Figure 4A Western blot analysis of selected protein markers , including ERα , EGFR , ERBB2 , EpCAM , K18 , K19 , K8 , E - cadherin , N - cadherin , vimentin , CD44 , CD24 , SNAIL , SLUG , TWIST and α - tubulin ( as a loading control ) ( n = 3 replicates ) . CTC - ITB - 01 was compared to more mesenchymal ER - Hs578t and epithelial ER + MCF - 7 breast cancer cell lines . B ICC staining of CTC - ITB - 01 for pan - keratin ( orange ) , ER ( green ) , and DAPI ( blue ) . The scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 ( 1 ) and ( 2 ) . MCF - 7 cells are depicted as reference cell line . C PAM50 classifier results showing probabilities of pooled CTC - ITB - 01 cells matching specific molecular breast cancer subtypes . Starting from lowest probability , CTC - ITB - 01 was classified as 1 . 02 % ( s = 2 . 5 % ) normal - like , 3 . 11 % ( s = 6 . 6 % ) Basal - like , 13 . 11 % ( s = 9 . 3 % ) ERBB2 - positive , 16 . 77 % ( s = 23 . 7 % ) luminal A , and 65 . 22 % ( s = 16 . 6 % ) luminal B breast cancer subtype . Data was generated from n = 3 replicates . D PAM50 classifier results showing probabilities of the non - adherent and adherent CTC - ITB - 01 fractions matching molecular subtypes . Selected bars are not visible due to extremely low probability ( close to zero ) . Both fractions show greatest alignment with a luminal B subtype . Starting from lowest probability , the adherent fraction of CTC - ITB - 01 was classified as 0 % ( ± 0 % ) normal - like , 6 . 22 % ( ± 9 . 0 % ) Basal - like , 17 . 85 % ( ± 8 . 6 % ) ERBB2 - positive , 0 % ( s = 0 % ) luminal A , and 75 . 92 % ( s = 0 . 7 % ) luminal B breast cancer subtype . The suspension cell fraction of CTC - ITB - 01 was classified as 2 . 04 % ( s = 3 . 5 % ) normal - like , 0 % ( s = 0 % ) Basal - like , 17 . 32 % ( s = 9 . 7 % ) ERBB2 - positive , 33 . 5 % ( s = 23 . 8 % ) luminal A , and 68 . 67 % ( s = 18 . 6 % ) luminal B breast cancer subtype , respectively . Data was generated from n = 3 replicates . ( E ) ALDH activity measurement on viable cells via flow cytometry using the ALDEFLUOR ™ assay . CTC - ITB - 01 is compared to A549 as recommended control cell line . An internal ( DEAB ) control is also depicted . 77 . 92 % of the gated CTC - ITB - 01 population are ALDH + .

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

Figure 4A Western blot analysis of selected protein markers, including ERα, EGFR, ERBB2, EpCAM, K18, K19, K8, E-cadherin, N-cadherin, vimentin, CD44, CD24, SNAIL, SLUG, TWIST and α-tubulin (as a loading control) (n=3 replicates). CTC-ITB-01 was compared to more mesenchymal ER- Hs578t and epithelial ER+ MCF-7 breast cancer cell lines.B ICC staining of CTC-ITB-01 for pan-keratin (orange), ER (green), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01 (1) and (2). MCF-7 cells are depicted as reference cell line.C PAM50 classifier results showing probabilities of pooled CTC-ITB-01 cells matching specific molecular breast cancer subtypes. Starting from lowest probability, CTC-ITB-01 was classified as 1.02% (s=2.5%) normal-like, 3.11% (s=6.6%) Basal-like, 13.11% (s=9.3%) ERBB2-positive, 16.77% (s=23.7%) luminal A, and 65.22% (s=16.6%) luminal B breast cancer subtype. Data was generated from n=3 replicates.D PAM50 classifier results showing probabilities of the non-adherent and adherent CTC-ITB-01 fractions matching molecular subtypes. Selected bars are not visible due to extremely low probability (close to zero). Both fractions show greatest alignment with a luminal B subtype. Starting from lowest probability, the adherent fraction of CTC-ITB-01 was classified as 0% (±0%) normal-like, 6.22% (±9.0%) Basal-like, 17.85% (±8.6%) ERBB2-positive, 0% (s=0%) luminal A, and 75.92% (s=0.7%) luminal B breast cancer subtype. The suspension cell fraction of CTC-ITB-01 was classified as 2.04% (s=3.5%) normal-like, 0% (s=0%) Basal-like, 17.32% (s=9.7%) ERBB2-positive, 33.5% (s=23.8%) luminal A, and 68.67% (s=18.6%) luminal B breast cancer subtype, respectively. Data was generated from n=3 replicates.(E) ALDH activity measurement on viable cells via flow cytometry using the ALDEFLUOR™ assay. CTC-ITB-01 is compared to A549 as recommended control cell line. An internal (DEAB) control is also depicted. 77.92% of the gated CTC-ITB-01 population are ALDH+.


Figure 5A ICC staining of CTC - ITB - 01 for ß - catenin ( green ) , E - cadherin ( orange ) , and DAPI ( blue ) . The scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 . B ICC staining of CTC - ITB - 01 for α - catenin ( green ) , visualization of actin filaments by phalloidin ( orange ) , and nuclei by DAPI ( blue ) . The scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 . C Generic EMT score of the adherent and non - adherently growing CTC - ITB - 01 fractions in relation to tumor samples from different cancer entities . Scaling ranges from 1 ( completely epithelial ) to - 1 ( entirely mesenchymal ) . The adherent fraction is shown with a black dot , the non - adherent fraction with a black triangle , and the mean score per cancer entity by a black line . D Generic EMT score of the adherent and non - adherently growing CTC - ITB - 01 fractions in relation to established cancer cell lines . Scaling ranges from 1 ( completely epithelial ) to - 1 ( entirely mesenchymal ) . The adherent fraction is shown with a black dot , the non - adherent fraction with a black triangle , and the mean score per cancer entity by a black line .

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Figure 5A ICC staining of CTC-ITB-01 for ß-catenin (green), E-cadherin (orange), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.B ICC staining of CTC-ITB-01 for α-catenin (green), visualization of actin filaments by phalloidin (orange), and nuclei by DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.C Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to tumor samples from different cancer entities. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line. D Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to established cancer cell lines. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line.


Figure 6A In vivo bioluminescence imaging of CTC - ITB - 01 in four NSG mice . Blue indicates low radiance , red indicates high radiance . B Logarithmic plotting of in vivo radiance increase over time . Curves represent means ± SEM of measurements performed on multiple glands ( n = 8 ) . C H & E staining of xenografted mouse mammary glands show formation of primary tumors . D Stereoscope image of a mammary gland of immunodeficient NSG mouse . Fluorescently tagged CTC - ITB - 01 cells in green . E IHC staining for ER , confirms retained in vivo ER - positivity of formed tumors . Representative images of ER staining in higher ( left panel ) and lower ( right panel ) magnification of 2 different areas of a xenografted mammary gland . F Representative ex vivo luminescence images of indicated organs from engrafted mice . Organs include brain ( 1 ) , lung ( 2 ) , liver ( 3 ) and bones ( 4 ) . G Scatter blot showing ex vivo radiance intensity of metastatic organs in three different mice ( M1 , M2 , M3 ) , indicating highest intensity in liver and bone . Data represent means ± SD of measurements performed on metastatic organs derived from three different mice .

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Figure 6A In vivo bioluminescence imaging of CTC-ITB-01 in four NSG mice. Blue indicates low radiance, red indicates high radiance. B Logarithmic plotting of in vivo radiance increase over time. Curves represent means ± SEM of measurements performed on multiple glands (n=8). C H&E staining of xenografted mouse mammary glands show formation of primary tumors.D Stereoscope image of a mammary gland of immunodeficient NSG mouse. Fluorescently tagged CTC-ITB-01 cells in green.E IHC staining for ER, confirms retained in vivo ER-positivity of formed tumors. Representative images of ER staining in higher (left panel) and lower (right panel) magnification of 2 different areas of a xenografted mammary gland.F Representative ex vivo luminescence images of indicated organs from engrafted mice. Organs include brain (1), lung (2), liver (3) and bones (4). G Scatter blot showing ex vivo radiance intensity of metastatic organs in three different mice (M1, M2, M3), indicating highest intensity in liver and bone. Data represent means ± SD of measurements performed on metastatic organs derived from three different mice.


