Figure 1Down regulation of SREBP target genes in Cideb - deficient liver . Microarray heatmap of fatty acids / cholesterol synthesis genes that were down - regulated in Cideb - / - mice and Scap liver - specific knockout mice , comparing to the respective WT controls ( WT VS Cideb - / - , WT VS L - Scap - / - , n = 2 per group ) . The expression level of each gene was linearly normalized to range from - 1 to 1 before plotting . Decreased SREBP processing and maturation in Cideb deficient liver . Immune blotting ( IB ) of the full length , precursor SREBP - 1 / 2 ( SREBP FL ) and the cleaved , active SREBP - 1 / 2 ( SREBP N ) from WT and Cideb - / - mice liver under ad - lib ( ND ) , fasting 12h ( F ) , and refeeding ( R ) conditions . Mice were refed with high - carbohydrate low - fat diet for 12h after 12h fasting . Defective SREBP transcriptional response to refeeding in Cideb - deficient liver . Transcript levels of lipogenic and cholesterol metabolism genes determined by qPCR from WT and Cideb - / - liver under ad - lib ( ND ) , fasting 12h ( F ) , and refeeding ( R ) conditions . Mice were refed with high - carbohydrate low - fat diet for 12h after 12h fasting ( n = 3 per group ) . Re - introduction of Cideb rescues SREBP processing in Cideb - / - liver . WT and Cideb - / - mice were injected with AAV - GFP or AAV - Cideb , and sacrificed for IB with the indicated antibodies 20 days after the injection . Re - introduction of Cideb rescues the expression of SREBP target genes in Cideb - / - liver . Transcript levels of lipogenic genes determined by qPCR from WT and Cideb - / - liver . WT and Cideb - / - mice were injected with AAV - GFP or AAV - Cideb , and sacrificed for qPCR 20 days after the injection ( n = 3 per group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Down regulation of SREBP target genes in Cideb-deficient liver. Microarray heatmap of fatty acids/cholesterol synthesis genes that were down-regulated in Cideb-/- mice and Scap liver-specific knockout mice, comparing to the respective WT controls (WT VS Cideb-/-, WT VS L-Scap-/-, n = 2 per group). The expression level of each gene was linearly normalized to range from -1 to 1 before plotting.Decreased SREBP processing and maturation in Cideb deficient liver. Immune blotting (IB) of the full length, precursor SREBP-1/2 (SREBP FL) and the cleaved, active SREBP-1/2 (SREBP N) from WT and Cideb-/- mice liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting.Defective SREBP transcriptional response to refeeding in Cideb-deficient liver. Transcript levels of lipogenic and cholesterol metabolism genes determined by qPCR from WT and Cideb-/- liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting (n = 3 per group).Re-introduction of Cideb rescues SREBP processing in Cideb-/- liver. WT and Cideb-/- mice were injected with AAV-GFP or AAV-Cideb, and sacrificed for IB with the indicated antibodies 20 days after the injection.Re-introduction of Cideb rescues the expression of SREBP target genes in Cideb-/- liver. Transcript levels of lipogenic genes determined by qPCR from WT and Cideb-/- liver. WT and Cideb-/- mice were injected with AAV-GFP or AAV-Cideb, and sacrificed for qPCR 20 days after the injection (n = 3 per group).


