are shown in ( C ) and ( D ) . Scale bar , 200 nm .

O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O

B-D. HRP-labeled small vesicles (filled arrows) and endosomes (arrowheads) in DRG neurons. Quantitative data are shown in (C) and (D). Scale bar, 200 nm.


E . Representative docked vesicles ( arrows ) in a DRG neuron . Quantitative data are on the right . Scale bar , 200 nm .

O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O

E. Representative docked vesicles (arrows) in a DRG neuron. Quantitative data are on the right. Scale bar, 200 nm.


F - H . Clathrin - coated pits ( CCPs , hatched arrows ) and bulk - endocytosis - related structures of invaginating membrane ( open arrowhead ) in DRGneurons . Quantitative data are shown in ( G ) and ( H ) . Scale bars , 200 nm .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O

F-H. Clathrin-coated pits (CCPs, hatched arrows) and bulk-endocytosis-related structures of invaginating membrane (open arrowhead) in DRGneurons. Quantitative data are shown in (G) and (H). Scale bars, 200 nm.