Figure 7A Equal numbers of CTC - ITB - 01 cells were seeded on 6 - well culture plates and grown in the presence or absence of different 17 - β - estradiol concentrations as indicated . After 10 days cells were fixed and stained with Coomassie blue staining ( right panel ) . Macroscopic photos are shown of the fixed cell colonies from cells grown in the absence or presence of the indicated E2 concentrations ( right panel ) . Scale bar 200 µm . B Stable knockdown of ER - alpha within CTC - ITB - 01 cells was performed by using lentiviral transfer of non - targeted ( scramble ) or two different targeted shRNAs against ER - alpha . CTC - ITB - 01 cells were seeded on 6 - well plates . After 10 days cells were fixed and stained ( left upper panel ) . Macroscopic photos are shown of the fixed cells ( left lower panel ) , scale bar 200 µm . Western blots of lysates from knockdown of ER - alpha expression in CTC - ITB - 01 cells transduced with the same amounts of lentiviral vectors were probed with the indicated antibodies . The extracts were obtained 72h after lentiviral transduction ( right panel ) . Where indicated , western blots were visualized at long exposure ( l . e . ) and / or short exposure ( s . e . ) . C CTC - ITB - 01 and MCF - 7 cells were grown under the same culture conditions in the presence or absence of E2 as indicated . 24h after E2 deprivation or E2 - treatment cells were harvested . Western blots of the protein lysates were probed with antibodies targeting ER - alpha , FOXM1 , Bcl - 2 , PR - A , PR - B and ID1 , actin was used as loading control , visualized at long exposure ( l . e . ) and / or short exposure ( s . e . ) . ( D ) Growth curves of CTC - ITB - 01 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . E ) Growth curves of MCF - 7 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . ( F ) Influence of Palbociclib on CTC - ITB - 01 growth after 300 hours Data was chosen from one representative time point during the exponential growth phase . Statistical significance was analyzed with a one - way ANOVA with Dunnett ' s multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide ( DMSO ) - . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . G ) Influence of Palbociclib on MCF - 7 after 80 hours . Data was chosen from one representative time point during the exponential growth phase . Statistical significance was analyzed with a one - way ANOVA with Dunnett ' s multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide ( DMSO ) - . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . ( H ) Effect of Palbociclib on the growth of CTC - ITB - 01 cells Concentrations were transformed to common logarithm . Three - parameter non - linear logistic regression was used to determine the IC50 . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . Error bars for standard deviation smaller than the symbols are not displayed . I ) Effect of Palbociclib on the growth of MCF - 7 cells . Concentrations were transformed to common logarithm . Three - parameter non - linear logistic regression was used to determine the IC50 . The mean values from three technical triplicates ( one experiment ) with standard deviation are shown . Error bars for standard deviation smaller than the symbols are not displayed .

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 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 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 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 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 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

Figure 7A Equal numbers of CTC-ITB-01 cells were seeded on 6-well culture plates and grown in the presence or absence of different 17-β-estradiol concentrations as indicated. After 10 days cells were fixed and stained with Coomassie blue staining (right panel). Macroscopic photos are shown of the fixed cell colonies from cells grown in the absence or presence of the indicated E2 concentrations (right panel). Scale bar 200 µm.B Stable knockdown of ER-alpha within CTC-ITB-01 cells was performed by using lentiviral transfer of non-targeted (scramble) or two different targeted shRNAs against ER-alpha. CTC-ITB-01 cells were seeded on 6-well plates. After 10 days cells were fixed and stained (left upper panel). Macroscopic photos are shown of the fixed cells (left lower panel), scale bar 200 µm. Western blots of lysates from knockdown of ER-alpha expression in CTC-ITB-01 cells transduced with the same amounts of lentiviral vectors were probed with the indicated antibodies. The extracts were obtained 72h after lentiviral transduction (right panel). Where indicated, western blots were visualized at long exposure (l.e.) and/or short exposure (s.e.).C CTC-ITB-01 and MCF-7 cells were grown under the same culture conditions in the presence or absence of E2 as indicated. 24h after E2 deprivation or E2-treatment cells were harvested. Western blots of the protein lysates were probed with antibodies targeting ER-alpha, FOXM1, Bcl-2, PR-A, PR-B and ID1, actin was used as loading control, visualized at long exposure (l.e.) and/or short exposure (s.e.).(D) Growth curves of CTC-ITB-01 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.E) Growth curves of MCF-7 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.(F) Influence of Palbociclib on CTC-ITB-01 growth after 300 hours Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.G) Influence of Palbociclib on MCF-7 after 80 hours. Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.(H) Effect of Palbociclib on the growth of CTC-ITB-01 cells Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.I) Effect of Palbociclib on the growth of MCF-7 cells. Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.


Figure 1Representative immunofluorescence images of SCOTIN - KO cells expressing Str - Ii - SCOTIN - SBP - EGFP with 40 μM D - biotin treatment for the indicated times . B Upper , localization of RUSH reporters retained on the ER membrane with Sec61β ( red ) . Bottom , localization of released RUSH reporters after 240 min of biotin treatment . The arrow indicates the EGFP signal on the ER . The arrowhead indicates the EGFP signal on vesicles . The empty arrowhead indicates the EGFP signal on the plasma membrane . Scale bar : 20 μm . Scale bars in the magnified images : 5 μm . Representative immunofluorescence images of SCOTIN - KO cells expressing Str - Ii - VSVG - SBP - EGFP ( green ) with 40 μM D - biotin treatment for the indicated times . , Upper , localization of RUSH reporters retained on the ER membrane with Sec61β ( red ) . Bottom , localization of released RUSH reporters after 240 min of biotin treatment . The arrow indicates the EGFP signal on the ER . The arrowhead indicates the EGFP signal on vesicles . The empty arrowhead indicates the EGFP signal on the plasma membrane . Scale bar : 20 μm . Scale bars in the magnified images : 5 μm . ( F ) The colocalization of EGFP and Sec61 or CD63 was calculated as the Pearson correlation coefficient from the experimental data in Figure 1E . The error bars indicate the SEMs . n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * < 0 . 01 and * * * * P < 0 . 0001 .

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Figure 1Representative immunofluorescence images of SCOTIN-KO cells expressing Str-Ii-SCOTIN-SBP-EGFP with 40 μM D-biotin treatment for the indicated times. B Upper, localization of RUSH reporters retained on the ER membrane with Sec61β (red). Bottom, localization of released RUSH reporters after 240 min of biotin treatment. The arrow indicates the EGFP signal on the ER. The arrowhead indicates the EGFP signal on vesicles. The empty arrowhead indicates the EGFP signal on the plasma membrane. Scale bar: 20 μm. Scale bars in the magnified images: 5 μm.Representative immunofluorescence images of SCOTIN-KO cells expressing Str-Ii-VSVG-SBP-EGFP (green) with 40 μM D-biotin treatment for the indicated times. , Upper, localization of RUSH reporters retained on the ER membrane with Sec61β (red). Bottom, localization of released RUSH reporters after 240 min of biotin treatment. The arrow indicates the EGFP signal on the ER. The arrowhead indicates the EGFP signal on vesicles. The empty arrowhead indicates the EGFP signal on the plasma membrane. Scale bar: 20 μm. Scale bars in the magnified images: 5 μm.(F) The colocalization of EGFP and Sec61 or CD63 was calculated as the Pearson correlation coefficient from the experimental data in Figure 1E. The error bars indicate the SEMs. n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, **<0.01 and ****P<0.0001.


Figure 2 ( A ) Immunoblot analysis of proteinase K protection assay . HeLa cells were transfected with SCOTIN - Myc ( Total ) and digitonin - permeabilized . The supernatants ( Cytosol ) and pellet ( PNSs ) were separated by centrifugation . The PNSs were incubated with PBS or with proteinase K ( + PK ) in the presence or absence of Triton X - 100 ( + PK + Triton X ) . ( B ) Representative high - resolution microscopy images of Rab5 Q79L - EGFP - and SCOTIN - DsRed - expressing HeLa cells . Magnified images show enlarged endosomes . Ⅰ : SCOTIN on the endosomal membrane , Ⅱ : SCOTIN on the endosomal membrane and luminal side . Scale bar : 10 μm . Scale bars in the magnified images : 5 μm . ( C ) Plot analysis of SCOTIN relative to the Rab5 Q79L signal shown . The graph indicates the fluorescence intensity of each channel on lines Ⅰ and Ⅱ from Figure 2B . The arrow indicates the boundary of the endosomal membrane . ( F ) Left : Representative images of Rab5 Q79L - mCherry - expressing SCOTIN - FP11 knock - in cells . Right : Plot analysis of SCOTIN - FP11 relative to the Rab5 Q79L signal shown . The graph indicates the fluorescence intensity of each channel on the lines . The arrow indicates the boundary of the endosomal membrane . Scale bars : 10 μm . Scale bars in the magnified images : 5 μm .