Figure 2Liver morphology of WT and Cideb - / - liver under chow diet and HFLF diet . H / E staining , oil red - O staining , immunohistochemistry and ultra - structure ( Electron Microscope ) were performed . Scale bar represented 50 µm in the upper three rows of images , and 2 µm in the bottom row of images . LD : lipid droplet , ER : endoplasmic reticulum , M : mitochondrial , N : nucleus . Total liver TAG levels ( B ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . Liver cholesterol ester levels ( C ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . D Loss of Cideb inhibits diet - induced SREBP activation . IB of hepatic SREBP processing and lipogenic enzyme levels ( Fasn , Scd1 ) of WT and Cideb - / - mice under chow diet and HFLF diet . E Serum AST and ALT levels of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . F Cideb deficiency alleviates the whole body inflammation response . Serum TNFα , MCP1 and IL6 levels of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . Serum fed glucose levels ( G ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . Serum fed insulin levels ( H ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . I Glucose tolerance test ( GTT ) of WT and Cideb - / - mice under HFLF diet ( n = 5 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . J Insulin tolerance test ( ITT ) of WT and Cideb - / - mice under HFLF diet ( n = 5 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Liver morphology of WT and Cideb-/- liver under chow diet and HFLF diet. H/E staining, oil red-O staining, immunohistochemistry and ultra-structure (Electron Microscope) were performed. Scale bar represented 50 µm in the upper three rows of images, and 2 µm in the bottom row of images. LD: lipid droplet, ER: endoplasmic reticulum, M: mitochondrial, N: nucleus.Total liver TAG levels (B) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.Liver cholesterol ester levels (C) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.D Loss of Cideb inhibits diet-induced SREBP activation. IB of hepatic SREBP processing and lipogenic enzyme levels (Fasn, Scd1) of WT and Cideb-/- mice under chow diet and HFLF diet.E Serum AST and ALT levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.F Cideb deficiency alleviates the whole body inflammation response. Serum TNFα, MCP1 and IL6 levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.Serum fed glucose levels (G) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.Serum fed insulin levels (H) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.I Glucose tolerance test (GTT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.J Insulin tolerance test (ITT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Figure 3Decreased SREBP - 1 / 2 and SCAP levels in Golgi apparatus in Cideb - / - mice . IB of organelle fractions of the indicated proteins and quantification of IB data from 3 independent experiments . Data information : Data represent the Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . Cideb deficiency impairs the packaging of SCAP / SREBP into COPII vesicles . Protein present in the vesicle or microsome fractions obtained from WT or Cideb - / - liver were detected by IB . Quantification of IB data from 3 independent experiments . Data information : Data represent the Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells . Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A - Flag - BirA * . Cells were cultured in sterol - depleted medium and treated with 80 µM biotin , prior to pulldown with streptavidin - conjugated beads . The biotinylated proteins were detected by IB with the indicated antibodies following SDS - PAGE . Cleaved SREBP bypasses Cideb deficiency . WT and Cideb - / - mice were injected with adenovirus expressing GFP , SREBP - 1c full length ( FL ) or SREBP - 1c active form ( N ) . Mice were sacrificed 7 days after injection , and liver TAG levels and hepatic expression SREBP - 1 target genes in the indicated groups were determined ( n = 5 per group ) . Data information : Data represent the Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Decreased SREBP-1/2 and SCAP levels in Golgi apparatus in Cideb-/- mice. IB of organelle fractions of the indicated proteins and quantification of IB data from 3 independent experiments. Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.Cideb deficiency impairs the packaging of SCAP/SREBP into COPII vesicles. Protein present in the vesicle or microsome fractions obtained from WT or Cideb-/- liver were detected by IB. Quantification of IB data from 3 independent experiments. Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells. Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A-Flag-BirA*. Cells were cultured in sterol-depleted medium and treated with 80 µM biotin, prior to pulldown with streptavidin-conjugated beads. The biotinylated proteins were detected by IB with the indicated antibodies following SDS-PAGE.Cleaved SREBP bypasses Cideb deficiency. WT and Cideb-/- mice were injected with adenovirus expressing GFP, SREBP-1c full length (FL) or SREBP-1c active form (N). Mice were sacrificed 7 days after injection, and liver TAG levels and hepatic expression SREBP-1 target genes in the indicated groups were determined (n=5 per group). Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Figure 4Cideb co - precipitates with SCAP . Flag - tagged Cideb was co - expressed with different HA - tagged proteins ( PLIN2 , Insig , SCAP , SREBP - 1 or SREBP - 2 ) in 293T cells and immuno - precipitated with an anti - Flag antibody , and the co - immunoprecipitated protein were detected by an anti - HA antibody following SDS - PAGE . Interaction between the endogenous SCAP and Cideb . Endogenous SCAP was immuno - precipitated from primary hepatocytes under normal medium , 25 - HC depletion or addition conditions . And the presence of Cideb in the immune - complex was detected by IB . SREBP - 1 was detected as the positive control . Grp94 was detected as the negative control . Cideb interacts with the 416 - 800 portion of SCAP . Flag - tagged Cideb was co - expressed with the indicated HA - tagged SCAP truncation mutants in 293T cells and immuno - precipitated with an anti - Flag antibody . Levels of the co - immunoprecipitated protein were detected by an anti - HA antibody following SDS - PAGE . Sterol - dependent interaction switch of SCAP . SCAP interacts with Insig in the presence of sterol , while switches to Cideb binding upon sterol deprivation . Flag - tagged Insig - 1 or Cideb was co - expressed with HA - tagged SREBP - 2 and SCAP or SCAP - Y298C in CHO - K1 cells in sterol - depleted medium . When indicated , 10 µg / ml 25 - HC were supplemented before cells were immuno - precipitated with an anti - Flag antibody , and the levels of co - immunoprecipitated proteins were detected by IB following SDS - PAGE . The dotted blue vertical line was drawn to separate the result between SCAP - WT and SCAP - Y298C . Cideb relieves sterol - dependent suppression of SREBP processing . Hepatocytes infected with adenovirus expressing GFP or Flag - Cideb were cultured in sterol deprived condition . When indicated , cells were treated with different doses of 25 - HC before subjected to IB with the indicated antibodies following SDS - PAGE . Quantification of IB data from 3 independent experiments . Data represents Mean ± SEM . Cideb promotes Golgi localization of SCAP . HepG2 cells transfected with GFP - SCAP alone or with HA - Cideb were cultured in sterol deprived conditions or treated with 1 µg / ml 25 - HC . Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA . Scale bars represent 10 µm ( Merge ) , and 2 µm ( Inlay ) . Bottom : quantification of the percentage of SCAP localized in Golgi - positive region per cell ( left ) and quantification of the percentage of cells containing Golgi - localized GFP - SCAP ( right ) under indicated conditions , from 3 independent experiments ; Data represent Mean ± SEM ; * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Cideb co-precipitates with SCAP. Flag-tagged Cideb was co-expressed with different HA-tagged proteins (PLIN2, Insig, SCAP, SREBP-1 or SREBP-2) in 293T cells and immuno-precipitated with an anti-Flag antibody, and the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.Interaction between the endogenous SCAP and Cideb. Endogenous SCAP was immuno-precipitated from primary hepatocytes under normal medium, 25-HC depletion or addition conditions. And the presence of Cideb in the immune-complex was detected by IB. SREBP-1 was detected as the positive control. Grp94 was detected as the negative control.Cideb interacts with the 416-800 portion of SCAP. Flag-tagged Cideb was co-expressed with the indicated HA-tagged SCAP truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.Sterol-dependent interaction switch of SCAP. SCAP interacts with Insig in the presence of sterol, while switches to Cideb binding upon sterol deprivation. Flag-tagged Insig-1 or Cideb was co-expressed with HA-tagged SREBP-2 and SCAP or SCAP-Y298C in CHO-K1 cells in sterol-depleted medium. When indicated, 10 µg/ml 25-HC were supplemented before cells were immuno-precipitated with an anti-Flag antibody, and the levels of co-immunoprecipitated proteins were detected by IB following SDS-PAGE. The dotted blue vertical line was drawn to separate the result between SCAP-WT and SCAP-Y298C.Cideb relieves sterol-dependent suppression of SREBP processing. Hepatocytes infected with adenovirus expressing GFP or Flag-Cideb were cultured in sterol deprived condition. When indicated, cells were treated with different doses of 25-HC before subjected to IB with the indicated antibodies following SDS-PAGE. Quantification of IB data from 3 independent experiments. Data represents Mean ± SEM.Cideb promotes Golgi localization of SCAP. HepG2 cells transfected with GFP-SCAP alone or with HA-Cideb were cultured in sterol deprived conditions or treated with 1 µg/ml 25-HC. Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA. Scale bars represent 10 µm (Merge), and 2 µm (Inlay). Bottom: quantification of the percentage of SCAP localized in Golgi-positive region per cell (left) and quantification of the percentage of cells containing Golgi-localized GFP-SCAP (right) under indicated conditions, from 3 independent experiments; Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Figure 5Cideb interacts with Sec12 . 293T cells transfected with Cideb and GFP - tagged Sec12 , Sec23 , Sec16S , or Sec16L were subjected to immuno - precipitation with an anti - Cideb antibody , and levels of the co - immunoprecipitated protein were detected by an anti - GFP antibody . Co - localization of SCAP and Cideb at the ER exit sites . HepG2 cells transfected with GFP - SCAP and HA - Cideb were arrested with 10 µM BFA and 1 µM Nocodazole for 2 hours , then fixed with methanol and stained with antibodies against Sec12 or HA . 3D reconstruction was obtained by processing 3D - original image with Imaris . Scale bars represent 10 µm ( Merge ) , and 1 µm ( Inlay and 3D reconstruction ) . Localization of Cideb at the ER exit sites . AML12 cells knocked - in with a GFP tag at the C - terminus of Cideb genome was treated with 10 µM BFA for 1 hour , then fixed with methanol and stained with antibodies against Sec12 . Scale bars represent 10 µm ( Merge ) , and 1 µm ( Inlay ) . Sec12 interacts with the 118 - 165 portion of Cideb . HA - tagged Sec12 was co - expressed with the indicated Flag - tagged Cideb truncation mutants in 293T cells and immuno - precipitated with an anti - Flag antibody . Levels of the co - immunoprecipitated protein were detected by an anti - HA antibody following SDS - PAGE . Lysine 128 of Cideb is required for the interaction with Sec12 . 293T cells transfected with HA - Sec12 and the indicated Flag - tagged Cideb constructs were subjected to immunoprecipitation with an anti - Flag antibody , and levels of the co - immunoprecipitated HA - Sec12 were detected with an anti - HA antibody . Recombinant Cideb interacts with Sec12 . GST - Sec12 and MBP - tagged Cideb or Cideb - K128A were purified from E . coli and subjected to in vitro binding . Cideb - K128A fails to promote SREBP processing . Cideb - / - mice were injected with adenovirus expressing GFP , Flag - Cideb , or Flag - Cideb - K128A , and sacrificed 7 days after injection . Liver lysates were subjected to IB with the indicated antibodies following SDS - PAGE and quantification of IB data from 3 independent experiments . Data represent Mean ± SEM ; * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . Cideb , but not Cideb - K128A , promotes SCAP / SREBP incorporation into COPII vesicles . Liver microsomes were isolated from mice expressing the indicated proteins and subjected to in vitro budding assay . Resulting vesicles and the donor microsomes were subject to IB with the indicated antibodies following SDS - PAGE .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Cideb interacts with Sec12. 293T cells transfected with Cideb and GFP-tagged Sec12, Sec23, Sec16S, or Sec16L were subjected to immuno-precipitation with an anti-Cideb antibody, and levels of the co-immunoprecipitated protein were detected by an anti-GFP antibody.Co-localization of SCAP and Cideb at the ER exit sites. HepG2 cells transfected with GFP-SCAP and HA-Cideb were arrested with 10 µM BFA and 1 µM Nocodazole for 2 hours, then fixed with methanol and stained with antibodies against Sec12 or HA. 3D reconstruction was obtained by processing 3D-original image with Imaris. Scale bars represent 10 µm (Merge), and 1 µm (Inlay and 3D reconstruction).Localization of Cideb at the ER exit sites. AML12 cells knocked-in with a GFP tag at the C-terminus of Cideb genome was treated with 10 µM BFA for 1 hour, then fixed with methanol and stained with antibodies against Sec12. Scale bars represent 10 µm (Merge), and 1 µm (Inlay).Sec12 interacts with the 118-165 portion of Cideb. HA-tagged Sec12 was co-expressed with the indicated Flag-tagged Cideb truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.Lysine 128 of Cideb is required for the interaction with Sec12. 293T cells transfected with HA-Sec12 and the indicated Flag-tagged Cideb constructs were subjected to immunoprecipitation with an anti-Flag antibody, and levels of the co-immunoprecipitated HA-Sec12 were detected with an anti-HA antibody.Recombinant Cideb interacts with Sec12. GST-Sec12 and MBP-tagged Cideb or Cideb-K128A were purified from E.coli and subjected to in vitro binding.Cideb-K128A fails to promote SREBP processing. Cideb-/- mice were injected with adenovirus expressing GFP, Flag-Cideb, or Flag-Cideb-K128A, and sacrificed 7 days after injection. Liver lysates were subjected to IB with the indicated antibodies following SDS-PAGE and quantification of IB data from 3 independent experiments. Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.Cideb, but not Cideb-K128A, promotes SCAP/SREBP incorporation into COPII vesicles. Liver microsomes were isolated from mice expressing the indicated proteins and subjected to in vitro budding assay. Resulting vesicles and the donor microsomes were subject to IB with the indicated antibodies following SDS-PAGE.


Figure 6Cideb enriches SCAP to ERESs via interacting with Sec12 . CHO - K1 cells transfected with GFP - SCAP , HA - Cideb or HA - Cideb - K128A were arrested with 5 µM Nocodazole for 30 min , then fixed with methanol and stained with antibodies against Sec12 and HA . Scale bars represent 10 µm ( left ) , and 2 . 5 µm ( Inlay ) . Number of SCAP - positive ER exit sites per cell . Normalized fluorescent intensity of SCAP at each individual ER exit site . Total fluorescent intensity of SCAP at ER exit sites per cell . Number of Sec12 - positive ER exit sites per cell . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . In panel C - F , quantification of images from 3 independent experiments .

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Figure 6Cideb enriches SCAP to ERESs via interacting with Sec12. CHO-K1 cells transfected with GFP-SCAP, HA-Cideb or HA-Cideb-K128A were arrested with 5 µM Nocodazole for 30 min, then fixed with methanol and stained with antibodies against Sec12 and HA. Scale bars represent 10 µm (left), and 2.5 µm (Inlay).Number of SCAP-positive ER exit sites per cell.   Normalized fluorescent intensity of SCAP at each individual ER exit site.   Total fluorescent intensity of SCAP at ER exit sites per cell.   Number of Sec12-positive ER exit sites per   cell. Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. In panel C-F, quantification of images from 3 independent experiments.


Figure 1A . Confocal microscopic image showing HeLa cells immunostained for Nup358 ( green , upper panel ) or Nup214 ( green , lower panel ) and RanGAP1 ( red ) using specific antibodies . DNA was stained with Hoechst 33342 ( blue ) . Scale bars , 10 μm . B . COS - 7 cells transfected with GFP - Nup358 and one of the cells was subjected for live imaging using confocal microscopy and the frames at indicated time points have been provided . Arrows denoted by different colors indicate individual AL structures . Note that one of the AL structures ( marked by yellow arrow ) originated from the NE ( dotted line ) and fused with another AL structure ( marked by red arrow ) , which later fused with a different AL structure ( marked by blue arrow ) . N , nucleus ; C , cytoplasm . Scale bar , 5 μm . C . Maximum intensity projection confocal image of a sodium arsenite treated HeLa cell immunostained for endogenous Nup358 ( green ) , P bodies ( red , Dcp1a as a marker ) and SGs ( blue , eIF3η as a marker ) using specific antibodies . Scale bar , 10 μm . The histograms represent fluorescence intensity profile along the dotted arrows . Adjacent graph represents quantitative data showing percentage of P bodies ( top ) or SGs ( bottom ) associated with nuclear envelope ( NE ) , Nup358 positive AL , with each other or unassociated with any of the other mentioned structures ( others ) . Data are presented as mean SD ( n = 3 ) . D . HeLa cells were transfected with Nup358 siRNA ( siNup358 ) or Nup214 siRNA ( siNup214 ) . Cells were fixed and stained for endogenous Nup358 ( green , upper panel ) or Nup214 ( green , lower panel ) and endogenous P body marker ( Dcp1a , red ) . Graph represents the quantitative data as mean ± SD . The data was obtained from three independent experiments , and in each experiment , 100 cells were counted from different fields for the presence of intact microscopically distinct P bodies and expressed as percentage . D . HeLa cells were transfected with Nup358 siRNA ( siNup358 ) or Nup214 siRNA ( siNup214 ) . Western blot ( WB ) indicates the extent of Nup358 depletion and the level of Dcp1a .

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Figure 1A. Confocal microscopic image showing HeLa cells immunostained for Nup358 (green, upper panel) or Nup214 (green, lower panel) and RanGAP1 (red) using specific antibodies. DNA was stained with Hoechst 33342 (blue). Scale bars, 10 μm.B. COS-7 cells transfected with GFP-Nup358 and one of the cells was subjected for live imaging using confocal microscopy and the frames at indicated time points have been provided. Arrows denoted by different colors indicate individual AL structures. Note that one of the AL structures (marked by yellow arrow) originated from the NE (dotted line) and fused with another AL structure (marked by red arrow), which later fused with a different AL structure (marked by blue arrow). N, nucleus; C, cytoplasm. Scale bar, 5 μm.C. Maximum intensity projection confocal image of a sodium arsenite treated HeLa cell immunostained for endogenous Nup358 (green), P bodies (red, Dcp1a as a marker) and SGs (blue, eIF3η as a marker) using specific antibodies. Scale bar, 10 μm. The histograms represent fluorescence intensity profile along the dotted arrows. Adjacent graph represents quantitative data showing percentage of P bodies (top) or SGs (bottom) associated with nuclear envelope (NE), Nup358 positive AL, with each other or unassociated with any of the other mentioned structures (others). Data are presented as mean SD (n = 3).D. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Cells were fixed and stained for endogenous Nup358 (green, upper panel) or Nup214 (green, lower panel) and endogenous P body marker (Dcp1a, red). Graph represents the quantitative data as mean ± SD. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact microscopically distinct P bodies and expressed as percentage.D. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Western blot (WB) indicates the extent of Nup358 depletion and the level of Dcp1a.


Figure 2A . Western analysis of HeLa cells , treated with control ( siControl ) , Dicer ( siDicer ) , Nup214 ( siNup214 ) or Nup358 ( siNup358 ) siRNA , for assessing the extent of protein depletion using indicated antibodies . Vinculin was used as loading control . B . HeLa cells were initially transfected with indicated siRNAs , followed by renilla luciferase ( RL ) reporter constructs ; RL - control ( no let - 7a binding site in the 3 ' UTR ) or RL - 3xBulge ( 3 imperfect let - 7a binding sites in the 3 ' - UTR ) . Firefly luciferase ( FL ) was co - transfected to serve as internal control . RL / FL luminescence ratio was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test . C . HEK293T cells were transfected with indicated siRNAs , followed by pCMV - FL - miR30 ( P ) reporter with either pSUPER - control or pSUPER - miR30 constructs . RL was co - transfected as internal control . FL / RL luminescence ratio was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test . D . HeLa cells were initially transfected with control ( siControl ) , Dicer ( siDicer ) or Nup358 ( siNup358 ) specific siRNAs , followed by FL constructs containing wild type ( HMGA2 - wt ) or mutant ( HMGA2 - mut ) HMGA2 3 ' - UTR , along with RL as internal control . Graph was plotted using data from three independent experiments . FL / RL luminescence ratio was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test .

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Figure 2A. Western analysis of HeLa cells, treated with control (siControl), Dicer (siDicer), Nup214 (siNup214) or Nup358 (siNup358) siRNA, for assessing the extent of protein depletion using indicated antibodies. Vinculin was used as loading control.B. HeLa cells were initially transfected with indicated siRNAs, followed by renilla luciferase (RL) reporter constructs; RL-control (no let-7a binding site in the 3'UTR) or RL-3xBulge (3 imperfect let-7a binding sites in the 3'-UTR). Firefly luciferase (FL) was co-transfected to serve as internal control. RL/FL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.C. HEK293T cells were transfected with indicated siRNAs, followed by pCMV-FL-miR30 (P) reporter with either pSUPER-control or pSUPER-miR30 constructs. RL was co-transfected as internal control. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.D. HeLa cells were initially transfected with control (siControl), Dicer (siDicer) or Nup358 (siNup358) specific siRNAs, followed by FL constructs containing wild type (HMGA2-wt) or mutant (HMGA2-mut) HMGA2 3'-UTR, along with RL as internal control. Graph was plotted using data from three independent experiments. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