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Figure 2(A) Immunoblot analysis of proteinase K protection assay. HeLa cells were transfected with SCOTIN-Myc (Total) and digitonin-permeabilized. The supernatants (Cytosol) and pellet (PNSs) were separated by centrifugation. The PNSs were incubated with PBS or with proteinase K (+ PK) in the presence or absence of Triton X-100 (+PK +Triton X).(B) Representative high-resolution microscopy images of Rab5 Q79L-EGFP- and SCOTIN-DsRed-expressing HeLa cells. Magnified images show enlarged endosomes. Ⅰ: SCOTIN on the endosomal membrane, Ⅱ: SCOTIN on the endosomal membrane and luminal side. Scale bar: 10 μm. Scale bars in the magnified images: 5 μm. (C) Plot analysis of SCOTIN relative to the Rab5 Q79L signal shown. The graph indicates the fluorescence intensity of each channel on lines Ⅰ and Ⅱ from Figure 2B. The arrow indicates the boundary of the endosomal membrane.(F) Left: Representative images of Rab5 Q79L-mCherry-expressing SCOTIN-FP11 knock-in cells. Right: Plot analysis of SCOTIN-FP11 relative to the Rab5 Q79L signal shown. The graph indicates the fluorescence intensity of each channel on the lines. The arrow indicates the boundary of the endosomal membrane. Scale bars : 10 μm. Scale bars in the magnified images: 5 μm.


Figure 3 ( B ) Representative images from a PLA with VAPA and Rab7 in WT and SCOTIN - Dsred - overexpressing HeLa cells . Only one primary antibody ( anti - Rab7 antibody alone ) served as a negative control . The PLA color legends are inverted . Asterisks indicate transfected cells . All scale bars : 20 μm . ( C ) The number of PLA signals in each cell was quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * < 0 . 05 and * * * * P < 0 . 0001 . ( E ) The number of PLA signals in each cell was quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * < 0 . 05 and * * * * P < 0 . 0001 . ( F ) Immunoblot analysis of WT and SCOTIN - KO HeLa cell lysates for VAPA , Rab7 , SCOTIN , and α - tubulin . ( G ) Representative images from a PLA with VAPA and Rab7 in SCOTIN - , TMPRD - , or PRD - Myc - transfected SCOTIN - KO HeLa cells . The PLA color legends are inverted . All scale bars : 20 μm . ( H ) The number of PLA signals in each cell was quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * < 0 . 05 and * * * * P < 0 . 0001 .

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 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 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

Figure 3(B) Representative images from a PLA with VAPA and Rab7 in WT and SCOTIN-Dsred-overexpressing HeLa cells. Only one primary antibody (anti-Rab7 antibody alone) served as a negative control. The PLA color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (C) The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, *<0.05 and ****P<0.0001.(E) The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, *<0.05 and ****P<0.0001.(F) Immunoblot analysis of WT and SCOTIN-KO HeLa cell lysates for VAPA, Rab7, SCOTIN, and α-tubulin.(G) Representative images from a PLA with VAPA and Rab7 in SCOTIN-, TMPRD-, or PRD-Myc-transfected SCOTIN-KO HeLa cells. The PLA color legends are inverted. All scale bars: 20 μm. (H) The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, *<0.05 and ****P<0.0001.


Figure 4SCOTIN PRD - mediated tethering of SUVs and GUVs . E , Representative confocal images of SCOTIN PRD - induced membrane tethering between GUVs and SUVs . PRD - SUV aligned to the PRD - GUV membrane ( arrowhead , type I in the model ) or accumulated at the surface of GUVs ( arrow , type Ⅱ in the model ) .

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Figure 4SCOTIN PRD-mediated tethering of SUVs and GUVs. E, Representative confocal images of SCOTIN PRD-induced membrane tethering between GUVs and SUVs. PRD-SUV aligned to the PRD-GUV membrane (arrowhead, type I in the model) or accumulated at the surface of GUVs (arrow, type Ⅱ in the model).


Figure 5 ( B ) Representative images of VAPA - Rab7 PLA signals taken from Str - Ii - SCOTIN - SBP - EGFP transfected SCOTIN - KO cells after untreated ( - Biotin ) or biotin treatment ( + Biotin 4 h ) . For the PLA signal , the color legends are inverted . Asterisks indicate transfected cells . All scale bars : 20 μm . ( C ) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * < 0 . 01 , * * * < 0 . 001 , and * * * * P < 0 . 0001 . ( D ) Representative images of Sec61β - Emerald - or Sec61β - PRD - Emerald - transfected SCOTIN - KO cells . Cells were stained with RTN4 ( red ) . Arrowheads indicate the accumulated ER membrane . All scale bars : 20 μm . ( G ) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * < 0 . 01 , * * * < 0 . 001 , and * * * * P < 0 . 0001 .

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 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 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

Figure 5(B) Representative images of VAPA-Rab7 PLA signals taken from Str-Ii-SCOTIN-SBP-EGFP transfected SCOTIN-KO cells after untreated (-Biotin) or biotin treatment (+ Biotin 4 h). For the PLA signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (C) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, **<0.01, ***<0.001, and ****P<0.0001.(D) Representative images of Sec61β-Emerald- or Sec61β-PRD-Emerald-transfected SCOTIN-KO cells. Cells were stained with RTN4 (red). Arrowheads indicate the accumulated ER membrane. All scale bars: 20 μm.(G) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, **<0.01, ***<0.001, and ****P<0.0001.


Figure 6 ( C ) HEK293 cells were transfected with SCOTIN - GST along with empty ( pcDNA3 . 1 - Myc ) , SCOTIN - Myc , or SCOTIN ( Δ150 - 177 ) - Myc plasmids for 48 h . SCOTIN - GST was pulled down from total cell lysates using glutathione - Sepharose beads , and the interacting proteins were analyzed by immunoblotting . ( D ) Representative images from a PLA with VAPA and Rab7 in SCOTIN - KO HeLa cells reconstituted with FL SCOTIN , SCOTIN ( ΔPRD ) , or SCOTIN ( Δ150 - 177 ) - DsRed . The PLA color legends are inverted . Asterisks indicate transfected cells . All scale bars : 20 μm . ( G ) Representative images of VAPA - VAPA PLA signals taken from Str - Ii - SCOTIN ( Δ150 - 177 ) - SBP - EGFP transfected SCOTIN - KO cells after untreated ( - Biotin ) or biotin - treated ( + Bition 4 h ) . For the PLA signal , the color legends are inverted . Asterisks indicate transfected cells . All scale bars : 20 μm . ( H ) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ . Data information : n : number of cells analyzed . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * * * P < 0 . 0001 .

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 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 O O O O O O O O O O O O O O O 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(C) HEK293 cells were transfected with SCOTIN-GST along with empty (pcDNA3.1-Myc), SCOTIN-Myc, or SCOTIN(Δ150-177)-Myc plasmids for 48 h. SCOTIN-GST was pulled down from total cell lysates using glutathione-Sepharose beads, and the interacting proteins were analyzed by immunoblotting.(D) Representative images from a PLA with VAPA and Rab7 in SCOTIN-KO HeLa cells reconstituted with FL SCOTIN, SCOTIN(ΔPRD), or SCOTIN(Δ150-177)-DsRed. The PLA color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm.(G) Representative images of VAPA-VAPA PLA signals taken from Str-Ii-SCOTIN(Δ150-177)-SBP-EGFP transfected SCOTIN-KO cells after untreated (-Biotin) or biotin-treated (+Bition 4 h). For the PLA signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (H) The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.


Figure 7 ( A - D ) TEM images of the ER and endosomes of HeLa ( A ) , SCOTIN - KO HeLa ( B ) , SCOTIN - DsRed transfected SCOTIN - KO HeLa ( C ) , and SCOTIN Δ150 - 177 - DsRed transfected SCOTIN - KO HeLa ( D ) . DsRed - positive cells were processed for EM quantification .

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Figure 7(A-D) TEM images of the ER and endosomes of HeLa (A), SCOTIN-KO HeLa (B), SCOTIN-DsRed transfected SCOTIN-KO HeLa (C), and SCOTIN Δ150-177-DsRed transfected SCOTIN-KO HeLa (D). DsRed-positive cells were processed for EM quantification.