Figure 3A . HeLa cells were transfected with control , Dicer or Nup358 specific siRNAs , as indicated . Upper panel , total RNA was isolated and analyzed by northern blotting for let - 7a using radio - labeled probe . Ethidium bromide ( EtBr ) stained gel indicates equal loading of RNA samples . A . HeLa cells were transfected with control , Dicer or Nup358 specific siRNAs , as indicated . Bottom panel , total RNA was isolated from control , Dicer or Nup358 siRNA treated cells and was reverse - transcribed using TaqMan microRNA reverse transcription kit . The levels of let - 7a were quantified by qPCR following manufacturer ' s instructions . Graph represents the relative levels of miRNAs as compared to U6 RNA control . The values were further normalized to let - 7a levels in control siRNA knockdown condition . Data are presented as mean SD ( n = 3 ) . B . HeLa cells were transfected with indicated siRNAs , followed by RL - 3xBulge construct . Immunoprecipitation ( IP ) was performed with control mouse IgG ( IgG IP ) or anti - AGO2 antibody ( AGO2 IP ) using lysates prepared from these cells . Bottom right panel ; AGO2 immunoprecipitates were analyzed by western blotting using AGO2 specific antibody . Vinculin was used as loading control . Data are presented as mean SD ( n = 3 ) . B . HeLa cells were transfected with indicated siRNAs , followed by RL - 3xBulge construct . Immunoprecipitation ( IP ) was performed with control mouse IgG ( IgG IP ) or anti - AGO2 antibody ( AGO2 IP ) using lysates prepared from these cells . Bottom left panel ; RNA isolated from IgG control or AGO2 immunoprecipitate samples was reverse - transcribed using oligo ( dT ) primer . RL - 3xBulge mRNA association with AGO2 was quantified by q - PCR and normalized to the level of GAPDH mRNA associated with AGO2 . B . HeLa cells were transfected with indicated siRNAs , followed by RL - 3xBulge construct . Immunoprecipitation ( IP ) was performed with control mouse IgG ( IgG IP ) or anti - AGO2 antibody ( AGO2 IP ) using lysates prepared from these cells . Total RNA was isolated from control and AGO2 immunoprecipitates and the levels of AGO2 associated let - 7a ( top left panel ) and miR - 17 ( top right panel ) were quantified by q - PCR . Graph represents the extent of miRNAs associated with AGO2 as compared to U6 RNA ( as a negative control ) with AGO2 . C . HeLa cells were transfected with control ( siControl ) or Nup358 specific siRNA ( siNup358 ) for 96 h . Western blots indicate the extent of Nup358 depletion and AGO2 immunoprecipitation in siControl and siNup358 samples . Vinculin was used as loading control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test . C . HeLa cells were transfected with control ( siControl ) or Nup358 specific siRNA ( siNup358 ) for 96 h . Cells were lysed and subjected to immunoprecipitation with control IgG ( IgG IP ) or anti - AGO2 ( AGO2 IP ) . Total RNA was extracted from initial lysate and IP samples and analyzed by q - PCR for validated miRNA targets , Serbp1 and Dnajb11 . GAPDH was considered as negative control . Data expressed as the relative amount of target mRNA associated with AGO2 as compared to GAPDH mRNA . D . HeLa cells were treated with indicated siRNAs for 96 h . Cells were lysed and analyzed for extent of Nup358 depletion and for the levels of validated miRNA targets , Ras and c - Myc by western analysis . Vinculin was used as loading control . E . HeLa cells were lysed and immunoprecipitation was performed with control ( IgG - IP ) or Nup358 ( Nup358 - IP , left panel ) or AGO2 ( AGO2 - IP , right panel ) using specific antibodies . The immunoprecipitates were washed with a buffer containing ( + ) or not containing ( - ) RNAse A and probed for the presence of indicated proteins by western blotting . F . HeLa cells were transfected with RL - 3xBulge construct and immunoprecipitation was performed using control ( IgG - IP ) or Nup358 ( Nup358 - IP ) antibodies . Total RNA was isolated from the immunoprecipitates and analyzed for let - 7a miRNA ( left panel ) , RL - 3xBulge mRNA and the endogenous miRNA target Dnajb11 using q - PCR ( right panel ) . Fold enrichment in Nup358 - IP as compared to IgG - control was calculated and the data are presented as mean SD ( n = 3 ) , P ‐ value was obtained using Student ' s t ‐ test .

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Figure 3A. HeLa cells were transfected with control, Dicer or Nup358 specific siRNAs, as indicated. Upper panel, total RNA was isolated and analyzed by northern blotting for let-7a using radio-labeled probe. Ethidium bromide (EtBr) stained gel indicates equal loading of RNA samples.A. HeLa cells were transfected with control, Dicer or Nup358 specific siRNAs, as indicated. Bottom panel, total RNA was isolated from control, Dicer or Nup358 siRNA treated cells and was reverse-transcribed using TaqMan microRNA reverse transcription kit. The levels of let-7a were quantified by qPCR following manufacturer's instructions. Graph represents the relative levels of miRNAs as compared to U6 RNA control. The values were further normalized to let-7a levels in control siRNA knockdown condition. Data are presented as mean SD (n = 3).B. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Bottom right panel; AGO2 immunoprecipitates were analyzed by western blotting using AGO2 specific antibody. Vinculin was used as loading control. Data are presented as mean SD (n = 3).B. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Bottom left panel; RNA isolated from IgG control or AGO2 immunoprecipitate samples was reverse-transcribed using oligo(dT) primer. RL-3xBulge mRNA association with AGO2 was quantified by q-PCR and normalized to the level of GAPDH mRNA associated with AGO2.B. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Total RNA was isolated from control and AGO2 immunoprecipitates and the levels of AGO2 associated let-7a (top left panel) and miR-17 (top right panel) were quantified by q-PCR. Graph represents the extent of miRNAs associated with AGO2 as compared to U6 RNA (as a negative control) with AGO2.C. HeLa cells were transfected with control (siControl) or Nup358 specific siRNA (siNup358) for 96 h. Western blots indicate the extent of Nup358 depletion and AGO2 immunoprecipitation in siControl and siNup358 samples. Vinculin was used as loading control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.C. HeLa cells were transfected with control (siControl) or Nup358 specific siRNA (siNup358) for 96 h. Cells were lysed and subjected to immunoprecipitation with control IgG (IgG IP) or anti-AGO2 (AGO2 IP). Total RNA was extracted from initial lysate and IP samples and analyzed by q-PCR for validated miRNA targets, Serbp1 and Dnajb11. GAPDH was considered as negative control. Data expressed as the relative amount of target mRNA associated with AGO2 as compared to GAPDH mRNA.D. HeLa cells were treated with indicated siRNAs for 96 h. Cells were lysed and analyzed for extent of Nup358 depletion and for the levels of validated miRNA targets, Ras and c-Myc by western analysis. Vinculin was used as loading control.E. HeLa cells were lysed and immunoprecipitation was performed with control (IgG-IP) or Nup358 (Nup358-IP, left panel) or AGO2 (AGO2-IP, right panel) using specific antibodies. The immunoprecipitates were washed with a buffer containing (+) or not containing (-) RNAse A and probed for the presence of indicated proteins by western blotting.F. HeLa cells were transfected with RL-3xBulge construct and immunoprecipitation was performed using control (IgG-IP) or Nup358 (Nup358-IP) antibodies. Total RNA was isolated from the immunoprecipitates and analyzed for let-7a miRNA (left panel), RL-3xBulge mRNA and the endogenous miRNA target Dnajb11 using q-PCR (right panel). Fold enrichment in Nup358-IP as compared to IgG-control was calculated and the data are presented as mean SD (n = 3), P‐value was obtained using Student's t‐test.


Figure 4B . HEK293T cells were co - transfected with GFP - maltose binding protein ( MBP ) - control or GFP - tagged version of indicated Nup358 fragments along with HA - AGO2 . Immunoprecipitation ( IP ) was performed using GFP antibodies and the presence of AGO2 was detected by western blotting ( WB ) using HA antibodies . C . Cells were transfected with indicated constructs and IP and WB were performed as described for B . D . HEK293T cells transfected with GFP - MBP ( control ) , GFP - Nup358 - C or GFP - Nup358 - CΔIR ( a mutant devoid of IR region ) and HA - AGO2 . IP and WB analyses were performed as indicated . E . Cells were transfected with HA - tagged version of indicated AGO subfamily member and GFP - Nup358 - IR . IP and WB analyses were performed using indicated antibodies . F . Lysates from cells expressing GFP - control , GFP - human ( h ) Nup358 - IR or GFP - zebrafish ( z ) Nup358 - IR and HA - AGO2 were immunoprecipitated using GFP antibodies and the immunoprecipitates were analyzed for the presence of HA - AGO2 by western blotting .

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Figure 4B. HEK293T cells were co-transfected with GFP-maltose binding protein (MBP)-control or GFP-tagged version of indicated Nup358 fragments along with HA-AGO2. Immunoprecipitation (IP) was performed using GFP antibodies and the presence of AGO2 was detected by western blotting (WB) using HA antibodies.C. Cells were transfected with indicated constructs and IP and WB were performed as described for B.D. HEK293T cells transfected with GFP-MBP (control), GFP-Nup358-C or GFP-Nup358-CΔIR (a mutant devoid of IR region) and HA-AGO2. IP and WB analyses were performed as indicated.E. Cells were transfected with HA-tagged version of indicated AGO subfamily member and GFP-Nup358-IR. IP and WB analyses were performed using indicated antibodies.F. Lysates from cells expressing GFP-control, GFP-human (h) Nup358-IR or GFP-zebrafish (z) Nup358-IR and HA-AGO2 were immunoprecipitated using GFP antibodies and the immunoprecipitates were analyzed for the presence of HA-AGO2 by western blotting.


Figure 5B . HEK293T cells were transfected with indicated constructs and immunoprecipitation ( IP ) was performed using GFP specific antibodies and probed for HA - AGO2 by western blotting ( WB ) using HA antibodies . Endogenous RanGAP and Ubc9 were probed with specific antibodies . C . Lysates prepared from cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies . Presence of endogenous RanGAP and Ubc9 was determined by WB . D . GFP - control , GFP - IR1 + 2 wild type or mutants were co - expressed with HA - AGO and IP and WB analysis were performed to detect the interaction using indicated antibodies . E . Lysates prepared from cells expressing the indicated proteins were immunoprecipitated with GFP specific antibodies and the presence of specific proteins in the immunoprecipitates was detected by WB with indicated antibodies .

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Figure 5B. HEK293T cells were transfected with indicated constructs and immunoprecipitation (IP) was performed using GFP specific antibodies and probed for HA-AGO2 by western blotting (WB) using HA antibodies. Endogenous RanGAP and Ubc9 were probed with specific antibodies.C. Lysates prepared from cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies. Presence of endogenous RanGAP and Ubc9 was determined by WB.D. GFP-control, GFP-IR1+2 wild type or mutants were co-expressed with HA-AGO and IP and WB analysis were performed to detect the interaction using indicated antibodies.E. Lysates prepared from cells expressing the indicated proteins were immunoprecipitated with GFP specific antibodies and the presence of specific proteins in the immunoprecipitates was detected by WB with indicated antibodies.


Figure 6A . Top panel ; depicts the amino acid sequence corresponding to the Nup358 - SIM1 region and substitutions introduced in the SIM1 mutant . Bottom panel : HEK293T cells were co - transfected with GFP - control , GFP - SIM1 or GFP - SIM1 - mut along with HA - AGO2 and immunoprecipitation ( IP ) was performed with GFP specific antibodies and western analysis ( WB ) of the input lysate and immunoprecipitates were carried out using indicated antibodies . B . Top panel ; depiction of amino acid sequence corresponding to the SIMs in the indicated proteins used in this study . Hydrophobic residues involved in SUMO binding are indicated in red . Bottom panel ; constructs expressing GFP - control or GFP - fused SIMs of indicated proteins were co - transfected with HA - AGO2 in HEK293T cells and IP and WB were performed to determine the extent of interaction . C . HA - MBP - control , HA - AGO1 or HA - AGO2 was co - expressed with GFP - SIM1 and the cell lysates were subjected to IP with HA antibodies and WB analysis with indicated antibodies to detect the presence of proteins in the immunoprecipitates . D . SIM1 directly interacts with AGO2 . Bacterial lysates expressing MBP control or MBP - AGO2 and GST or GST - SIM1 were mixed and GST pull down assay was performed . The presence of proteins in the pull down samples was analyzed by WB using specific antibodies . The extent of GST pull down was monitored by coomassie staining of the membrane ( coomassie ) . E . SIM1 interacts with PIWI subfamily of proteins . HEK293T cells were co - transfected with indicated constructs expressing HA - tagged version of PIWI proteins and GFP - SIM1 . IP was performed using HA antibodies . The immunoprecipitates were probed for the presence of GFP - SIM1 using GFP specific antibodies by WB . F . Putative SIM - binding regions in AGO2 . Three distinct superimpositions of AGO2 ( grey ) and SUMO ( blue ) , in complex with Nup358 - SIM1 ( red ) , are shown in cartoon representation rendered with Chimera [ 90 ] . The residue numbers on the AGO2 that could be the putative SIM - binding regions are labelled . To identify possible Nup358 binding sites on AGO2 , the 3D structure of AGO2 ( PDB ID : 4W5O ) [ 89 ] was compared with that of SUMO ( PDB ID : 1Z5S ) [ 47 ] .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Top panel; depicts the amino acid sequence corresponding to the Nup358-SIM1 region and substitutions introduced in the SIM1 mutant. Bottom panel: HEK293T cells were co-transfected with GFP-control, GFP-SIM1 or GFP-SIM1-mut along with HA-AGO2 and immunoprecipitation (IP) was performed with GFP specific antibodies and western analysis (WB) of the input lysate and immunoprecipitates were carried out using indicated antibodies.B. Top panel; depiction of amino acid sequence corresponding to the SIMs in the indicated proteins used in this study. Hydrophobic residues involved in SUMO binding are indicated in red. Bottom panel; constructs expressing GFP-control or GFP-fused SIMs of indicated proteins were co-transfected with HA-AGO2 in HEK293T cells and IP and WB were performed to determine the extent of interaction.C. HA-MBP-control, HA-AGO1 or HA-AGO2 was co-expressed with GFP-SIM1 and the cell lysates were subjected to IP with HA antibodies and WB analysis with indicated antibodies to detect the presence of proteins in the immunoprecipitates.D. SIM1 directly interacts with AGO2. Bacterial lysates expressing MBP control or MBP-AGO2 and GST or GST-SIM1 were mixed and GST pull down assay was performed. The presence of proteins in the pull down samples was analyzed by WB using specific antibodies. The extent of GST pull down was monitored by coomassie staining of the membrane (coomassie).E. SIM1 interacts with PIWI subfamily of proteins. HEK293T cells were co-transfected with indicated constructs expressing HA-tagged version of PIWI proteins and GFP-SIM1. IP was performed using HA antibodies. The immunoprecipitates were probed for the presence of GFP-SIM1 using GFP specific antibodies by WB.F. Putative SIM-binding regions in AGO2. Three distinct superimpositions of AGO2 (grey) and SUMO (blue), in complex with Nup358-SIM1(red), are shown in cartoon representation rendered with Chimera [90]. The residue numbers on the AGO2 that could be the putative SIM-binding regions are labelled. To identify possible Nup358 binding sites on AGO2, the 3D structure of AGO2 (PDB ID: 4W5O) [89] was compared with that of SUMO (PDB ID: 1Z5S) [47].


Figure 7A . Right panel ; Western blots showing the expression of transfected constructs using indicated antibodies . a - tubulin was used as loading control . A . Left panel ; HeLa cells were co - transfected with GFP - MBP ( control ) , GFP - SIM1 or GFP - SIM1 - mut ( AGO interaction defective ) along with RL - control or RL - 3xbulge . Firefly luciferase was used as transfection control . Dual luciferase assay was performed . RL / FL luminescence ration was plotted after normalization with GFP - control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test . B . Bottom right panel ; Western blot for monitoring the expression levels of indicated proteins using antibodies . a - tubulin was used as loading control . B . Bottom left panel ; HEK293T cells were co - transfected with indicated expression constructs and the RL reporter . Firefly luciferase ( FL ) was used as transfection control . Dual luciferase assay was performed . The ratio of RL / FL luminescence was plotted after normalization with N - HA - MBP control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test . C . Ectopic expression of AGO2 restores P body formation , but fails to rescue the impairment in miRNA function in Nup358 depleted cells . HeLa cells were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Left panel : Quantitative representation showing the number of cells showing microscopically distinct P bodies in different indicated conditions . The data was obtained from three independent experiments , and in each experiment , 100 cells were counted from different fields for the presence of intact P bodies and expressed as percentage . C . Ectopic expression of AGO2 restores P body formation , but fails to rescue the impairment in miRNA function in Nup358 depleted cells . HeLa cells were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Middle panel : HeLa cells were transfected with indicated siRNAs , followed by expression constructs and miRNA RL reporters . FL was used as transfection control . The ratio of RL / FL luminescence was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was obtained using Student ' s t ‐ test . C . Ectopic expression of AGO2 restores P body formation , but fails to rescue the impairment in miRNA function in Nup358 depleted cells . HeLa cells were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Right panel : Western blot analysis for the relative expression of indicated constructs in HEK293T cells . a - tubulin is used as loading control . D . HeLa cells were transfected with siControl or siNup358 and subjected to membrane flotation assay . Fractions were collected and analyzed for the presence of indicated proteins by western blotting .

O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. Right panel; Western blots showing the expression of transfected constructs using indicated antibodies. a-tubulin was used as loading control.A. Left panel; HeLa cells were co-transfected with GFP-MBP (control), GFP-SIM1 or GFP-SIM1-mut (AGO interaction defective) along with RL-control or RL-3xbulge. Firefly luciferase was used as transfection control. Dual luciferase assay was performed. RL/FL luminescence ration was plotted after normalization with GFP-control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.B. Bottom right panel; Western blot for monitoring the expression levels of indicated proteins using antibodies. a-tubulin was used as loading control.B. Bottom left panel; HEK293T cells were co-transfected with indicated expression constructs and the RL reporter. Firefly luciferase (FL) was used as transfection control. Dual luciferase assay was performed. The ratio of RL/FL luminescence was plotted after normalization with N-HA-MBP control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Left panel: Quantitative representation showing the number of cells showing microscopically distinct P bodies in different indicated conditions. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact P bodies and expressed as percentage.C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Middle panel: HeLa cells were transfected with indicated siRNAs, followed by expression constructs and miRNA RL reporters. FL was used as transfection control. The ratio of RL/FL luminescence was calculated. Data are presented as mean SD (n = 3), P‐value was obtained using Student's t‐test.C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Right panel: Western blot analysis for the relative expression of indicated constructs in HEK293T cells. a-tubulin is used as loading control.D. HeLa cells were transfected with siControl or siNup358 and subjected to membrane flotation assay. Fractions were collected and analyzed for the presence of indicated proteins by western blotting.