Figure 8 ( A ) Distribution of late endosomes ( CD63 ) in WT or SCOTIN - KO HeLa cells . Representative Z projections of confocal images are shown . The color legends are inverted . Scale bars : 20 μm . ( B ) Spatial distribution of late endosomes ( CD63 ) expressed as the fractional distance of fluorescent pixels along a straight line drawn through the endolysosomal cloud from the nuclear center ( 0 ) to the plasma membrane ( 1 . 0 ) from the experimental data in Figure 8A . ( C ) Quantification of the number of CD63 - positive vesicles per cell from the experimental data in Figure 8A . Data information : n : number of cells analyzed , with # of data points analyzed per condition given in parentheses . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * * * P < 0 . 0001 . ( D ) Live - image analysis of WT and SCOTIN - KO HeLa cells expressing Rab7 - EGFP . Left : confocal images taken at time 0 . Right : time - lapse images of Rab7 - EGFP in WT and SCOTIN - KO HeLa cells , as shown by temporal color - coded representations . Each confocal image of late endosomes was color coded according to its acquisition time point , with images taken at 12 - sec intervals over 5 min . A total of 24 color - coded images were projected to form a single image that demonstrates the dynamics of Rab7 - labeled late endosomes . White = static late endosomes . Multicolored = motile late endosomes . Boxed , magnified images highlight selected perinuclear and peripheral regions . Scale bar : 30 μm . Scale bars in the magnified images : 3 μm . ( E ) Quantification of the fractional distance of Rab7 - EGFP - positive late endosomes in WT and SCOTIN - KO HeLa cells from the experimental data in Figure 8D . Error bars indicate the SEMs . ( F ) Quantification of Rab7 - EGFP - positive late endosome vesicles per cell from the experimental data in Figure 8D . Data information : n : number of cells analyzed , with # of data points analyzed per condition given in parentheses . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * * * P < 0 . 0001 . ( G , H ) Live - image analysis of Rab7 - EGFP in SCOTIN - KO HeLa cells reconstituted with SCOTIN - DsRed ( G ) or SCOTIN ( Δ150 - 177 ) - DsRed ( H ) taken at time 0 or for 5 min ( color - coded images ) . Lower , boxed , magnified images of selected perinuclear and peripheral regions . Scale bar : 20 μm . Scale bars in the magnified images : 2 μm . ( I ) Quantification of the fractional distance of Rab7 - EGFP in SCOTIN - KO cells reconstituted with SCOTIN - DsRed or SCOTIN ( Δ150 - 177 ) - DsRed from the experimental data in Figure 8G and 8H . Data information : n : number of cells analyzed , with # of data points analyzed per condition given in parentheses . Data are shown as bars with dots and represent the means ± SEMs . P values were determined using a two - tailed unpaired t test . Significant differences are labeled , * * * * P < 0 . 0001 .

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 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 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

Figure 8(A) Distribution of late endosomes (CD63) in WT or SCOTIN-KO HeLa cells. Representative Z projections of confocal images are shown. The color legends are inverted. Scale bars: 20 μm. (B) Spatial distribution of late endosomes (CD63) expressed as the fractional distance of fluorescent pixels along a straight line drawn through the endolysosomal cloud from the nuclear center (0) to the plasma membrane (1.0) from the experimental data in Figure 8A. (C) Quantification of the number of CD63-positive vesicles per cell from the experimental data in Figure 8A. Data information: n: number of cells analyzed, with # of data points analyzed per condition given in parentheses. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.(D) Live-image analysis of WT and SCOTIN-KO HeLa cells expressing Rab7-EGFP. Left: confocal images taken at time 0. Right: time-lapse images of Rab7-EGFP in WT and SCOTIN-KO HeLa cells, as shown by temporal color-coded representations. Each confocal image of late endosomes was color coded according to its acquisition time point, with images taken at 12-sec intervals over 5 min. A total of 24 color-coded images were projected to form a single image that demonstrates the dynamics of Rab7-labeled late endosomes. White = static late endosomes. Multicolored = motile late endosomes. Boxed, magnified images highlight selected perinuclear and peripheral regions. Scale bar: 30 μm. Scale bars in the magnified images: 3 μm. (E) Quantification of the fractional distance of Rab7-EGFP-positive late endosomes in WT and SCOTIN-KO HeLa cells from the experimental data in Figure 8D. Error bars indicate the SEMs. (F) Quantification of Rab7-EGFP-positive late endosome vesicles per cell from the experimental data in Figure 8D. Data information: n: number of cells analyzed, with # of data points analyzed per condition given in parentheses. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.(G, H) Live-image analysis of Rab7-EGFP in SCOTIN-KO HeLa cells reconstituted with SCOTIN-DsRed (G) or SCOTIN(Δ150-177)-DsRed (H) taken at time 0 or for 5 min (color-coded images). Lower, boxed, magnified images of selected perinuclear and peripheral regions. Scale bar: 20 μm. Scale bars in the magnified images: 2 μm. (I) Quantification of the fractional distance of Rab7-EGFP in SCOTIN-KO cells reconstituted with SCOTIN-DsRed or SCOTIN(Δ150-177)-DsRed from the experimental data in Figure 8G and 8H. Data information: n: number of cells analyzed, with # of data points analyzed per condition given in parentheses. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.


figf1 ( b ) MAPPIT screen identified RAL - interacting proteins . A MAPPIT array covering close to 8 , 500 preys was screened with RALA - G23V as bait . Data are shown in a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus P value Source data can be found in Supplementary Table S1 . ( c ) MAPPIT assay confirmed interaction between the RAL proteins and USP33 . RALA - G23V or RALB - G23V baits were introduced into HEK293T cells together with the indicated preys . The results are expressed as a mean of normalized luciferase activity ± s . d ( leptin - treated cells versus leptin - untreated cells ) for three independent experiments . Statistics source data can be found in Supplementary Table S1 . ( d , e ) USP33 interacts with both RALA and RALB . At 48 h post - transfection with tagged USP33 and RAL expression constructs , the indicated proteins were then immunoprecipitated with anti - Flag ( M2 ) or anti - HA - agarose , followed by immunoblotting using anti - Flag or anti - HA antibodies . WCL , whole - cell lysate ; IP , immunoprecipitates . ( f ) Endogenous USP33 interacts with RALB . Flag - RALB was immunoprecipitated using anti - Flag ( M2 ) agarose and analysed for co - precipitation with endogenous USP33 using anti - USP33 antibody . Cells expressing USP33 shRNA were used as a negative control . ( g ) GTP binding is not required for interaction between USP33 and the RAL proteins . The GST - tagged RALA and RALB were isolated with glutathione - agarose and mixed with lysates derived from HEK293T cells expressing Flag - USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag - specific antibody . ( h ) USP33 affects ubiquitylation of RALB , but not RALA . 6xHis - tagged ubiquitin and Flag - RAL - G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA . Ubiquitylated RAL proteins were purified by Co2 + metal affinity chromatography and detected by antibodies specific to RALA or RALB . Uncropped images of blots are shown in Supplementary Fig . S7 . ( g ) GTP binding is not required for interaction between USP33 and the RAL proteins . The USP33 - tagged RALA and RALB were isolated with glutathione - agarose and mixed with lysates derived from HEK293T cells expressing Flag - USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag - specific antibody .