Figure 1 ( A , B ) Localization of oskar mRNA in wild - type ( A ) and in Tm1eg9 / Tm1eg9 ( B ) egg - chambers . ( C ) Kymograph of oskMS2 - GFP mRNPs ( cyan ) travelling along polarity marked MTs ( red , EB1 protein ) in an ex vivo ooplasmic preparation . White dashed arrow shows the growing plus tip of the MT . Scale bars represent 1 s and 1 µm , respectively . ( D ) Distribution of oskMS2 - GFP mRNP runs towards plus ( white ) and minus ends ( grey ) . Numbers within the bars indicate number of runs . P value of Chi2 test against wild - type is indicated above each bar . ( E , F ) oskar mRNA ( green ) and Oskar protein ( magenta ) distribution in Tm1eg1 / Tm1eg9 egg - chambers expressing oskarMS2 ( 6x ) ( E ) or oskarMS2 ( 6x ) and Khc401 - MCP ( F ) . ( G ) oskar mRNA ( magenta ) distribution in Tm1eg1 / Tm1eg9 egg - chambers rescued with EmGFP - Tm1 - I ( green ) . Scale bars represent 50 µm .

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Figure 1(A, B) Localization of oskar mRNA in wild-type (A) and in Tm1eg9/Tm1eg9 (B) egg-chambers.(C) Kymograph of oskMS2-GFP mRNPs (cyan) travelling along polarity marked MTs (red, EB1 protein) in an ex vivo ooplasmic preparation. White dashed arrow shows the growing plus tip of the MT. Scale bars represent 1 s and 1 µm, respectively.(D) Distribution of oskMS2-GFP mRNP runs towards plus (white) and minus ends (grey). Numbers within the bars indicate number of runs. P value of Chi2 test against wild-type is indicated above each bar.(E, F) oskar mRNA (green) and Oskar protein (magenta) distribution in Tm1eg1/Tm1eg9 egg-chambers expressing oskarMS2(6x) (E) or oskarMS2(6x) and Khc401-MCP (F).(G) oskar mRNA (magenta) distribution in Tm1eg1/Tm1eg9 egg-chambers rescued with EmGFP-Tm1-I (green). Scale bars represent 50 µm.


Figure 2 ( A - B " ) Colocalization of oskMS2 - mCherry ( red , A , B ) ( A ' , B ' ) with Khc - EGFP ( green , A ) ( A ' ) or with EmGFP - Tm1 - I ( green B ) ( B ' ) in ex vivo ooplasmic preparations . ( C ) Fraction of oskMS2 - MCP - mCherry mRNPs located non - randomly within a 200 nm distance of one of the indicated GFP tagged protein particles in ex vivo ooplasmic preparations . MCP indicates MCP - EGFP which , like MCP - mCherry , can bind to MS2 loops . Staufen ( Stau ) is a dsRNA binding protein and bona fide partner of oskar mRNA ( St Johnston et al . , 1991 ; St Johnston et al . , 1992 ) . All values are significantly different from zero ( p < 10 - 3 , one sample t - test ) . ( D ) Fraction of oskMS2 - mCherry mRNPs co - localizing ( max . 200 nm ) non - randomly with Khc - EGFP particles in wild - type and in Tm1eg1 / Tm1eg9 ooplasms in presence of two ( white ) or one ( grey ) copy of endogenous Khc . ( C , D ) P values of two sample t - tests are indicated above the relevant bar pairs . Numbers indicate the number of particle clusters ( 160 mRNPs in each ) and the number of preparations ( in brackets ) analysed . Error bars represent 95 % confidence intervals .

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Figure 2(A-B") Colocalization of oskMS2-mCherry (red, A, B) (A', B') with Khc-EGFP (green, A) (A') or with EmGFP-Tm1-I (green B) (B') in ex vivo ooplasmic preparations.(C) Fraction of oskMS2-MCP-mCherry mRNPs located non-randomly within a 200 nm distance of one of the indicated GFP tagged protein particles in ex vivo ooplasmic preparations. MCP indicates MCP-EGFP which, like MCP-mCherry, can bind to MS2 loops. Staufen (Stau) is a dsRNA binding protein and bona fide partner of oskar mRNA (St Johnston et al., 1991; St Johnston et al., 1992). All values are significantly different from zero (p<10-3, one sample t-test).(D) Fraction of oskMS2-mCherry mRNPs co-localizing (max. 200 nm) non-randomly with Khc-EGFP particles in wild-type and in Tm1eg1/Tm1eg9 ooplasms in presence of two (white) or one (grey) copy of endogenous Khc.(C, D) P values of two sample t-tests are indicated above the relevant bar pairs. Numbers indicate the number of particle clusters (160 mRNPs in each) and the number of preparations (in brackets) analysed. Error bars represent 95% confidence intervals.


Figure 3 ( A , B ) Kymographs of oskMS2 - GFP mRNPs ( green ) associated with Khc - mKate2 ( A , red ) and mCherry - Tm1 - I / C ( B , red ) ex vivo . Arrows indicate motile RNPs in stable complex with Khc ( A ) or Tm1 - I / C ( B ) , the arrowheads point to non - motile oskMS2 - RNPs showing no obvious accumulation of the tagged protein . Note that mCherry - Tm1 - I was exposed twice as long as Khc - mKate to obtain comparable red fluorescence signals . Scale bars represent 1 µm and 1 second , respectively . ( C - E ) Relative Khc - mKate2 ( C ) , mCherry - Tm1 - I / C ( D ) coverage of motile ( white ) and non - motile ( grey ) oskMS2 - GFP trajectories . Numbers within the boxes indicate the number of trajectories and the number of ooplasms ( in brackets ) analysed . Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein ( for at least half of the duration of the trajectory , p < 0 . 01 , binominal distribution , see also Fig EV2B ) . P values of pairwise Mann - Whitney U tests are indicated above the boxplots . ( C - E ) Relative GFP - Staufen ( E ) coverage of motile ( white ) and non - motile ( grey ) oskMS2 - GFP trajectories . Numbers within the boxes indicate the number of trajectories and the number of ooplasms ( in brackets ) analysed . Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein ( for at least half of the duration of the trajectory , p < 0 . 01 , binominal distribution , see also Fig EV2B ) . P values of pairwise Mann - Whitney U tests are indicated above the boxplots . ( F ) Example kymograph of Khc molecules ( red ) associating with an oskMS2 - GFP mRNP ( green ) before and during its run . White arrowheads indicate two association events , yellow arrow indicates the onset of motility . Scale bars represent 5 µm and 1 second , respectively . ( G ) Frequency of Khc - mKate2 appearance on motile ( white ) , before ( motility - primed , checked ) and after ( dotted ) the onset of motility , and non - motile ( grey ) oskMS2 - GFP trajectories . Fractions within the bars indicate number of association events that lasted longer than a single frame over the total number of frames analysed . Indicated P values show results of pairwise Fisher ' s exact test . The Khc association frequency observed on RNPs before ( checked ) and during ( dotted ) their motility is not significantly different from wild - type motile RNP controls ( p > 0 . 01 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, B) Kymographs of oskMS2-GFP mRNPs (green) associated with Khc-mKate2 (A, red) and mCherry-Tm1-I/C (B, red) ex vivo. Arrows indicate motile RNPs in stable complex with Khc (A) or Tm1-I/C (B), the arrowheads point to non-motile oskMS2-RNPs showing no obvious accumulation of the tagged protein. Note that mCherry-Tm1-I was exposed twice as long as Khc-mKate to obtain comparable red fluorescence signals. Scale bars represent 1 µm and 1 second, respectively.(C-E) Relative Khc-mKate2 (C), mCherry-Tm1-I/C (D) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots.(C-E) Relative GFP-Staufen (E) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots.(F) Example kymograph of Khc molecules (red) associating with an oskMS2-GFP mRNP (green) before and during its run. White arrowheads indicate two association events, yellow arrow indicates the onset of motility. Scale bars represent 5 µm and 1 second, respectively.(G) Frequency of Khc-mKate2 appearance on motile (white), before (motility-primed, checked) and after (dotted) the onset of motility, and non-motile (grey) oskMS2-GFP trajectories. Fractions within the bars indicate number of association events that lasted longer than a single frame over the total number of frames analysed. Indicated P values show results of pairwise Fisher's exact test. The Khc association frequency observed on RNPs before (checked) and during (dotted) their motility is not significantly different from wild-type motile RNP controls (p>0.01).


Figure 4 ( A ) Western blots of oskar mRNP components ( Staufen , Bruno and Y14 ) and motor associated proteins ( Khc , BicD , Dic ) co - immunoprecipitated with EmGFP - Tm1 - I from ovarian lysates . Protein marker bands and their molecular weight in kDa are indicated . ( B ) Signal developed with oligo ( dT ) 25 - TexasRed probes on GFP - Trap _ M beads binding EmGFP - Tm1 - I or FlagMycGFP after denaturing washes . 594 nm reflection marks bead boundaries . ( B ' ) Quantification of mean oligo ( dT ) signal measured on beads . ( C ) qRT - PCR of EmGFP - Tm1 - I and control Flag - Myc - GFP and bead alone ( w1118 ) bound oskar and gapdh RNAs after stringent washes ( mean ± s . e . m . , N = 3 ) . ( D ) Images of beads binding to EmGFP - Tm1 - I ( green ) and DIG labelled in vitro transcribed RNA fragments ( magenta ) . ( E , F ) Mean DIG - Cy5 fluorescence measured on beads capturing the RNA fragment , with or without UV cross - linking , indicated below the charts . P values of pairwise Mann - Whitney U tests are indicated . In panel F , none of the non - cross - linked samples differ significantly from the no RNA control ( p > 0 . 05 ) . Numbers below the plots indicate the number of beads analysed .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Western blots of oskar mRNP components (Staufen, Bruno and Y14) and motor associated proteins (Khc, BicD, Dic) co-immunoprecipitated with EmGFP-Tm1-I from ovarian lysates. Protein marker bands and their molecular weight in kDa are indicated.(B) Signal developed with oligo(dT)25-TexasRed probes on GFP-Trap_M beads binding EmGFP-Tm1-I or FlagMycGFP after denaturing washes. 594 nm reflection marks bead boundaries.(B') Quantification of mean oligo(dT) signal measured on beads.(C) qRT-PCR of EmGFP-Tm1-I and control Flag-Myc-GFP and bead alone (w1118) bound oskar and gapdh RNAs after stringent washes (mean ± s.e.m., N = 3).(D) Images of beads binding to EmGFP-Tm1-I (green) and DIG labelled in vitro transcribed RNA fragments (magenta).(E, F) Mean DIG-Cy5 fluorescence measured on beads capturing the RNA fragment, with or without UV cross-linking, indicated below the charts. P values of pairwise Mann-Whitney U tests are indicated. In panel F, none of the non-cross-linked samples differ significantly from the no RNA control (p>0.05). Numbers below the plots indicate the number of beads analysed.


Figure 5 ( A , B , C ) Confocal image of a Tm1eg9 homozygous egg - chamber expressing EmGFP - Tm1 - I ( green ) and Khc - mKate2 ( magenta ) . oskar mRNA labelled with osk1 - 5 FIT probes ( Hovelmann et al . , 2014 ) is in yellow . ( B ' , C ' ) oskar mRNPs co - localizing with both EmGFP - Tm1 - I and Khc - mKate2 . Colors indicate the maximal co - localization distance ( C ' ) . Panels B - C ' represent the boxed regions in panel A . ( D , E ) Localization of Khc - mKate2 ( green ) and oskar mRNA ( magenta ) in wild - type and Tm1gs mutant nurse cells . ( F , G ) Fraction of oskar mRNPs co - localizing with Khc - mKate ( dark grey ) , EmGFP - Tm1 - I ( white ) , or both of these proteins ( light grey ) in the oocyte ( F ) or in the nurse cells ( G ) ( max . colocalization distance is 250 nm ) . None of the values are significantly different from each other ( one - way ANOVA , p > 10 - 3 ) . Horizontal dashed lines indicate the expected value of observing both proteins in an oskar mRNP if the interactions are independent ( see Fig EV3C and D ) . Significance of the observed co - localization values versus the expected values are shown . ( H ) Fraction of oskar mRNPs co - localizing with Khc - mKate in wild - type and Tm1gs mutant nurse cells when half or all Khc molecules are labelled ( as indicated above the graph ) . P values of pair - wise t - tests are indicated . ( I ) Fraction of oskar mRNPs co - localizing with free mCherry in wild - type nurse cells used as negative control . The measured fraction ( ~ 1 . 6 % ) is significantly different from zero ( one sample t - test ) . All other measured co - localization values are significantly different form this negative control ( p < 0 . 001 , one - way ANOVA ) . ( F - I ) Numbers indicate the number of particle clusters ( 100 oskar mRNPs in each ) and the number of egg - chambers ( in brackets ) analysed . Error bars represent 95 % confidence intervals . All values ( F - I ) are significantly different from zero ( p < 10 - 3 , one sample t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A, B, C) Confocal image of a Tm1eg9 homozygous egg-chamber expressing EmGFP-Tm1-I (green) and Khc-mKate2 (magenta). oskar mRNA labelled with osk1-5 FIT probes(Hovelmann et al., 2014) is in yellow.(B', C') oskar mRNPs co-localizing with both EmGFP-Tm1-I and Khc-mKate2. Colors indicate the maximal co-localization distance (C'). Panels B-C' represent the boxed regions in panel A.(D, E) Localization of Khc-mKate2 (green) and oskar mRNA (magenta) in wild-type and Tm1gs mutant nurse cells.(F, G) Fraction of oskar mRNPs co-localizing with Khc-mKate (dark grey), EmGFP-Tm1-I (white), or both of these proteins (light grey) in the oocyte (F) or in the nurse cells (G) (max. colocalization distance is 250 nm). None of the values are significantly different from each other (one-way ANOVA, p>10-3). Horizontal dashed lines indicate the expected value of observing both proteins in an oskar mRNP if the interactions are independent (see Fig EV3C and D). Significance of the observed co-localization values versus the expected values are shown.(H) Fraction of oskar mRNPs co-localizing with Khc-mKate in wild-type and Tm1gs mutant nurse cells when half or all Khc molecules are labelled (as indicated above the graph). P values of pair-wise t-tests are indicated.(I) Fraction of oskar mRNPs co-localizing with free mCherry in wild-type nurse cells used as negative control. The measured fraction (~1.6%) is significantly different from zero (one sample t-test). All other measured co-localization values are significantly different form this negative control (p<0.001, one-way ANOVA).(F-I) Numbers indicate the number of particle clusters (100 oskar mRNPs in each) and the number of egg-chambers (in brackets) analysed. Error bars represent 95% confidence intervals. All values (F-I) are significantly different from zero (p<10-3, one sample t-test).