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figf1(b) MAPPIT screen identified RAL-interacting proteins. A MAPPIT array covering close to 8,500 preys was screened with RALA-G23V as bait. Data are shown in a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus P value Source data can be found in Supplementary Table S1.(c) MAPPIT assay confirmed interaction between the RAL proteins and USP33. RALA-G23V or RALB-G23V baits were introduced into HEK293T cells together with the indicated preys. The results are expressed as a mean of normalized luciferase activity ± s.d (leptin-treated cells versus leptin-untreated cells) for three independent experiments. Statistics source data can be found in Supplementary Table S1.(d,e) USP33 interacts with both RALA and RALB. At 48 h post-transfection with tagged USP33 and RAL expression constructs, the indicated proteins were then immunoprecipitated with anti-Flag (M2) or anti-HA-agarose, followed by immunoblotting using anti-Flag or anti-HA antibodies. WCL, whole-cell lysate; IP, immunoprecipitates.(f) Endogenous USP33 interacts with RALB. Flag-RALB was immunoprecipitated using anti-Flag (M2) agarose and analysed for co-precipitation with endogenous USP33 using anti-USP33 antibody. Cells expressing USP33 shRNA were used as a negative control.(g) GTP binding is not required for interaction between USP33 and the RAL proteins. The GST-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.(h) USP33 affects ubiquitylation of RALB, but not RALA. 6xHis-tagged ubiquitin and Flag-RAL-G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA. Ubiquitylated RAL proteins were purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALA or RALB. Uncropped images of blots are shown in Supplementary Fig. S7.(g) GTP binding is not required for interaction between USP33 and the RAL proteins. The USP33-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.


figf2 ( a ) Tandem affinity purification of ubiquitylated RALB . 6xHis - ubiquitin and Flag - RALB - G23V were co - transfected into HEK293T cells , and ubiquitylated RALB was purified using anti - Flag resin followed by Co2 + metal affinity chromatography . Isolated RALB was visualized by immunoblotting using anti - RALB antibody . ( b ) RALB undergoes ubiquitylation on multiple sites . Schematic diagram of the RALB domains ( upper panel ) . RALB ubiquitylation sites were identified in two independent tandem mass spectrometry analyses with a total coverage of 63 % of the RALB amino acid sequence ( Supplementary Fig . S4a ) . The major identified ubiquitylation sites , Lys 47 and Lys 160 , are located in the effector - interacting domain ( green ) and the G5 box ( red ) , respectively . ( c ) Substitution of arginine for lysine at position 47 significantly inhibits ubiquitylation of RALB . ( ( d ) USP33 regulates RALB ubiquitylation at Lys 47 . ( e ) The GTP - GDP status of RALB affects RALB ubiquitylation at Lys 47 . For c - e , 6xHis - tagged ubiquitin and the indicated RALB mutants were introduced into HEK293T cells expressing GFP shRNA or USP33 shRNA . Ubiquitylated RALB was purified by Co2 + metal affinity chromatography and detected by antibodies specific to RALB . WT , wild type . ( f ) Lack of ubiquitylation at Lys 47 inhibits RALB binding to SEC5 . The indicated Flag - tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - SEC5 antibody . g ) Ubiquitylation at Lys 47 impairs RALB binding to EXO84 . Flag - tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - EXO84 antibody . ( h ) Ubiquitylation at Lys 47 does not affect RALB activity . The activity of RALB mutants was determined by a RALBP1 - RBD binding assay . ( i ) The Rosetta v3 protocol described in ref . was used for ubiquitin - docking simulations . The plot represents the energies of the produced decoys distributed by the root - mean - square deviation ( RMSD ) of ubiquitin position relative to RALA from an arbitrarily chosen decoy . ( j ) Rosetta modelling of ubiquitylated - RALA - SEC5 - RBD and ubiquitylated - RALA - EXO84 - RBD complexes . Superposition of the top ten refined solutions of ubiquitylated - RALA - SEC5 - RBD complex , selected by combined scores of two independent docking simulations of ubiquitylated - RALA - SEC5 - RBD and ubiquitylated - RALA ( left ) . Simulated ubiquitin positions linked to Lys 47 of RALA were combined with the RALA - EXO84 complex ( right ) . Uncropped images of blots are shown in Supplementary Fig . S7 .

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figf2(a) Tandem affinity purification of ubiquitylated RALB. 6xHis-ubiquitin and Flag-RALB-G23V were co-transfected into HEK293T cells, and ubiquitylated RALB was purified using anti-Flag resin followed by Co2+ metal affinity chromatography. Isolated RALB was visualized by immunoblotting using anti-RALB antibody.(b) RALB undergoes ubiquitylation on multiple sites. Schematic diagram of the RALB domains (upper panel). RALB ubiquitylation sites were identified in two independent tandem mass spectrometry analyses with a total coverage of 63% of the RALB amino acid sequence (Supplementary Fig. S4a). The major identified ubiquitylation sites, Lys 47 and Lys 160, are located in the effector-interacting domain (green) and the G5 box (red), respectively.(c) Substitution of arginine for lysine at position 47 significantly inhibits ubiquitylation of RALB. ((d) USP33 regulates RALB ubiquitylation at Lys 47. (e) The GTP-GDP status of RALB affects RALB ubiquitylation at Lys 47. For c-e, 6xHis-tagged ubiquitin and the indicated RALB mutants were introduced into HEK293T cells expressing GFP shRNA or USP33 shRNA. Ubiquitylated RALB was purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALB. WT, wild type.(f) Lack of ubiquitylation at Lys 47 inhibits RALB binding to SEC5. The indicated Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-SEC5 antibody.g) Ubiquitylation at Lys 47 impairs RALB binding to EXO84. Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-EXO84 antibody.(h) Ubiquitylation at Lys 47 does not affect RALB activity. The activity of RALB mutants was determined by a RALBP1-RBD binding assay.(i) The Rosetta v3 protocol described in ref. was used for ubiquitin-docking simulations. The plot represents the energies of the produced decoys distributed by the root-mean-square deviation (RMSD) of ubiquitin position relative to RALA from an arbitrarily chosen decoy.(j) Rosetta modelling of ubiquitylated-RALA-SEC5-RBD and ubiquitylated-RALA-EXO84-RBD complexes. Superposition of the top ten refined solutions of ubiquitylated-RALA-SEC5-RBD complex, selected by combined scores of two independent docking simulations of ubiquitylated-RALA-SEC5-RBD and ubiquitylated-RALA (left). Simulated ubiquitin positions linked to Lys 47 of RALA were combined with the RALA-EXO84 complex (right). Uncropped images of blots are shown in Supplementary Fig. S7.


figf3 ( a ) USP33 does not affect RALB activity . The activity of RALB was determined in HEK293T cells expressing GFP shRNA or USP33 shRNA and empty vector ( V ) or HA - tagged USP33 by RALBP1 - RBD binding assay . ( b ) USP33 modulates interaction between RALB and SEC5 . Flag - tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA , USP33 shRNA , empty vector ( V ) or HA - tagged USP33 . The indicated proteins were then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - Flag or anti - SEC5 antibodies . ( c ) USP33 regulates RALB binding to EXO84 . Flag - tagged RALB was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells expressing the indicated constructs . The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti - HA or anti - EXO84 antibody . ( d ) Activated RALB co - localizes with both EXO84 and SEC5 . At 24 h after co - transfection with RALB - G23V - EGFP and HA - EXO84 , HeLa cells were immunostained with anti - HA and anti - SEC5 antibodies . Scale bars , 10 μm . The outlined areas are shown at higher magnification in the bottom panels . ( e ) RALB ubiquitylation controls interaction between EXO84 and SEC5 . Flag - tagged SEC5 was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells overexpressing RALB - G23V or RALB - G23V - K47R . The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti - EXO84 antibody . ( f ) USP33 regulates EXO84 - SEC5 complex formation . Flag - tagged SEC5 was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti - EXO84 antibody . ( g ) Suppression of SEC5 affects RALB binding to EXO84 . HA - tagged RALB - G23V was immunoprecipitated from HEK293T cells expressing the indicated constructs . The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti - EXO84 antibody . For a - c , f , g immunoblotting using antibodies specific for USP33 , anti - Flag ( M2 ) or anti - HA determined the levels of USP33 suppression or overexpression . Immunoblot analysis was performed using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S7 .

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figf3(a) USP33 does not affect RALB activity. The activity of RALB was determined in HEK293T cells expressing GFP shRNA or USP33 shRNA and empty vector (V) or HA-tagged USP33 by RALBP1-RBD binding assay.(b) USP33 modulates interaction between RALB and SEC5. Flag-tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA, USP33 shRNA, empty vector (V) or HA-tagged USP33. The indicated proteins were then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-Flag or anti-SEC5 antibodies.(c) USP33 regulates RALB binding to EXO84. Flag-tagged RALB was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-HA or anti-EXO84 antibody.(d) Activated RALB co-localizes with both EXO84 and SEC5. At 24 h after co-transfection with RALB-G23V-EGFP and HA-EXO84, HeLa cells were immunostained with anti-HA and anti-SEC5 antibodies. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels.(e) RALB ubiquitylation controls interaction between EXO84 and SEC5. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing RALB-G23V or RALB-G23V-K47R. The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti-EXO84 antibody.(f) USP33 regulates EXO84-SEC5 complex formation. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti-EXO84 antibody.(g) Suppression of SEC5 affects RALB binding to EXO84. HA-tagged RALB-G23V was immunoprecipitated from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-EXO84 antibody. For a-c,f,g immunoblotting using antibodies specific for USP33, anti-Flag (M2) or anti-HA determined the levels of USP33 suppression or overexpression. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.


figf4 ( a ) Suppression of USP33 facilitates TBK1 - SEC5 complex formation . Flag - tagged SEC5 was introduced into HEK293T cells expressing empty vector ( V ) or HA - tagged RALB - G23V together with GFP shRNA or USP33 shRNA as indicated , and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using antibody specific for TBK1 . ( c ) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB - SEC5 complex in response to poly ( I : C ) treatment . Flag - tagged SEC5 and the indicated HA - tagged RALB mutants were introduced into 293 - hTLR3 cells . At 48 h after transfection cells were treated 100 μg ml − 1 of poly ( I : C ) for 6 h . Flag - tagged SEC5 was immunoprecipitated with anti - Flag ( M2 ) , and analysed for co - precipitation by immunoblotting using anti - HA antibody . ( c ) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB - SEC5 complex in response to poly ( I : C ) treatment . Flag - tagged SEC5 and the indicated HA - tagged RALB mutants were introduced into 293 - hTLR3 cells . At 48 h after transfection cells were treated 100 μg ml − 1 of poly ( I : C ) for 6 h . Flag - tagged SEC5 was immunoprecipitated with anti - Flag ( M2 ) , and analysed for co - precipitation by immunoblotting using anti - HA antibody . ( d ) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3 . At 48 h after transfection with empty vector ( V ) , Flag - RALB - G23V and Flag - RALB - G23V - K47R , 293 - hTLR3 cells were treated with 10 μg ml − 1 of poly ( I : C ) for 6 h . Immunoblot analysis was performed using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S7 .

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figf4(a) Suppression of USP33 facilitates TBK1-SEC5 complex formation. Flag-tagged SEC5 was introduced into HEK293T cells expressing empty vector (V) or HA-tagged RALB-G23V together with GFP shRNA or USP33 shRNA as indicated, and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using antibody specific for TBK1.(c) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB-SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody.(c) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB-SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody.(d) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3. At 48 h after transfection with empty vector (V), Flag-RALB-G23V and Flag-RALB-G23V-K47R, 293-hTLR3 cells were treated with 10 μg ml−1 of poly(I:C) for 6 h. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.


figf5 ( a - d ) USP33 inhibits assembly of SEC5 - beclin - 1 complexes . The indicated proteins were immunoprecipitated from cells expressing GFP shRNA or USP33 shRNA with anti - Flag ( M2 ) , anti - HA or anti - SEC5 antibody and analysed for co - precipitation by immunoblotting using specific antibodies as indicated . ( e ) USP33 potentiates the interaction between EXO84 and beclin 1 . Endogenous beclin 1 was immunoprecipitated from HEK293T cells , expressing either empty vector ( V ) , HA - USP33 wild type ( WT ) or the catalytically inactive mutant C194S - H683Q , using anti - beclin 1 antibody . Immunoprecipitates were analysed for co - precipitation by immunoblotting using anti - EXO84 or anti - beclin 1 antibodies . ( f , g ) RALB but not RALA undergoes deubiquitylation under nutrient deprivation . 6xHis - tagged ubiquitin and the indicated RAL mutants were overexpressed in HEK293T cells . At 48 h after transfection cells were deprived of nutrients and incubated in Hank ' s buffered salt solution ( HBSS ) medium for 90 min . Ubiquitylated RALproteins were purified by Co2 + metal affinity chromatography and analysed by immunoblotting using anti - RALA or anti - RALB antibodies . ( h ) Nutrient starvation results in the accumulation of USP33 . HEK TE cells were incubated in HBSS medium for the indicated periods of time . Immunoblotting was performed using anti - USP33 antibody . ( i ) USP33 co - localizes with RALB - positive vesicles in the absence of nutrients . At 48 h after co - transfection with RALB - G23V - EGFP and USP33 - mCherry , HeLa cells were incubated in HBSS medium for 90 min . Cells were imaged for EGFP and mCherry fluorescence . Scale bars , 10 μm . The outlined areas are shown at higher magnification in the bottom panels . Arrows indicate USP33 co - localization to RALB - positive vesicles . ( j ) Beclin 1 co - localizes with USP33 . HeLa cells were transfected with beclin - 1 - HA and USP33 - mCherry . At 48 h after transfection , cells were incubated in HBSS medium for 90 minutes and treated as in i . Scale bars , 10 μm . The outlined areas are shown at higher magnification in the bottom panels . Uncropped images of blots are shown in Supplementary Fig . S7 .

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figf5(a-d) USP33 inhibits assembly of SEC5-beclin-1 complexes. The indicated proteins were immunoprecipitated from cells expressing GFP shRNA or USP33 shRNA with anti-Flag (M2), anti-HA or anti-SEC5 antibody and analysed for co-precipitation by immunoblotting using specific antibodies as indicated.(e) USP33 potentiates the interaction between EXO84 and beclin 1. Endogenous beclin 1 was immunoprecipitated from HEK293T cells, expressing either empty vector (V), HA-USP33 wild type (WT) or the catalytically inactive mutant C194S-H683Q, using anti-beclin 1 antibody. Immunoprecipitates were analysed for co-precipitation by immunoblotting using anti-EXO84 or anti-beclin 1 antibodies.(f,g) RALB but not RALA undergoes deubiquitylation under nutrient deprivation. 6xHis-tagged ubiquitin and the indicated RAL mutants were overexpressed in HEK293T cells. At 48 h after transfection cells were deprived of nutrients and incubated in Hank's buffered salt solution (HBSS) medium for 90 min. Ubiquitylated RALproteins were purified by Co2+ metal affinity chromatography and analysed by immunoblotting using anti-RALA or anti-RALB antibodies.(h) Nutrient starvation results in the accumulation of USP33. HEK TE cells were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using anti-USP33 antibody.(i) USP33 co-localizes with RALB-positive vesicles in the absence of nutrients. At 48 h after co-transfection with RALB-G23V-EGFP and USP33-mCherry, HeLa cells were incubated in HBSS medium for 90 min. Cells were imaged for EGFP and mCherry fluorescence. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Arrows indicate USP33 co-localization to RALB-positive vesicles.(j) Beclin 1 co-localizes with USP33. HeLa cells were transfected with beclin-1-HA and USP33-mCherry. At 48 h after transfection, cells were incubated in HBSS medium for 90 minutes and treated as in i. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Uncropped images of blots are shown in Supplementary Fig. S7.


figf6 ( a ) USP33 depletion inhibits accumulation of LC3 - lipid conjugates . HEK TE cells stably expressing GFP shRNA or USP33 shRNA were incubated with DMEM or HBSS medium for 4 h , in the presence or absence of 50 μM of chloroquine ( CQ ) . Cells were then assayed for the relative accumulation of LC3 - I and LC3 - II by immunoblotting analysis using anti - LC3 antibody . LC3 - II / GAPDH ratios have been calculated using ImageJ densitometric analysis . ( b - d ) USP33 expression levels affect the accumulation of LC3punctae . Wild - type USP33 or a USP33 - shRNA - resistant USP33 mutant ( USP33R ) was introduced into HEK TE cells stably expressing GFP shRNA or USP33 shRNA . The generated cells were incubated in HBSS medium for 4 h . USP33 was analysed by immunoblotting using anti - USP33 antibody ( b ) . Representative images of LC3immunostaining are shown ( c ) . Endogenous LC3punctae were quantified using the In Cell Analyser 2000 automatic imaging system and IN Cell Investigator software . The mean distribution of LC3punctae per cell is represented as the mean of two independent experiments . Statistics source data for d can be found in Supplementary Table S1 ( d ) . ( e ) USP33 depletion leads to increased levels of p62 . HEK TE cells expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for the indicated periods of time . Immunoblotting was performed using the indicated antibodies . ( f ) Overexpression of the RALB - G23V - K47R mutant leads to accumulation of LC3 - lipid conjugates . HEK293T cells overexpressing the RALB - G23V or RALB - G23V - K47R mutants were assayed for the relative accumulation of LC3 - I and LC3 - II by immunoblotting using anti - LC3 antibody .

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figf6(a) USP33 depletion inhibits accumulation of LC3-lipid conjugates. HEK TE cells stably expressing GFP shRNA or USP33 shRNA were incubated with DMEM or HBSS medium for 4 h, in the presence or absence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting analysis using anti-LC3 antibody. LC3-II/GAPDH ratios have been calculated using ImageJ densitometric analysis.(b-d) USP33 expression levels affect the accumulation of LC3punctae. Wild-type USP33 or a USP33-shRNA-resistant USP33 mutant (USP33R) was introduced into HEK TE cells stably expressing GFP shRNA or USP33 shRNA. The generated cells were incubated in HBSS medium for 4 h. USP33 was analysed by immunoblotting using anti-USP33 antibody (b). Representative images of LC3immunostaining are shown (c). Endogenous LC3punctae were quantified using the In Cell Analyser 2000 automatic imaging system and IN Cell Investigator software. The mean distribution of LC3punctae per cell is represented as the mean of two independent experiments. Statistics source data for d can be found in Supplementary Table S1 (d).(e) USP33 depletion leads to increased levels of p62. HEK TE cells expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using the indicated antibodies.(f) Overexpression of the RALB-G23V-K47R mutant leads to accumulation of LC3-lipid conjugates. HEK293T cells overexpressing the RALB-G23V or RALB-G23V-K47R mutants were assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting using anti-LC3 antibody.


Figure 1Demonstration of translational readthrough of AGO1 using luciferase - based reporter assay . Plasmid containing in - frame AGO1 - TGA ( or GCA ) - ISR - FLuc were transfected in HEK293 cells and translational readthrough was detected as FLuc activity normalized to the activity of co - transfected renilla luciferase ( RLuc ) . FLuc mRNA levels determined by RT - PCR are shown below . * * * , P = 0 . 0001 . Significance of the identity of the canonical stop codon in AGO1 translational readthrough . AGO1 - ISR - FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 cells and translational readthrough was quantified as described above . FLuc mRNA levels determined by RT - PCR are shown below . * * * , P = 0 . 0002 ; * * , P = 0 . 0004 . First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough . AGO1 - TGA - FLuc constructs with different lengths of ISR ( all in - frame with AGO1 and FLuc ) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate . FLuc activity reflects readthrough activity . * * , P = 0 . 012 ; * , P = 0 . 017 . Demonstration of translational readthrough of AGO1 using fluorescence - based reporter assay . Plasmid containing in - frame AGO1 - TGA ( or GCA ) - ISR - GFP were transfected in HEK293 cells and translational readthrough was detected as fluorescence . Scale bar , 50 um . The bar graph shows mean fluorescence intensities in cells transfected with indicated constructs . Fluorescence intensity was measured by flowcytometry . * * * , P = 0 . 007 .

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Figure 1Demonstration of translational readthrough of AGO1 using luciferase-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-FLuc were transfected in HEK293 cells and translational readthrough was detected as FLuc activity normalized to the activity of co-transfected renilla luciferase (RLuc). FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0001.Significance of the identity of the canonical stop codon in AGO1 translational readthrough. AGO1-ISR-FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 cells and translational readthrough was quantified as described above. FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0002; **, P = 0.0004.First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough. AGO1-TGA-FLuc constructs with different lengths of ISR (all in-frame with AGO1 and FLuc) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate. FLuc activity reflects readthrough activity. **, P = 0.012; *, P = 0.017.Demonstration of translational readthrough of AGO1 using fluorescence-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-GFP were transfected in HEK293 cells and translational readthrough was detected as fluorescence. Scale bar, 50 um. The bar graph shows mean fluorescence intensities in cells transfected with indicated constructs. Fluorescence intensity was measured by flowcytometry. ***, P = 0.007.


Figure 2Detection of endogenous Ago1x by Western blot in HEK293 cells using anti - Ago1x antibody and pre - immune serum . Western blot of lysates from HEK293 and HeLa cells using anti - Ago1x antibody pre - treated with Ago1x - specific peptide . Expression of Ago1x in HEK293 cells transfected with three AGO1 - specific siRNAs ( 1 , 2 , 3 ) . qRT - PCR analysis of AGO1 transcript expression in these cells is shown below . Proportion of Ago1x isoform in HEK293 cells . To separate Ago1x and Ago1 ( ~ 3 kDa difference ) , the cell lysate was electrophoresed for 3 . 5 h in 10 % polyacrylamide gel . Proportion of Ago1x and canonical isoform ( Ago1 ) was calculated by densitometric analysis . Results from three independent experiments are shown as a bar graph ( mean ± S . E . ) . Expression of Ago1x in mouse organs . Presence of ribosome footprint in the Inter stop codon region of AGO1 transcript in U2 - OS cells . Ribosome profiling data was taken from NCBI ' s sequence read archive ( SRA Accession no . SRR1257257 ) and ribosome footprints on AGO1 transcript were analyzed as described in Methods section . Zoomed area near the inter - stop codon region is shown in the inset . Position of canonical stop codon and the downstream in - frame stop codon are shown . Detection of Ago1x by Western blot in U2 - OS cells is shown ( right ) .

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Figure 2Detection of endogenous Ago1x by Western blot in HEK293 cells using anti-Ago1x antibody and pre-immune serum.Western blot of lysates from HEK293 and HeLa cells using anti-Ago1x antibody pre-treated with Ago1x-specific peptide.Expression of Ago1x in HEK293 cells transfected with three AGO1-specific siRNAs (1,2,3). qRT-PCR analysis of AGO1 transcript expression in these cells is shown below.Proportion of Ago1x isoform in HEK293 cells. To separate Ago1x and Ago1 (~ 3 kDa difference), the cell lysate was electrophoresed for 3.5 h in 10% polyacrylamide gel. Proportion of Ago1x and canonical isoform (Ago1) was calculated by densitometric analysis. Results from three independent experiments are shown as a bar graph (mean ± S.E.).Expression of Ago1x in mouse organs.Presence of ribosome footprint in the Inter stop codon region of AGO1 transcript in U2-OS cells. Ribosome profiling data was taken from NCBI's sequence read archive (SRA Accession no. SRR1257257) and ribosome footprints on AGO1 transcript were analyzed as described in Methods section. Zoomed area near the inter-stop codon region is shown in the inset. Position of canonical stop codon and the downstream in-frame stop codon are shown. Detection of Ago1x by Western blot in U2-OS cells is shown (right).


Figure 3Effect of let - 7a inhibitor on translational readthrough of AGO1 . let - 7a inhibitor or control inhibitor was transfected in HeLa cells and the expression of Ago1x was determined by Western blot after 48 h . Densitometric analysis of three independent experiments is shown as a bar diagram ( mean ± S . E . ) . * , P = 0 . 01 . Effect of let - 7a overexpression on translational readthrough of AGO1 . HeLa cells were stably transfected with pri - let - 7a overexpression construct . Ago1x expression was determined by Western blot . Densitometric analysis of three independent experiments is shown as a bar diagram ( mean ± S . E . ) . * , P = 0 . 007 . let - 7a inhibitor reduces the AGO1 readthrough efficiency . HeLa cells were transfected with let - 7a inhibitor or control inhibitor along with AGO1 - TGA ( or GCA ) - ISR - FLuc construct . Readthrough was quantified by measuring FLuc activity , which was normalized to co - transfected renilla luciferase activity . FLuc mRNA expression in HeLa cells treated with miRNA inhibitors is shown ( Right ) . * , P = 0 . 027 . AGO1 ISR with mutated let - 7a binding site shows reduced translational readthrough activity . Assay was done as described above in HeLa cells . Mutated sequence is shown ( top ) . * * * , P = 0 . 008 . AGO1 ISR with displaced let - 7a binding site ( AGO1 - TGA - disISR - FLuc ) shows reduced translational readthrough activity . let - 7a binding site was moved 18 nucleotides downstream of the stop codon . Readthrough assay was done as described above in HeLa cells . * * * , P < 0 . 0001 .

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Figure 3Effect of let-7a inhibitor on translational readthrough of AGO1. let-7a inhibitor or control inhibitor was transfected in HeLa cells and the expression of Ago1x was determined by Western blot after 48 h. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.01.Effect of let-7a overexpression on translational readthrough of AGO1. HeLa cells were stably transfected with pri-let-7a overexpression construct. Ago1x expression was determined by Western blot. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.007.let-7a inhibitor reduces the AGO1 readthrough efficiency. HeLa cells were transfected with let-7a inhibitor or control inhibitor along with AGO1-TGA (or GCA)-ISR-FLuc construct. Readthrough was quantified by measuring FLuc activity, which was normalized to co-transfected renilla luciferase activity. FLuc mRNA expression in HeLa cells treated with miRNA inhibitors is shown (Right). *, P = 0.027.AGO1 ISR with mutated let-7a binding site shows reduced translational readthrough activity. Assay was done as described above in HeLa cells. Mutated sequence is shown (top). ***, P = 0.008.AGO1 ISR with displaced let-7a binding site (AGO1-TGA-disISR-FLuc) shows reduced translational readthrough activity. let-7a binding site was moved 18 nucleotides downstream of the stop codon. Readthrough assay was done as described above in HeLa cells. ***, P < 0.0001.


Figure 4AGO1 ISR can drive translational readthrough in a heterologous context of RHOA . AGO1 coding sequence in AGO1 - TGA - ISR - FLuc construct was replaced by RHOA coding sequence ( 582 nucleotides ) and the readthrough assay was done as described above in HeLa cells . 3 ′ UTRRho , 159 nucleotides in the proximal 3 ′ UTR of RHOA ( NM _ 001664 ) ; ISRAgo1 , Inter - stop codon region of AGO1 . * * * , P < 0 . 0001 . AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase ( RLuc ) . AGO1 coding sequence in AGO1 - TGA - ISR - FLuc construct was replaced by RLuc coding sequence and the readthrough assay was done as described above in HeLa cells ( left ) and in vitro using rabbit reticulocyte lysate system ( right ) . * * , P = 0 . 006 ; * * * , P = 0 . 0001 . Tethering of N - peptide - tagged Ago1 or Ago1x can induce readthrough of AGO1 . Two BoxB elements were cloned downstream of the AGO1 and upstream of the in - frame FLuc as shown in the schematic . This construct was transfected in HeLa cells expressing N - peptide - tagged or FLAG - tagged Ago proteins as indicated . Readthrough was measured as described above . * , P = 0 . 035 ( with Welch correction ) ; * * , P = 0 . 016 .

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Figure 4AGO1 ISR can drive translational readthrough in a heterologous context of RHOA. AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RHOA coding sequence (582 nucleotides) and the readthrough assay was done as described above in HeLa cells. 3′UTRRho, 159 nucleotides in the proximal 3′UTR of RHOA (NM_001664); ISRAgo1, Inter-stop codon region of AGO1. ***, P < 0.0001.AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase (RLuc). AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RLuc coding sequence and the readthrough assay was done as described above in HeLa cells (left) and in vitro using rabbit reticulocyte lysate system (right). **, P = 0.006; ***, P = 0.0001.Tethering of N-peptide-tagged Ago1 or Ago1x can induce readthrough of AGO1. Two BoxB elements were cloned downstream of the AGO1 and upstream of the in-frame FLuc as shown in the schematic. This construct was transfected in HeLa cells expressing N-peptide-tagged or FLAG-tagged Ago proteins as indicated. Readthrough was measured as described above. *, P = 0.035 (with Welch correction); **, P = 0.016.


Figure 5Linear regression analysis of miRNAs co - immunoprecipiated with Ago1 and those co - immunoprecipiated with Ago1x in HeLa cells . Reads per million ( RPM ) values of 395 miRNAs , which were common to Ago1 and Ago1x are shown . qRT - PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells . Bars show mean of three experiments ± S . E . RT - PCR results showing PTEN and MYC mRNAs co - immunoprecipitated with Ago1 or Ago1x in HeLa cells . Result of co - immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells .

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Figure 5Linear regression analysis of miRNAs co-immunoprecipiated with Ago1 and those co-immunoprecipiated with Ago1x in HeLa cells. Reads per million (RPM) values of 395 miRNAs, which were common to Ago1 and Ago1x are shown.qRT-PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells. Bars show mean of three experiments ± S.E.RT-PCR results showing PTEN and MYC mRNAs co-immunoprecipitated with Ago1 or Ago1x in HeLa cells.Result of co-immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells.


Figure 6Result of co - immunoprecipitation experiment in HeLa cells shows that GW182 is co - immunoprecipitated with Ago1 , but not with Ago1x . Confocal immunofluoresence images of HeLa cells show co - localization of GW182 with Ago1 , but not with Ago1x . White circles indicate zoomed areas . Confocal microscopy images of proximity ligation assay ( PLA ) in HeLa cells showing interaction of GW182 with Ago1 , but not with Ago1x . Rabbit anti - GW182 and Mouse anti - FLAG antibodies were used to achieve proximity ligation . Interaction is indicated by red dots that results from proximity ligation . Quantification of dots per cell is shown ( right ) . Each black circle represents a microscopic field . Mean ± S . E . are shown as horizontal lines ( n = 10 fields for vector control ; n = 12 fields for Ago1 ; n = 11 fields for Ago1x ) . At least 100 cells were counted for dots in each category . * * * , P < 0 . 0001 ( Mann - Whitney test ) .

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Figure 6Result of co-immunoprecipitation experiment in HeLa cells shows that GW182 is co-immunoprecipitated with Ago1, but not with Ago1x.Confocal immunofluoresence images of HeLa cells show co-localization of GW182 with Ago1, but not with Ago1x. White circles indicate zoomed areas.Confocal microscopy images of proximity ligation assay (PLA) in HeLa cells showing interaction of GW182 with Ago1, but not with Ago1x. Rabbit anti-GW182 and Mouse anti-FLAG antibodies were used to achieve proximity ligation. Interaction is indicated by red dots that results from proximity ligation. Quantification of dots per cell is shown (right). Each black circle represents a microscopic field. Mean ± S.E. are shown as horizontal lines (n=10 fields for vector control; n=12 fields for Ago1; n=11 fields for Ago1x). At least 100 cells were counted for dots in each category. ***, P < 0.0001 (Mann-Whitney test).


Figure 7Activity of RLuc relative to co - transfected FLuc in HeLa cells expressing N - HA - tagged or untagged Ago proteins as indicated . Bar graph ( mean ± S . E . ) is a representative of four independent experiments done in triplicates . Expression of HA - tagged Ago proteins was confirmed by Western blot ( bottom ) . * , P = 0 . 018 ; # , P = 0 . 041 ; $ , P = 0 . 013 . Numbers on the bars represent % change in relative luciferase activity . Expression of RLuc protein in HeLa cells expressing N - HA - tagged or untagged Ago proteins as indicated . Densitometric analysis of three independent experiments is shown as bar graph ( mean ± S . E . ) . RT - PCR analysis for RLuc mRNA level is shown ( bottom ) . * * , P = 0 . 001 . Activity of RLuc relative to co - transfected FLuc in HeLa cells expressing N - HA - tagged Ago2 ( N - HA - Ago2 ) or N - HA - tagged chimeric Ago2 appended with the ISR region of Ago1x ( N - HA - Ago2 - ISRAgo1 ) as indicated . Bar graph ( mean ± S . E . ) is a representative of three independent experiments done in triplicates . Expression of HA - tagged Ago proteins was confirmed by Western blot ( bottom ) . * * * , P < 0 . 0001 ; * * , P = 0 . 0004 .

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Figure 7Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Bar graph (mean ± S.E.) is a representative of four independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). *, P = 0.018; #, P = 0.041; $, P = 0.013. Numbers on the bars represent % change in relative luciferase activity.Expression of RLuc protein in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). RT-PCR analysis for RLuc mRNA level is shown (bottom). **, P = 0.001.Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged Ago2 (N-HA-Ago2) or N-HA-tagged chimeric Ago2 appended with the ISR region of Ago1x (N-HA-Ago2-ISRAgo1) as indicated. Bar graph (mean ± S.E.) is a representative of three independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). ***, P < 0.0001; **, P = 0.0004.


Figure 8Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG - HA - Ago1 or FLAG - HA - Ago1x . Densitometric analysis of four independent experiments is shown as bar graph ( mean ± S . E . ) . Puromycin densities were normalized to ponceau S stain densities . * , P < 0 . 029 ( Mann - Whitney test ) . [ 35S ] - methionine autoradiogram showing global translation profile of HeLa cells overexpressing FLAG - HA - Ago1 or FLAG - HA - Ago1x . Densitometric analysis of three independent experiments is shown as bar graph ( mean ± S . E . ) . Autoradiogram densities were normalized to Coomassie stain densities . * , P < 0 . 029 ( Mann - Whitney test ) .

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Figure 8Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of four independent experiments is shown as bar graph (mean ± S.E.). Puromycin densities were normalized to ponceau S stain densities. *, P < 0.029 (Mann-Whitney test).[35S]-methionine autoradiogram showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). Autoradiogram densities were normalized to Coomassie stain densities. *, P < 0.029 (Mann-Whitney test).