Figure 6 ( A - C ) Localization of EmGFP - Tm1 - I ( A ) oskar mRNA ( B ) and Khc ( C ) around the nurse cell nuclear envelope ( magenta , WGA staining ) . ( D - F ) Mean distribution profile of Khc ( D ) mKate2 - Tm1 - I , EmGFP - Tm1 - I ( E ) and oskar mRNA ( F ) around the NE of nurse cells ( marked with a dotted vertical line ) . Genotypes are indicated as follows : wild - type control with solid black line ( D - F ) , oskar RNA null with solid grey line ( D , E ) , Tm1eg9 / Tm1eg9 with dotted black line ( D , F ) Lines indicate mean and 95 % confidence intervals . We analysed the accumulation of mKate2 - Tm1 - I instead of EmGFP - Tm1 - I due to unexpected GFP expression from the oskar0 allele . ( G ) Khc accumulation around the NE . To calculate accumulation the signal intensity measured at the position of the peak observed in the wild - type control ( D , arrowhead , 356 ± 17 . 6 nm away from NE ) was divided by the signal intensity 2 s . d . away ( d , arrow , at 356 + 2 * 410 nm ) . P values of pairwise Mann - Whitney U tests against wild - type control or Tm1gs rescued with EmGFP - Tm1 - I are indicated above the boxplots . Numbers indicate the number of nuclei and the number of egg - chambers ( in brackets ) analysed . ( H - K ) Khc accumulation around the NE of nurse cells over - expressing EmGFP - Tm1 - I within Tm1eg9 / Tm1eg9 ( H ) , and oskar RNA null egg - chambers ( I ) expressing either the oskar 3 ' UTR ( J ) or oskar Δi ( 1 , 2 , 3 ) ( K ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A-C) Localization of EmGFP-Tm1-I (A) oskar mRNA (B) and Khc (C) around the nurse cell nuclear envelope (magenta, WGA staining).(D-F) Mean distribution profile of Khc (D) mKate2-Tm1-I, EmGFP-Tm1-I (E) and oskar mRNA (F) around the NE of nurse cells (marked with a dotted vertical line). Genotypes are indicated as follows: wild-type control with solid black line (D-F), oskar RNA null with solid grey line (D, E), Tm1eg9/Tm1eg9 with dotted black line (D, F) Lines indicate mean and 95% confidence intervals. We analysed the accumulation of mKate2-Tm1-I instead of EmGFP-Tm1-I due to unexpected GFP expression from the oskar0 allele.(G) Khc accumulation around the NE. To calculate accumulation the signal intensity measured at the position of the peak observed in the wild-type control (D, arrowhead, 356±17.6 nm away from NE) was divided by the signal intensity 2 s.d. away (d, arrow, at 356+2*410 nm). P values of pairwise Mann-Whitney U tests against wild-type control or Tm1gs rescued with EmGFP-Tm1-I are indicated above the boxplots. Numbers indicate the number of nuclei and the number of egg-chambers (in brackets) analysed.(H-K) Khc accumulation around the NE of nurse cells over-expressing EmGFP-Tm1-I within Tm1eg9/Tm1eg9 (H), and oskar RNA null egg-chambers (I) expressing either the oskar 3' UTR (J) or oskar Δi(1,2,3) (K).


Figure 7 ( A - E ) Mean oskar mRNA distribution ( green , detected by conventional FISH ) within oocytes in which oskar mRNA is substituted by oskar Δi ( 1 , 2 , 3 ) mRNA ( A , B ) , oskar 3 ' UTR mRNA ( D ) and that ( in addition ) over - express EmGFP - Tm1 - I ( B , D and E ) . Wild - type control ( C ) and oocytes expressing Khc RNAi and EmGFP - Tm1 - I ( E ) . Magenta indicates standard deviation of the distribution of oskar mRNA . Scale bar is 10 % of total oocyte length ( C ) . ( F ) Position of the oskar mRNA center of mass relative to the geometric center of the oocyte ( dotted horizontal line ) along the anteroposterior ( AP ) axis . Posterior pole is the top of the chart . P values of pairwise Mann - Whitney U tests are indicated . Numbers indicate number of oocytes analysed .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A-E) Mean oskar mRNA distribution (green, detected by conventional FISH) within oocytes in which oskar mRNA is substituted by oskar Δi(1,2,3) mRNA (A, B), oskar 3'UTR mRNA (D) and that (in addition) over-express EmGFP-Tm1-I (B, D and E). Wild-type control (C) and oocytes expressing Khc RNAi and EmGFP-Tm1-I (E). Magenta indicates standard deviation of the distribution of oskar mRNA. Scale bar is 10% of total oocyte length (C).(F) Position of the oskar mRNA center of mass relative to the geometric center of the oocyte (dotted horizontal line) along the anteroposterior (AP) axis. Posterior pole is the top of the chart. P values of pairwise Mann-Whitney U tests are indicated. Numbers indicate number of oocytes analysed.


Figure 1C . HDo - TRaMs were engineered with vectors relevant to treatment of Fabry , Gaucher , Farber and Pompe diseases . Intracellular lysosomal enzyme specific activities were measured in transduced T - Rapa and controls . Transductions varied across vectors / donors , as indicated . D , E . Enzyme activity was assessed in transduced T - Rapa supernatants compared to controls in activated / dividing ( D ) and quiescent / resting ( E ) states . F . Cells from all FDo were stably transduced , as indicated . Intracellular α - galactosidase A ( α - gal A ) specific activities were determined . G , H . Secreted α - gal A activity was measured from FDo - derived TRaMs in activated ( G ) , and quiescent / resting ( H ) states . II . α - Gal A specific activity in Fabry patient - derived skin fibroblasts after 6 hours of exposure to conditioned media from TRaMs in the presence or absence of 1mM M6P .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C. HDo-TRaMs were engineered with vectors relevant to treatment of Fabry, Gaucher, Farber and Pompe diseases. Intracellular lysosomal enzyme specific activities were measured in transduced T-Rapa and controls. Transductions varied across vectors/donors, as indicated. D, E. Enzyme activity was assessed in transduced T-Rapa supernatants compared to controls in activated/dividing (D) and quiescent/resting (E) states.F. Cells from all FDo were stably transduced, as indicated. Intracellular α-galactosidase A (α-gal A) specific activities were determined. G, H. Secreted α-gal A activity was measured from FDo-derived TRaMs in activated (G), and quiescent/resting (H) states. II. α-Gal A specific activity in Fabry patient-derived skin fibroblasts after 6 hours of exposure to conditioned media from TRaMs in the presence or absence of 1mM M6P.


Figure 2B . Engraftment was evaluated by flow cytometry for human CD3 expression . C , D . α - Gal A activity in plasma , and specific activities in liver , spleen , heart and kidneys were measured after transplant of transduced healthy donor ( HDo ) - derived ( C ) or Fabry donor ( FDo ) - derived ( D ) TRaMs , non - transduced ( NT ) cells , or in sham - treated NOD / SCID ( NS ) and NSF mice .

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Figure 2B. Engraftment was evaluated by flow cytometry for human CD3 expression.C, D. α-Gal A activity in plasma, and specific activities in liver, spleen, heart and kidneys were measured after transplant of transduced healthy donor (HDo)-derived (C) or Fabry donor (FDo)-derived (D) TRaMs, non-transduced (NT) cells, or in sham-treated NOD/SCID (NS) and NSF mice.


Figure 3A , B . Levels of globotriaosylceramide ( Gb3 ) were measured by LC / MS in plasma and extracts of liver , spleen , heart , and kidneys of mice engrafted with healthy donor ( HDo ) - derived ( A ) or Fabry donor ( FDo ) - derived ( B ) TRaMs , non - transduced ( NT ) cells , or in sham - treated NOD / SCID ( NS ) and NSF mice . C , D . Levels of lyso - Gb3 were measured by LC / MS in plasma and extracts of tissues of mice engrafted with HDo - derived ( C ) or FDo - derived ( D ) TRaMs .

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Figure 3A, B. Levels of globotriaosylceramide (Gb3) were measured by LC/MS in plasma and extracts of liver, spleen, heart, and kidneys of mice engrafted with healthy donor (HDo)-derived (A) or Fabry donor (FDo)-derived (B) TRaMs, non-transduced (NT) cells, or in sham-treated NOD/SCID (NS) and NSF mice.C, D. Levels of lyso-Gb3 were measured by LC/MS in plasma and extracts of tissues of mice engrafted with HDo-derived (C) or FDo-derived (D) TRaMs.


Figure 1RAW264 . 7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h , Ca WT or Ca YL , and Lm WT or Lm Δhly for 60 min . ( A ) Levels of Rab8A and Rab8A pT72 phosphorylation were analysed by Western blot and quantified by densitometry . Data represent the mean ± SEM of three independent biological experiments . One - way ANOVA followed by Dunnett ' s test compared to WT strains . ns = non - significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 . ( B ) Recruitment of endogenous LRRK2 and EGFP - Rab8A to M . tuberculosis ( Mtb ) was visualised by confocal microscopy . Scale bar = 5 μm . ( C ) C . albicans ( Ca ) was analysed by confocal microscopy . Scale bar = 5 μm . Bottom panels show quantitative analysis of marker association to the respective pathogen . Data represents the mean ± SEM of two to four independent biological replicates . ( D ) L . monocytogenes ( Lm ) was analysed by confocal microscopy . Scale bar = 5 μm . Bottom panels show quantitative analysis of marker association to the respective pathogen . Data represents the mean ± SEM of two to four independent biological replicates .

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Figure 1RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h, Ca WT or Ca YL, and Lm WT or Lm Δhly for 60 min. (A) Levels of Rab8A and Rab8A pT72 phosphorylation were analysed by Western blot and quantified by densitometry. Data represent the mean ± SEM of three independent biological experiments. One-way ANOVA followed by Dunnett's test compared to WT strains. ns= non-significant; *p ≤0.05; **p ≤0.01.(B) Recruitment of endogenous LRRK2 and EGFP-Rab8A to M. tuberculosis (Mtb) was visualised by confocal microscopy. Scale bar = 5 μm.(C) C. albicans (Ca) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates.(D) L. monocytogenes (Lm) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates.