Figure 8 ( A ) HeLa cells transfected with control or ALIX siRNA were fixed , permeabilized and stained with anti - MAP1S ( red ) , anti - γ - tubulin ( white ) and Hoechst ( blue ) . Interphase cells are shown and the insets show close ups of the centrosomes . Scale bars , 5 μm . ( B ) The fluorescence intensity of centrosomal MAP1S was determined and the graph shows the average of three independent experiments ( ± S . E ) , in which in total 52 control and 36 ALIX siRNA cells were analyzed . Significant difference is indicated as * p < 0 . 05 ( Student ' s t - test ) ( C ) HeLa cells were transfected with control , ALIX , MAP1S or ALIX + MAP1S siRNA and the expression of ALIX , MAP1S and GAPDH ( loading control ) was determined by Western blotting analysis . Representative results are presented ( D , E ) Cells treated as in C were immunostained with anti - α - tubulin ( white ) , Hoechst ( blue ) ( D Scale bars , 5 μm . The intensities of astral MTs relative to total MTs were determined from confocal microscopy images and the graph in ( D ) shows the average of four separate experiments ( 10 cells per condition / experiment ) ( ± S . E ) . * p < 0 . 05 and * * p < 0 . 01 , Student ' s t - test ( D , E ) Cells treated as in C were immunostained wit anti - acetylated tubulin ( green ) ( E ) The relative intensity of acetylated MT relative to total MT was determined in ( E ) The average of four separate experiments ( 10 cells per condition / experiment ) is graphically presented ( ± S . E ) . Significant differences are indicated as * p < 0 . 05 ( Student ' s t - test ) ( F ) The regrowth of MTs ( i . e total tubulin intensity ) was assessed in cells incubated at 37 ° C for 0 , 60 and 150 seconds after ice - induced depolymerization ( for 30 minutes ) . The graph represents the average of three independent experiments ( calculating 10 cells per time point for each condition ) ( ± S . E ) . Significant differences are noted as * p < 0 . 05 , Student ' s t - test ( G ) HeLa cells transfected with control or ALIX siRNA grown on coverslips were transiently transfected with pEYFP - C1 ( green ) , pEYFP - MAP1S ( green ) , pEGFP - C1 ( green ) or pEGFP - CLASP2α and immunostained with anti - α - tubulin ( white ) , anti - Pericentrin ( red ) and Hoechst ( blue ) . Scale bars , 5 μm ( H ) The lengths and numbers of astral MTs of HeLa cells treated as in ( G ) were measured ( LSM software , version 3 . 2 ) . The graphs show the average numbers ( ± S . E ) based on calculating 30 control siRNA + YFP cells , 23 control siRNA + YFP - MAP1S cells , 30 ALIX siRNA + YFP cells , 30 ALIX siRNA + YFP - MAP1S cells , 28 control siRNA + GFP cells , 30 control siRNA + GFP - CLASP2α cells , 30 ALIX siRNA + GFP cells and 29 ALIX siRNA + GFP - CLASP2α cells from three separate experiments ( * p < 0 . 05 , Student ' s 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 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 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 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 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

Figure 8 (A) HeLa cells transfected with control or ALIX siRNA were fixed, permeabilized and stained with anti-MAP1S (red), anti-γ-tubulin (white) and Hoechst (blue). Interphase cells are shown and the insets show close ups of the centrosomes. Scale bars, 5 μm. (B) The fluorescence intensity of centrosomal MAP1S was determined and the graph shows the average of three independent experiments (± S.E), in which in total 52 control and 36 ALIX siRNA cells were analyzed. Significant difference is indicated as *p<0.05 (Student's t-test) (C) HeLa cells were transfected with control, ALIX, MAP1S or ALIX+MAP1S siRNA and the expression of ALIX, MAP1S and GAPDH (loading control) was determined by Western blotting analysis. Representative results are presented(D, E) Cells treated as in C were immunostained with anti-α-tubulin (white), Hoechst (blue) (D Scale bars, 5 μm. The intensities of astral MTs relative to total MTs were determined from confocal microscopy images and the graph in (D) shows the average of four separate experiments (10 cells per condition/experiment) (±S.E). *p<0.05 and **p<0.01, Student's t-test(D, E) Cells treated as in C were immunostained wit anti-acetylated tubulin (green) (E) The relative intensity of acetylated MT relative to total MT was determined in (E) The average of four separate experiments (10 cells per condition/experiment) is graphically presented (±S.E). Significant differences are indicated as *p<0.05 (Student's t-test)(F) The regrowth of MTs (i.e total tubulin intensity) was assessed in cells incubated at 37°C for 0, 60 and 150 seconds after ice-induced depolymerization (for 30 minutes). The graph represents the average of three independent experiments (calculating 10 cells per time point for each condition) (±S.E). Significant differences are noted as *p<0.05, Student's t-test(G) HeLa cells transfected with control or ALIX siRNA grown on coverslips were transiently transfected with pEYFP-C1 (green), pEYFP-MAP1S (green), pEGFP-C1 (green) or pEGFP-CLASP2α and immunostained with anti-α-tubulin (white), anti-Pericentrin (red) and Hoechst (blue). Scale bars, 5 μm(H) The lengths and numbers of astral MTs of HeLa cells treated as in (G) were measured (LSM software, version 3.2). The graphs show the average numbers (±S.E) based on calculating 30 control siRNA+YFP cells, 23 control siRNA+YFP-MAP1S cells, 30 ALIX siRNA+YFP cells, 30 ALIX siRNA+YFP-MAP1S cells, 28 control siRNA+GFP cells, 30 control siRNA+GFP-CLASP2α cells, 30 ALIX siRNA+GFP cells and 29 ALIX siRNA+GFP-CLASP2α cells from three separate experiments (*p<0.05, Student's t-test), respectively


Figure 1A : Immunoblot analysis of wild type ( WT ) , FAM83D - / - knockout ( KO ) and FAM83DGFP / GFP knockin ( KI ) U2OS cell lines . Data information : All blots are representative of at least 3 independent experiments . D : AS or nocodazole - synchronised M KI cells were lysed and subjected to GFP TRAP immunoprecipitations ( IP ) . Extracts ( input ) and IP samples were analysed by immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . E : KI cells synchronised in mitosis with either nocodazole ( M Noc . ) or STLC ( M STLC ) were collected by shake - off , and drug - treated cells that remained adherent after shake - off ( AS Noc . ; AS STLC ) were lysed and subjected to GFP TRAP IP . AS cells and free - GFP - expressing 2G - PAI1 U2OS cells were included as controls . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . F : Propidium iodide staining analyses revealing cell cycle distribution profiles for the samplesG , H : AS or nocodazole - synchronised ( M ) WT U2OS cells were subjected to IP with IgG and either anti - FAM83D - coupled sepharose beads ( G ) , or anti - CK1α - coupled sepharose beads ( H ) . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . I : KI cells were synchronised in G2 with RO - 3306 , or arrested in mitosis ( M ) using STLC . STLC - treated shake - off cells were washed and re - plated , and cells lysed at the indicated time points after STLC wash - out . Cell lysates were subjected to GFP TRAP IP and input and IP extracts analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . J : Propidium iodide staining analyses revealing cell cycle distribution profiles for the samplesK : AS or STLC - synchronised ( M ) HeLa , A549 and HaCaT cells were subjected to IP with either IgG - or anti - FAM83D - coupled sepharose beads . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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Figure 1A: Immunoblot analysis of wild type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cell lines. Data information: All blots are representative of at least 3 independent experiments.D: AS or nocodazole-synchronised M KI cells were lysed and subjected to GFP TRAP immunoprecipitations (IP). Extracts (input) and IP samples were analysed by immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.E: KI cells synchronised in mitosis with either nocodazole (M Noc.) or STLC (M STLC) were collected by shake-off, and drug-treated cells that remained adherent after shake-off (AS Noc.; AS STLC) were lysed and subjected to GFP TRAP IP. AS cells and free-GFP-expressing 2G-PAI1 U2OS cells were included as controls. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.F: Propidium iodide staining analyses revealing cell cycle distribution profiles for the samplesG, H: AS or nocodazole-synchronised (M) WT U2OS cells were subjected to IP with IgG and either anti-FAM83D-coupled sepharose beads (G), or anti-CK1α-coupled sepharose beads (H). Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.I: KI cells were synchronised in G2 with RO-3306, or arrested in mitosis (M) using STLC. STLC-treated shake-off cells were washed and re-plated, and cells lysed at the indicated time points after STLC wash-out. Cell lysates were subjected to GFP TRAP IP and input and IP extracts analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.J: Propidium iodide staining analyses revealing cell cycle distribution profiles for the samplesK: AS or STLC-synchronised (M) HeLa, A549 and HaCaT cells were subjected to IP with either IgG- or anti-FAM83D-coupled sepharose beads. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


Figure 2A : STLC - synchronised mitotic ( M ) wild - type ( WT ) , FAM83D - / - knockout ( KO ) and FAM83DGFP / GFP knockin ( KI ) U2OS cells were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20 µm . B : STLC - synchronised mitotic ( M ) FAM83D - / - knockout ( KO ) , FAM83DGFP / GFP knockin ( KI ) and FAM83DGFP / GFP ( F283A ) knockin ( FA ) U2OS cells were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20 µC : Quantification of CK1α spindle localisation for the cells Cell images denote the measured regions used to calculate the ratios on the box plot . Box plot whiskers denote the minimum and maximum measured values . The middle line represents the median , and the box ranges depict the 25th / 75th percentiles . * * * , P < 0 . 0001 ; ANOVA . Analysis was performed on the indicated number of cells , n = 2 . D : The cell lines were STLC - synchronised and mitotic cells ( M ) isolated by shake off . Asynchronous ( AS ) cells were included as a control . Cells were lysed and subjected to GFP TRAP immunoprecipitation ( IP ) and subsequent immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . F : KI cells were infected with retroviruses encoding either VHL , aGFP . 16 , or VHL - aGFP . 16 . Uninfected cells were used as a control . Cells were lysed and subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . G : The cell lines were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20 µm .

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Figure 2A: STLC-synchronised mitotic (M) wild-type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.B: STLC-synchronised mitotic (M) FAM83D-/- knockout (KO), FAM83DGFP/GFP knockin (KI) and FAM83DGFP/GFP(F283A) knockin (FA) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µC: Quantification of CK1α spindle localisation for the cells Cell images denote the measured regions used to calculate the ratios on the box plot. Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***, P<0.0001; ANOVA. Analysis was performed on the indicated number of cells, n=2.D: The cell lines were STLC-synchronised and mitotic cells (M) isolated by shake off. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to GFP TRAP immunoprecipitation (IP) and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.F: KI cells were infected with retroviruses encoding either VHL, aGFP.16, or VHL-aGFP.16. Uninfected cells were used as a control. Cells were lysed and subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.G: The cell lines were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.


Figure 3B : FAM83D - / - ( KO ) , wild - type ( WT ) , and two independent clones from a CRISPR / Cas9 - mediated knockin rescue of FAM83D cDNA into FAM83D - / - cells ( c . 6 and c . 11 ) , were synchronized in mitosis ( M ) with STLC . Asynchronous ( AS ) cells were included as a control . Cells were lysed and subjected to immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . The cell lines were STLC - synchronized in mitosis , fixed , and stained with antibodies recognizing FAM83D ( C ) Representative images of mitotic cells are included . Scale bars , 20 µm . The cell lines were STLC - synchronized in mitosis , fixed , and stained with antibodies recognizing CK1 ⍺ ( D ) . Representative images of mitotic cells are included . Scale bars , 20 µm . E : Quantification of CK1 ⍺ spindle localization Box plot whiskers denote the minimum and maximum measured values . The middle line represents the median , and the box ranges depict the 25th / 75th percentiles . * * * , P < 0 . 0001 ; ANOVA . Analysis was performed on the indicated number of cells , n = 1 . F : The cell lines were STLC - synchronized in mitosis ( M ) or left AS , lysed and subjected to immunoprecipitation ( IP ) with anti - FAM83D - coupled sepharose beads , before IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . G : Asynchronous ( AS ) FAM83DGFP / GFP / mCherry / mCherryCSNK1A1 knockin U2OS cells were fixed and imaged . Representative images from the indicated cell cycle stages are included . Scale bars , 10 µm .

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Figure 3B: FAM83D-/- (KO), wild-type (WT), and two independent clones from a CRISPR/Cas9-mediated knockin rescue of FAM83D cDNA into FAM83D-/- cells (c. 6 and c. 11), were synchronized in mitosis (M) with STLC. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing FAM83D (C) Representative images of mitotic cells are included. Scale bars, 20 µm.The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing CK1⍺ (D). Representative images of mitotic cells are included. Scale bars, 20 µm.E: Quantification of CK1⍺ spindle localization Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***, P<0.0001; ANOVA. Analysis was performed on the indicated number of cells, n=1.F: The cell lines were STLC-synchronized in mitosis (M) or left AS, lysed and subjected to immunoprecipitation (IP) with anti-FAM83D-coupled sepharose beads, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.G: Asynchronous (AS) FAM83DGFP/GFP/ mCherry/mCherryCSNK1A1 knockin U2OS cells were fixed and imaged. Representative images from the indicated cell cycle stages are included. Scale bars, 10 µm.


Figure 4A : Nocodazole - synchronised mitotic FAM83DGFP / GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation ( IP ) , followed by incubation ± λ - phosphatase . Asynchronous ( AS ) cells were used as a control . Whole cell extracts ( input ) and IP samples were subjected to SDS - PAGE and subsequent immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ( B ) AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± MG132 ( C ) AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± ProTAME ( D ) . AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . E : Wild - type U2OS cells were either left asynchronous ( AS ) , or arrested in mitosis with STLC and collected by shake - off ( M ) . AS and M cells were incubated in media containing combinations of RO - 3306 and MG132 as indicated , prior to lysis . MG132 was applied for 1 . 5 h , whereas RO - 3306 was applied for the last 1 h of incubation prior to lysis . Samples were lysed and extracts were subjected to SDS - PAGE , before IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . F : STLC - synchronised mitotic wild - type U2OS cells were subjected to qRT - PCR analysis using primers for FAM83D , HMMR , CSNK1A1 , and CCNB1 . AS cells were used as a control . Error bars , SEM ; * , P < 0 . 01 ; Student ' s T - test . n = 3H : STLC - synchronised mitotic wild - type ( WT ) and HMMR knockout ( KO ) mouse embryonic fibroblasts ( MEFs ) were lysed and subjected to IB with the indicated antibodies . AS cells were included as a control . Data information : All blots are representative of at least 3 independent experiments . The cells were STLC - synchronised in mitosis and subjected to immunofluorescence microscopy with an anti - HMMR antibody ( I ) DNA is stained with DAPI . Scale bars , 20 µm . The cells were STLC - synchronised in mitosis and subjected to immunofluorescence microscopy with an anti - CK1α antibody ( J ) . DNA is stained with DAPI . Scale bars , 20 µm .

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Figure 4A: Nocodazole-synchronised mitotic FAM83DGFP/GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation (IP), followed by incubation ± λ-phosphatase. Asynchronous (AS) cells were used as a control. Whole cell extracts (input) and IP samples were subjected to SDS-PAGE and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium (B) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± MG132 (C) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± ProTAME (D). AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.E: Wild-type U2OS cells were either left asynchronous (AS), or arrested in mitosis with STLC and collected by shake-off (M). AS and M cells were incubated in media containing combinations of RO-3306 and MG132 as indicated, prior to lysis. MG132 was applied for 1.5 h, whereas RO-3306 was applied for the last 1 h of incubation prior to lysis. Samples were lysed and extracts were subjected to SDS-PAGE, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.F: STLC-synchronised mitotic wild-type U2OS cells were subjected to qRT-PCR analysis using primers for FAM83D, HMMR, CSNK1A1, and CCNB1. AS cells were used as a control. Error bars, SEM; *, P<0.01; Student's T-test. n=3H: STLC-synchronised mitotic wild-type (WT) and HMMR knockout (KO) mouse embryonic fibroblasts (MEFs) were lysed and subjected to IB with the indicated antibodies. AS cells were included as a control. Data information: All blots are representative of at least 3 independent experiments.The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-HMMR antibody (I) DNA is stained with DAPI. Scale bars, 20 µm.The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-CK1α antibody (J). DNA is stained with DAPI. Scale bars, 20 µm.


Figure 5B : STLC - synchronised mitotic FAM83DGFP / GFP knockin ( KI ) , FAM83DGFP / GFP ( F283A ) ( FA ) , and FAM83DGFP / GFP ( F283A ) stably expressing aGFP . 16 - CK1α ( FA + aGFP . 16 - CK1α ) U2OS cells were subjected to GFP TRAP immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) with the indicated antibodies . Asynchronous ( AS ) cells were used as controls . Data information : All blots are representative of at least 3 independent experiments . C : FA cells were infected with retroviruses encoding wild - type aGFP . 16 - CK1α ( WT ) , or aGFP . 16 - CK1α with one of two distinct CK1α kinase - inactive mutants ( D136N or K46D ) . Uninfected cells ( - ) were included as a control . Cells were lysed , subjected to anti - GFP IP and IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . D : Immunofluorescence analysis for the cells described in ( B ) following synchronisation with STLC . Cells were stained using anti - CK1α antibody , and DNA is stained with DAPI . Scale bars , 20 µm . F : FAM83D - / - knockout ( KO ) , KI , and FA cells were synchronised in mitosis ( M ) using STLC . Lysed extracts were subjected to anti - GFP IPs , followed by ATP [ γ - 32P ] kinase assays , using an optimised CK1 substrate peptide ( CK1tide ) . AS cells were used as controls . Error bars , SEM ; P < 0 . 01 ; ANOVA . n = 3 . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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Figure 5B: STLC-synchronised mitotic FAM83DGFP/GFP knockin (KI), FAM83DGFP/GFP (F283A) (FA), and FAM83DGFP/GFP(F283A) stably expressing aGFP.16-CK1α (FA + aGFP.16-CK1α) U2OS cells were subjected to GFP TRAP immunoprecipitation (IP), followed by immunoblotting (IB) with the indicated antibodies. Asynchronous (AS) cells were used as controls. Data information: All blots are representative of at least 3 independent experiments.C: FA cells were infected with retroviruses encoding wild-type aGFP.16-CK1α (WT), or aGFP.16-CK1α with one of two distinct CK1α kinase-inactive mutants (D136N or K46D). Uninfected cells (-) were included as a control. Cells were lysed, subjected to anti-GFP IP and IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.D: Immunofluorescence analysis for the cells described in (B) following synchronisation with STLC. Cells were stained using anti-CK1α antibody, and DNA is stained with DAPI. Scale bars, 20 µm.F: FAM83D-/- knockout (KO), KI, and FA cells were synchronised in mitosis (M) using STLC. Lysed extracts were subjected to anti-GFP IPs, followed by ATP [γ-32P] kinase assays, using an optimised CK1 substrate peptide (CK1tide). AS cells were used as controls. Error bars, SEM; P<0.01; ANOVA. n=3. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


Figure 6A . Representative images initiating at metaphase for mitotic U2OS cell lines stained with Hoechst , taken every 5 min as they progress through division . Mitotic stage was determined by chromosome condensation and is indicated by the coloured boxes . Scale bar , 20 μm . B . Graphical representation for the kinetics of transition from metaphase alignment ( yellow ) to anaphase ( green ) , and cytokinesis ( blue ) . 100 mitotic cells per genotype are plotted ; n = 2 . C . Length of time needed to transition from metaphase to anaphase . Mean of 100 mitotic cells per genotype are plotted ; n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA . D . Representative bright field images for mitotic U2OS cells indicating the long axis during interphase ( white line ) preceding mitosis , and the division axis determined at anaphase ( yellow line ) . Scale bar , 20 μm . E . Percentage of oriented divisions for U2OS cells grown in subconfluent cultures . An oriented division axis was defined as being less than 30 ° removed from the long axis of the interphase cell . Mean of 50 cells per genotype are plotted , n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA . F . Representative images of mitotic U2OS cells stained with Hoechst and grown on L - shaped micropatterns previously coated with fibronectin . The position of metaphase chromosomes is indicated ( yellow line ) as is the presence of cortical blebbing ( red arrows ) . Scale bar , 20 μm . G . Circular graphs , superimposed on L - shaped micropattern , show the distribution of cell division angles measured at anaphase . Angles for 100 U2OS cells are plotted per genotype , n = 2 . Metaphase position is indicated ( yellow line ) and the percentages of division angles ± 15 ° from the expected axis ( red line ) are indicated . H . Percentage of metaphase U2OS cells that align chromosomes outside of the expected axis ( angles ± 15 ° ) . Mean of 100 cells per genotype are plotted , n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA . I . Representative images of RFP - actin localization in mitotic U2OS cells grown on fibronectin - coated , L - shaped micropatterns , which is superimposed . Arrowheads indicate polarized cortical actin . Heatmap shows the intensity of RFP - actin localization ( Image J z - projection standard deviation ) as the cell progresses from prophase to metaphase ( 12 - 24 min ) . Scale bar , 20 μm . J . Heatmap additive intensities of RFP - actin localization in two representative mitotic U2OS cells for each genotype grown on fibronectin - coated , L - shaped micropatterns . Arrowheads indicate polarized cortical actin .

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Figure 6A. Representative images initiating at metaphase for mitotic U2OS cell lines stained with Hoechst, taken every 5 min as they progress through division. Mitotic stage was determined by chromosome condensation and is indicated by the coloured boxes. Scale bar, 20 μm.B. Graphical representation for the kinetics of transition from metaphase alignment (yellow) to anaphase (green), and cytokinesis (blue). 100 mitotic cells per genotype are plotted; n=2.C. Length of time needed to transition from metaphase to anaphase. Mean of 100 mitotic cells per genotype are plotted; n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.D. Representative bright field images for mitotic U2OS cells indicating the long axis during interphase (white line) preceding mitosis, and the division axis determined at anaphase (yellow line). Scale bar, 20 μm.E. Percentage of oriented divisions for U2OS cells grown in subconfluent cultures. An oriented division axis was defined as being less than 30° removed from the long axis of the interphase cell. Mean of 50 cells per genotype are plotted, n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.F. Representative images of mitotic U2OS cells stained with Hoechst and grown on L-shaped micropatterns previously coated with fibronectin. The position of metaphase chromosomes is indicated (yellow line) as is the presence of cortical blebbing (red arrows). Scale bar, 20 μm.G. Circular graphs, superimposed on L-shaped micropattern, show the distribution of cell division angles measured at anaphase. Angles for 100 U2OS cells are plotted per genotype, n=2. Metaphase position is indicated (yellow line) and the percentages of division angles ± 15° from the expected axis (red line) are indicated.H. Percentage of metaphase U2OS cells that align chromosomes outside of the expected axis (angles ± 15°). Mean of 100 cells per genotype are plotted, n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.I. Representative images of RFP-actin localization in mitotic U2OS cells grown on fibronectin-coated, L-shaped micropatterns, which is superimposed. Arrowheads indicate polarized cortical actin. Heatmap shows the intensity of RFP-actin localization (Image J z-projection standard deviation) as the cell progresses from prophase to metaphase (12- 24 min). Scale bar, 20 μm.J. Heatmap additive intensities of RFP-actin localization in two representative mitotic U2OS cells for each genotype grown on fibronectin-coated, L-shaped micropatterns. Arrowheads indicate polarized cortical actin.


Figure 4B . Pull - down assay of Sec62 domain with different biotin - labeled Sec63 C - terminal peptides . Sec63 - C2P denotes di - phosphorylated Sec63 - C2 . C . ITC - thermogram obtained from ITC after titration of Sec62 domain with increasing amounts of Sec63 - C2P . F . Pull - down assay of the Sec62 domain and a mutant in the interaction interface ( Sec62R51E ) with biotin - labeled Sec63 - C2P .

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Figure 4B. Pull-down assay of Sec62 domain with different biotin-labeled Sec63 C-terminal peptides. Sec63-C2P denotes di-phosphorylated Sec63-C2.C. ITC-thermogram obtained from ITC after titration of Sec62 domain with increasing amounts of Sec63-C2P.F. Pull-down assay of the Sec62 domain and a mutant in the interaction interface (Sec62R51E) with biotin-labeled Sec63-C2P.


Figure 1E . The endouc and ela3l ( negative control ) mRNAs were separately microinjected into zebrafish embryos from huORFZ at a one - cell stage . Upper panels : observation of GFP signal expressed in huORFZ embryos at 72 hpf . Lower panels : magnification of pictures corresponding to the upper panels .

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Figure 1E. The endouc and ela3l (negative control) mRNAs were separately microinjected into zebrafish embryos from huORFZ at a one-cell stage. Upper panels: observation of GFP signal expressed in huORFZ embryos at 72 hpf. Lower panels: magnification of pictures corresponding to the upper panels.


Figure 2C - E . Western blot analysis of proteins , as indicated , in ( C ) zebrafish embryos , ( D ) HEK 293T cells and ( E ) HeLa cells . GAPDH and α - tubulin served as internal controls . Protein levels relative to each internal control are presented below each lane .

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Figure 2C-E. Western blot analysis of proteins, as indicated, in (C) zebrafish embryos, (D) HEK 293T cells and (E) HeLa cells. GAPDH and α-tubulin served as internal controls. Protein levels relative to each internal control are presented below each lane.


Figure 3D . Zebrafish embryos at one - cell stage were microinjected with plasmids as indicated , followed by extraction of embryonic proteins at the 96 - hpf stage . Protein levels of CHOP , p - eIF2α , total eIF2α , Endou - Flag , and its variants were detected using Western blot analysis . GAPDH served as an internal control . E . HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP , p - eIF2α , total eIF2α , Endou - Flag , and its variants using Western blot . The α - tubulin served as an internal control . Protein levels relative to each internal control are presented in each lane .

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Figure 3D. Zebrafish embryos at one-cell stage were microinjected with plasmids as indicated, followed by extraction of embryonic proteins at the 96-hpf stage. Protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants were detected using Western blot analysis. GAPDH served as an internal control. E. HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control are presented in each lane.


Figure 4D . Zebrafish embryos at one - cell stage were microinjected with plasmids as indicated followed by extraction of embryonic proteins at the 96 - hpf stage . Protein levels of CHOP , p - eIF2α , total eIF2α , Endou - Flag , and its variants were detected using Western blot analysis . GAPDH served as an internal control . E . HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP , p - eIF2α , total eIF2α , Endou - Flag , and its variants using Western blot . Here , α - tubulin served as an internal control . Protein levels relative to each internal control were presented in each lane .

Figure 4D. Zebrafish embryos at one-cell stage were microinjected with plasmids as indicated followed by extraction of embryonic proteins at the 96-hpf stage. Protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants were detected using Western blot analysis. GAPDH served as an internal control. E. HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot . Here, α-tubulin served as an internal control. Protein levels relative to each internal control were presented in each lane.


Figure 5B . HEK 293T cells were transfected with plasmids as indicated for 24 hr and treated with DMSO , thapsigargin ( TH ) , ISRIB , or TH plus ISRIB for 2 hr followed by analysis of the protein level of CHOP , p - eIF2α , total eIF2α , Endou - Flag , and its variants using Western blot . The α - tubulin served as an internal control . Protein levels relative to each internal control were presented at each lane . D , E . MEF WT and mutant S51A cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of CHOP , p - eIF2α , total eIF2α , GADD34 , and Endouc - Flag using Western blot . GAPDH served as an internal control . Protein levels relative to each internal control were presented at each lane .

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Figure 5B. HEK 293T cells were transfected with plasmids as indicated for 24 hr and treated with DMSO, thapsigargin (TH), ISRIB, or TH plus ISRIB for 2 hr followed by analysis of the protein level of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control were presented at each lane.D, E. MEF WT and mutant S51A cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, GADD34, and Endouc-Flag using Western blot. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane.


Figure 6A . MEF wild - type ( WT ) cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or Thapsigargin ( TH ) for 4 hr followed by analysis of the protein level of p - PKR , total PKR , and Endouc - Flag . GAPDH served as an internal control . Protein levels relative to each internal control were presented at each lane . The results obtained from the two trials are shown on the left and right . B . MEF WT cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of p - eIF2α , total eIF2α , CHOP , and Endouc - Flag . GAPDH served as an internal control . Protein levels relative to each internal control were presented at each lane . The results obtained from the two trials are shown on the left and right .

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Figure 6A. MEF wild-type (WT) cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or Thapsigargin (TH) for 4 hr followed by analysis of the protein level of p-PKR, total PKR, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right.B. MEF WT cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of p-eIF2α, total eIF2α, CHOP, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right.


Figure 7D , E . The cleavage activity of Endouc was studied using materials presented at each lane . EndoucK242A : Endouc mutant ; Extract : incubated in cell extracts ; and Buf : reaction buffer . RNA probe without adding reaction buffer served as a negative control . The predicted cleavage sites ( U ) were mutated to generate mutant huORFchop transcript ( unlabelled huORF - mt ) and labelled by biotin ( biotin - huORF - mt ) . Unlabelled competitor RNA was added in reactions at five times higher than that of the biotinylated probe .

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Figure 7D, E. The cleavage activity of Endouc was studied using materials presented at each lane. EndoucK242A: Endouc mutant; Extract: incubated in cell extracts; and Buf: reaction buffer. RNA probe without adding reaction buffer served as a negative control. The predicted cleavage sites (U) were mutated to generate mutant huORFchop transcript (unlabelled huORF-mt) and labelled by biotin (biotin-huORF-mt). Unlabelled competitor RNA was added in reactions at five times higher than that of the biotinylated probe.


Figure 8A . HEK 293T cells were transfected with pCS2 vector ( black ) , pCS2 - Endouc ( red ) , and pCS2 - EndoucK242A ( green ) for 24 hr and subjected to either DMSO ( control ) or thapsigargin ( TH ; stress ) treatment for 1 hr . The lysates of transfected cells were applied to polysome profile analysis ( PPA ) . The sucrose gradient from top to bottom was shown from left to right , respectively . The sedimentation of 40S , 60S , 80S , and polysomes was indicated . Lysate from control , Endouc - expressing , and EndoucK242A - expressing cells were applied to PPA . The resulting fractions were TCA - precipitated , analysed by SDS - PAGE , and subjected to Western blot using antibodies against Flag and S6 .

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Figure 8A. HEK 293T cells were transfected with pCS2 vector (black), pCS2-Endouc (red), and pCS2-EndoucK242A (green) for 24 hr and subjected to either DMSO (control) or thapsigargin (TH; stress) treatment for 1 hr. The lysates of transfected cells were applied to polysome profile analysis (PPA). The sucrose gradient from top to bottom was shown from left to right, respectively. The sedimentation of 40S, 60S, 80S, and polysomes was indicated. Lysate from control, Endouc-expressing, and EndoucK242A-expressing cells were applied to PPA. The resulting fractions were TCA-precipitated, analysed by SDS-PAGE, and subjected to Western blot using antibodies against Flag and S6.


Figure 9B . The luc activity of Fluc ( solid column ) versus Rluc ( blank column ) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures A was presented as an increase in fold over that obtained from control group of RF which was normalized as 1 . These data suggested that the change of Fluc / Rluc ratio shown in Figures A was entirely dependent on the change of Fluc activity . The data were averaged from three independent experiments and presented as mean ± S . D . ( n = 3 ) . Student ' s t test was used to determine significant differences between each group ( * * : P < 0 . 005 ) . D . The luc activity of Fluc ( solid column ) versus Rluc ( blank column ) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures C was presented as an increase in fold over that obtained from control group of hpRF which was normalized as 1 . These data suggested that the change of Fluc / Rluc ratio shown in Figures C was entirely dependent on the change of Fluc activity . The data were averaged from three independent experiments and presented as mean ± S . D . ( n = 3 ) . Student ' s t test was used to determine significant differences between each group ( * * * : P < 0 . 001 ) .

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Figure 9B. The luc activity of Fluc (solid column) versus Rluc (blank column) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures A was presented as an increase in fold over that obtained from control group of RF which was normalized as 1. These data suggested that the change of Fluc/Rluc ratio shown in Figures A was entirely dependent on the change of Fluc activity. The data were averaged from three independent experiments and presented as mean ± S.D. (n = 3). Student's t test was used to determine significant differences between each group (**: P<0.005).D. The luc activity of Fluc (solid column) versus Rluc (blank column) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures C was presented as an increase in fold over that obtained from control group of hpRF which was normalized as 1. These data suggested that the change of Fluc/Rluc ratio shown in Figures C was entirely dependent on the change of Fluc activity. The data were averaged from three independent experiments and presented as mean ± S.D. (n = 3). Student's t test was used to determine significant differences between each group (***: P <0.001).


Figure 10A - F . Schematic representation of capped ( m7G - capped ) and A - capped ( non - functional capped ) monocistronic mRNAs with poly ( A ) - tail - encoded firefly luciferase ( Fluc ) and fused upstream with ( A ) c - myc IRES , ( B ) no insertion , ( C ) non - specific 105 - nt luc RNA ( Non105 ) , ( D ) huORFchop - 1 - 105 - nt , ( E ) huORFchop - 69 - 105 - nt , and ( F ) huORFchop - 81 - 105 - nt as shown above each bar graph . The RNA transfection assay of each transcript equipped with m7G - capped or A - capped monocistronic mRNA in HEK 293T cells was compared . To normalize the luc activities of capped and A - capped Fluc transcripts , the m7G - capped Renilla luc ( Rluc ) mRNA was used as an internal control . The luc activity of cells transfected with Cap - monocistronic mRNA in each graph was set as 100 % .

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Figure 10A-F. Schematic representation of capped (m7G-capped) and A-capped (non-functional capped) monocistronic mRNAs with poly(A)-tail-encoded firefly luciferase (Fluc) and fused upstream with (A) c-myc IRES, (B) no insertion, (C) non-specific 105-nt luc RNA (Non105), (D) huORFchop-1-105-nt, (E) huORFchop-69-105-nt, and (F) huORFchop-81-105-nt as shown above each bar graph. The RNA transfection assay of each transcript equipped with m7G-capped or A-capped monocistronic mRNA in HEK 293T cells was compared. To normalize the luc activities of capped and A-capped Fluc transcripts, the m7G-capped Renilla luc (Rluc) mRNA was used as an internal control. The luc activity of cells transfected with Cap-monocistronic mRNA in each graph was set as 100%.


Figure 1 ( b ) Representative electron micrograph of a viroplasm ( V ) formed in NSP5 - EGFP cells infected with rotaviruses 8 hours post infection . Inset - double - layered particles emerging from the surface of a viroplasm . Scale bar = 300 nm .

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Figure 1(b) Representative electron micrograph of a viroplasm (V) formed in NSP5-EGFP cells infected with rotaviruses 8 hours post infection. Inset - double-layered particles emerging from the surface of a viroplasm. Scale bar = 300 nm.


Figure 2 ( a ) Fluorescence recovery after photobleaching ( FRAP ) of EGFP - tagged replication factories after 4 HPI ( early infection ) and 12 HPI ( late infection ) . Scale bar , 10 μm .

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Figure 2(a) Fluorescence recovery after photobleaching (FRAP) of EGFP-tagged replication factories after 4 HPI (early infection) and 12 HPI (late infection). Scale bar, 10 μm.


Figure 3 ( b ) Sensitivity of RV replication factories to aliphatic alcohols at 4 HPI . Left to right - 1 , 6 - hexanediol ( 1 , 6HD ) ; 1 , 3 - propylene diol ( 1 , 3PD ) ; 1 , 2 - propylene diol , or propylene glycol ( PG ) ; glycerol ( Gly ) ; polyethylene glycol 200 ( PEG200 ) . Top panels - before application and bottom panels - 1 min after application of these compounds ( 4 . 5 % v / v ) . Scale bar , 30 µm .

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Figure 3(b) Sensitivity of RV replication factories to aliphatic alcohols at 4 HPI. Left to right -1,6-hexanediol (1,6HD); 1,3-propylene diol (1,3PD); 1,2-propylene diol, or propylene glycol (PG); glycerol (Gly); polyethylene glycol 200 (PEG200). Top panels - before application and bottom panels - 1 min after application of these compounds (4.5% v/v). Scale bar, 30 µm.


Figure 4 ( a ) Recombinantly expressed , Atto647 - dye labelled NSP5 , Atto488 - dye labelled NSP2 and unlabeled NSP5 protein samples were used for investigating their phase - separation properties . All labelled protein samples were mixed with the unlabeled NSP5 ( 1 : 10 molar ratio ) to minimise the effect of labelling on LLPS . Top panel , left to right : NSP5 - 647 / NSP2 - 488 droplets formed upon mixing of both proteins ( 10 μM each ) . Both channels are shown , along with an image of both channels overlaid . 4 % ( v / v ) 1 , 6 - hexanediol ( 1 , 6HD ) dissolves these condensates . Scale bar , 10 µm . Middle panel , left to right : NSP5 - 647 ( 50 μM ) + poly - arginine ( polyArg , 5 μΜ ) ; NSP5 - polyArg condensate + 1 , 6 - HD ; NSP5 - polyArg condensates + 0 . 5 M NaCl ; NSP5 + R9 peptide ( Arg - 9 , 5 μΜ ) . Bottom row , left to right : NSP5 - 647 sample ( 35 μM ) alone ; after addition of 10 % v / v PEG - 20K ; NSP2 - 488 ( 25 μM ) alone ; after addition of 10 % v / v PEG - 20K .

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

Figure 4(a) Recombinantly expressed, Atto647-dye labelled NSP5, Atto488-dye labelled NSP2 and unlabeled NSP5 protein samples were used for investigating their phase-separation properties. All labelled protein samples were mixed with the unlabeled NSP5 (1:10 molar ratio) to minimise the effect of labelling on LLPS. Top panel, left to right: NSP5-647/NSP2-488 droplets formed upon mixing of both proteins (10 μM each). Both channels are shown, along with an image of both channels overlaid. 4% (v/v) 1,6-hexanediol (1,6HD) dissolves these condensates. Scale bar, 10 µm. Middle panel, left to right: NSP5-647 (50 μM) + poly-arginine (polyArg, 5 μΜ); NSP5-polyArg condensate + 1,6-HD; NSP5-polyArg condensates + 0.5 M NaCl; NSP5 + R9 peptide (Arg-9, 5 μΜ). Bottom row, left to right: NSP5-647 sample (35 μM) alone; after addition of 10% v/v PEG-20K; NSP2-488 (25 μM) alone; after addition of 10% v/v PEG-20K.


Figure 5Top panel , left to right : NSP5 - ∆ CTR labelled with Atto - 647 ( 25 μM ) incubated with unlabeled NSP2 ( 10 μΜ ) ; NSP5 - ∆ CTR in the presence of 10 % v / v PEG - 20K ; and with 5 μΜ of poly - arginine ( polyArg ) . Bottom panel : Atto - 647 - labelled NSP5 - ∆ CTR ( 5 μΜ ) incubated with unlabeled NSP5 ( 25 μΜ ) and Atto - 488 labelled NSP2 ( 10 μΜ ) . NSP5 / NSP2 droplets containing labelled NSP2 ( green ) also contain NSP5 - ∆ CTR - Atto647 ( magenta ) , shown along with an image of both 488 / 647 channels overlaid . Scale bar , 10 µm .

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Figure 5Top panel, left to right: NSP5-∆CTR labelled with Atto-647 (25 μM) incubated with unlabeled NSP2 (10 μΜ); NSP5-∆CTR in the presence of 10% v/v PEG-20K; and with 5 μΜ of poly-arginine (polyArg). Bottom panel: Atto-647-labelled NSP5-∆CTR (5 μΜ) incubated with unlabeled NSP5 (25 μΜ) and Atto-488 labelled NSP2 (10 μΜ). NSP5/NSP2 droplets containing labelled NSP2 (green) also contain NSP5-∆CTR-Atto647 (magenta), shown along with an image of both 488/647 channels overlaid. Scale bar, 10 µm.


Figure 6 ( a ) RV - infected MA - NSP5 - EGFP cells at 6 HPI . NSP5 - EGFP - tagged viral factories ( green ) are dissolved in the presence of 4 . 7 % ( v / v ) propylene glycol ( PG , middle ) . Viral RNA - protein condensates rapidly reform ( < 10 min ) after replacing the PG - containing cell culture medium ( PG removed , right ) . Seg3 ( magenta ) and Seg4 ( cyan ) transcripts are detected by smFISH , and colocalising Seg3 and Seg4 RNA signals ( white ) . Scale bars : 50 µm , zoomed - in regions : 10 µm . Effects of PG treatments on the viral production . PG - containing medium ( 2 % or 4 . 7 % PG ) , or standard cell culture medium , were applied to RV - infected cells ( MOI of 10 ) 2 HPI ( 1 hour after virus absorption ) . ( e ) Effects of PG treatments on virus production at different infection points . 4 . 7 % ( v / v ) PG was applied between 2 - 7 HPI ( ' early infection ' ) , and then diluted to 2 % between 7 - 11 HPI , or 4 . 7 % PG was applied between 7 - 11 HPI ( ' late infection ' ) , and diluted prior to harvesting the virus . Each group was compared to the control group ( 10 h treatment with 2 % PG ) . Each group was compared to the control group ( 10 h treatment with 2 % PG ) . Application of 4 . 7 % PG for 5 h significantly reduced virus replication ( P = 0 . 0016 ) between 2 - 7 HPI compared to the control group . PG treatment between 7 - 12 HPI did not significantly reduce the viral titre compared to the control group ( P = 0 . 1141 ) . Infectious titres represent mean ± SD values estimated for four independent biological replicates . Statistical analyses were performed using a Kruskal - Wallis test , followed by uncorrected Dunn ' s multiple comparisons test ( * * P < 0 . 002 ) . ( f , g ) 4 . 7 % PG treatment of RV - infected cells results in NSP5 dephosphorylation . ( f ) Western blot analysis of RV - infected cells harvested at 7 and 12 HPI , treated with 2 % or 4 . 7 % PG at different infection time points indicated in the figure . Multiple phosphorylated forms of NSP5 can be seen as higher MW bands . Treatment with 4 . 7 % PG reduces phosphorylation , which is not perturbed in the presence of 2 % PG . Treatments administered between 2 - 7 and 7 - 12 hpi reduce NSP5 phosphorylation , albeit to a considerably lower degree compared to the 2 - 12 hpi treatment . ( g ) Okadaic acid ( 5 μM ) applied to 4 . 7 % PG - treated infected cells blocks NSP5 dephosphorylation restoring its phosphorylation pattern .

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 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 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

Figure 6(a) RV-infected MA-NSP5-EGFP cells at 6 HPI. NSP5-EGFP-tagged viral factories (green) are dissolved in the presence of 4.7% (v/v) propylene glycol (PG, middle). Viral RNA-protein condensates rapidly reform (<10 min) after replacing the PG-containing cell culture medium (PG removed, right). Seg3 (magenta) and Seg4 (cyan) transcripts are detected by smFISH, and colocalising Seg3 and Seg4 RNA signals (white). Scale bars: 50 µm, zoomed-in regions: 10 µm.Effects of PG treatments on the viral production. PG-containing medium (2% or 4.7% PG), or standard cell culture medium, were applied to RV-infected cells (MOI of 10) 2 HPI (1 hour after virus absorption). (e) Effects of PG treatments on virus production at different infection points. 4.7% (v/v) PG was applied between 2-7 HPI ('early infection'), and then diluted to 2% between 7-11 HPI, or 4.7% PG was applied between 7-11 HPI ('late infection'), and diluted prior to harvesting the virus. Each group was compared to the control group (10 h treatment with 2% PG). Each group was compared to the control group (10 h treatment with 2% PG). Application of 4.7% PG for 5 h significantly reduced virus replication (P = 0.0016) between 2-7 HPI compared to the control group. PG treatment between 7-12 HPI did not significantly reduce the viral titre compared to the control group (P = 0.1141). Infectious titres represent mean±SD values estimated for four independent biological replicates. Statistical analyses were performed using a Kruskal-Wallis test, followed by uncorrected Dunn's multiple comparisons test (**P < 0.002).(f, g) 4.7% PG treatment of RV-infected cells results in NSP5 dephosphorylation. (f) Western blot analysis of RV-infected cells harvested at 7 and 12 HPI, treated with 2% or 4.7% PG at different infection time points indicated in the figure. Multiple phosphorylated forms of NSP5 can be seen as higher MW bands. Treatment with 4.7% PG reduces phosphorylation, which is not perturbed in the presence of 2% PG. Treatments administered between 2-7 and 7-12 hpi reduce NSP5 phosphorylation, albeit to a considerably lower degree compared to the 2-12 hpi treatment. (g) Okadaic acid (5 μM) applied to 4.7% PG-treated infected cells blocks NSP5 dephosphorylation restoring its phosphorylation pattern.


Figure 7 ( e ) 3D DNA - PAINT analysis of Seg3 RNA foci in RV - infected cells at 4 and 6 HPI . Scale bars , 2 µm ( left ) and 200 nm ( zoomed - in , right ) . ( f - g ) Distribution of calculated volumes and sphericities of the Seg3 RNA - containing granules in RV - infected cells at 4 HPI ( N = 704 ) and 6 hpi ( N = 698 ) , shown in ( e ) . Box plots represent the 25th / 75th interquartile range , with whiskers representing the 5th / 95th percentile values . Medians shown as central bands , and means shown as squares . Symbol ' x ' denotes 1 % and 99 % percentile values , and min and max values are shown as ' - ' . At the 0 , 001 level , the two distributions are significantly different between 4 and 6 hpi , assessed by the two - sample Kolmogorov - Smirnov test .

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Figure 7(e) 3D DNA-PAINT analysis of Seg3 RNA foci in RV-infected cells at 4 and 6 HPI. Scale bars, 2 µm (left) and 200 nm (zoomed-in, right). (f-g) Distribution of calculated volumes and sphericities of the Seg3 RNA-containing granules in RV-infected cells at 4 HPI (N=704) and 6 hpi (N=698), shown in (e). Box plots represent the 25th/75th interquartile range, with whiskers representing the 5th/95th percentile values. Medians shown as central bands, and means shown as squares. Symbol 'x' denotes 1% and 99% percentile values, and min and max values are shown as '-'. At the 0,001 level, the two distributions are significantly different between 4 and 6 hpi, assessed by the two-sample Kolmogorov-Smirnov test.


Figure 1 ( A ) mRNA expressions of PINK1 in CSCs and non - CSCs . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( B - C ) Immunoblot analysis of PINK1 protein in non - CSCs and CSCs . Representative and mean ± SEM from 9 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( D - E ) Immunoblot analysis of p - PINK1 protein in non - CSCs and CSCs . Representative and mean ± SEM from 7 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . Immunoblot analysis of Parkin protein in non - CSCs and CSCs . analysis of Parkin protein in non - CSCs and CSCs . Representative and mean ± SEM from 8 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( H - I ) PINK1 and Parkin protein levels in mitochondrial fractions and whole cell lyses of CSCs and non - CSCs . Representative and mean ± SEM from 8 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( J - K ) Intracellular levels of p - Parkin protein in non - CSCs and CSCs were analyzed using flow cytometry . Representative histograms and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . Immunoblot analysis of p - Parkin protein in non - CSCs and CSCs . analysis of p - Parkin protein in non - CSCs and CSCs . Representative and mean ± SEM from 11 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( N - P ) CSCs were treated with Mdivi - 1 ( 50 μM ) and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Scale bar 20 mm . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( Q ) CSCs transfected with PINK1 shRNA or the control were analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( R ) CSCs were treated with DAPT ( 10 μM ) and analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( S ) CSCs were transfected with PINK1 shRNA or control , and analysed for activated Notch1 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( T ) CSCs with or without transfection of Notch1 ICD were treated with Mdivi - 1 ( 50 μM ) and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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 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 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 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 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 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 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 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 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

Figure 1(A) mRNA expressions of PINK1 in CSCs and non-CSCs. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(B-C) Immunoblot analysis of PINK1 protein in non-CSCs and CSCs. Representative and mean±SEM from 9 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(D-E) Immunoblot analysis of p-PINK1 protein in non-CSCs and CSCs. Representative and mean±SEM from 7 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.Immunoblot analysis of Parkin protein in non-CSCs and CSCs.analysis of Parkin protein in non-CSCs and CSCs. Representative and mean±SEM from 8 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(H-I) PINK1 and Parkin protein levels in mitochondrial fractions and whole cell lyses of CSCs and non-CSCs. Representative and mean±SEM from 8 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(J-K) Intracellular levels of p-Parkin protein in non-CSCs and CSCs were analyzed using flow cytometry. Representative histograms and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.Immunoblot analysis of p-Parkin protein in non-CSCs and CSCs.analysis of p-Parkin protein in non-CSCs and CSCs. Representative and mean±SEM from 11 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(N-P) CSCs were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Scale bar 20 mm. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(Q) CSCs transfected with PINK1 shRNA or the control were analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(R) CSCs were treated with DAPT (10 μM) and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(S) CSCs were transfected with PINK1 shRNA or control, and analysed for activated Notch1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(T) CSCs with or without transfection of Notch1 ICD were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Figure 2 ( A - B ) Lysosomal DNA was identified with anti - LAMP1 and anti - DNA antibodies in non - CSCs and CSCs . Scale bar 5μm . Representative and mean ± SEM with 50 cells from 5 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( C ) Lysosomal content of mtDNA in CSCs and non - CSCs was determined with qPCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( D ) CSCs were treated with Mdivi - 1 ( 50 μM ) and analysed for lysosomal mtDNA . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( E ) CSCs with or without transfection of PINK1 shRNA were analysed for lysosomal mtDNA . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( F - G ) Protein expressions of TLR9 and MyD88 in CSCs and non - CSCs . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( H ) TLR9 proteins were immunoprecipitated from CSCs and non - CSCs and analyzed for the binding load of mtDNA . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( I ) CSCs were transfected with mtDNA and analysed for expressions of TLR9 plus MyD88 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( J - K ) CSCs treated with EtBr ( 50 ng / ml ) were analysed for lysosomal mtDNA , TLR9 , and MyD88 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( L ) CSCs transfected with DNase II expression vector or the control were analyzed for lysosomal DNA plus MyD88 protein . Mean ± SEM from 6 patients . ( M ) CSCs transfected with DNase II shRNA or the control were analyzed for lysosomal DNA plus MyD88 protein . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2(A-B) Lysosomal DNA was identified with anti-LAMP1 and anti-DNA antibodies in non-CSCs and CSCs. Scale bar 5μm. Representative and mean±SEM with 50 cells from 5 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(C) Lysosomal content of mtDNA in CSCs and non-CSCs was determined with qPCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(D) CSCs were treated with Mdivi-1 (50 μM) and analysed for lysosomal mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(E) CSCs with or without transfection of PINK1 shRNA were analysed for lysosomal mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(F-G) Protein expressions of TLR9 and MyD88 in CSCs and non-CSCs. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(H) TLR9 proteins were immunoprecipitated from CSCs and non-CSCs and analyzed for the binding load of mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(I) CSCs were transfected with mtDNA and analysed for expressions of TLR9 plus MyD88. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(J-K) CSCs treated with EtBr (50 ng/ml) were analysed for lysosomal mtDNA, TLR9, and MyD88. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(L) CSCs transfected with DNase II expression vector or the control were analyzed for lysosomal DNA plus MyD88 protein. Mean±SEM from 6 patients. (M) CSCs transfected with DNase II shRNA or the control were analyzed for lysosomal DNA plus MyD88 protein. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Figure 3 ( A ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) or control and analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . CSCs transfected with mtDNA were analyzed for sphere formation Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . CSCs transfected with mtDNA were analyzed for tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( D ) CSCs were transfected with 2 different TLR9 shRNAs or the control , and analysed for sphere formation and tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( E - F ) CSCs transfected with DNase II expression vector , DNase II shRNA , or the control was analyzed for sphere formation . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( G ) CpG promoted tumor growth of PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( H ) TLR9 inhibitor chloroquine abrogated the effect of CpG on tumor growth in PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( I ) CpG treatment increased the frequency of CD133 - expressing CSCs in PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( J ) Chloroquine impeded the function of CpG in elevating CD133 - expressing CSCs of PDX chimeras . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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 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 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 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 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 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

Figure 3(A) CSCs were stimulated with CpG ODN (10 μg/ml) or control and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.CSCs transfected with mtDNA were analyzed for sphere formation Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.CSCs transfected with mtDNA were analyzed for tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(D) CSCs were transfected with 2 different TLR9 shRNAs or the control, and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(E-F) CSCs transfected with DNase II expression vector, DNase II shRNA, or the control was analyzed for sphere formation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(G) CpG promoted tumor growth of PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(H) TLR9 inhibitor chloroquine abrogated the effect of CpG on tumor growth in PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(I) CpG treatment increased the frequency of CD133-expressing CSCs in PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(J) Chloroquine impeded the function of CpG in elevating CD133-expressing CSCs of PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Figure 4 ( A ) CSCs transfected with TLR9 shRNA or control were analysed for protein expressions of activated Notch1 plus HES1 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( B ) CSCs treated with or without EtBr ( 50 ng / ml ) were analyzed for activated Notch1 . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( C ) CSCs transfected with DNase II expression vector or control were analyzed for activated Notch1 expression . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( D ) CSCs with or without CpG treatment were analysed for interaction between TLR9 and Notch1 . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( E ) CSCs were treated with CpG ODN ( 10 μg / ml ) in the presence or absence of DAPT ( 10 μM ) , and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . ( F ) CSCs were transfected with 2 different Notch1 shRNAs or control , treated with CpG ODN ( 10 μg / ml ) , and analyzed for sphere formation plus tumorgenicity . Mean ± SEM from 6 experiments . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . PDX chimeras treated by CpG with or without DAPT were analysed for tumor growth Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 . PDX chimeras treated by CpG with or without DAPT were analysed for CD133 - expressing CSCs . Mean ± SEM from 6 mice . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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 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 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 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 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

Figure 4(A) CSCs transfected with TLR9 shRNA or control were analysed for protein expressions of activated Notch1 plus HES1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(B) CSCs treated with or without EtBr (50 ng/ml) were analyzed for activated Notch1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(C) CSCs transfected with DNase II expression vector or control were analyzed for activated Notch1 expression. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(D) CSCs with or without CpG treatment were analysed for interaction between TLR9 and Notch1. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(E) CSCs were treated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.(F) CSCs were transfected with 2 different Notch1 shRNAs or control, treated with CpG ODN (10 μg/ml), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.PDX chimeras treated by CpG with or without DAPT were analysed for tumor growth Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.PDX chimeras treated by CpG with or without DAPT were analysed for CD133-expressing CSCs. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Figure 5 ( A ) CSCs and non - CSCs were analyzed for mitochondrial metabolism using seahorse assay . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method ( A , * * p < 0 . 01 , * * * p < 0 . 001 . ( B ) CSCs were treated with 2 - DG ( 10 mM ) or Malonate ( 10 mM ) , and assayed for sphere formation plus tumorgenicity . Mean ± SEM from 6 patients . Data information : ANOVA test with Turkey ' s method ( B ) . * * p < 0 . 01 , * * * p < 0 . 001 . ( C ) CSCs treated with DAPT ( 10 μM ) were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 . ( D ) CSCs transfected with Notch1 shRNA or control shRNA were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 . ( E ) CSCs treated with EtBr ( 50 ng / ml ) were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 . ( F ) CSCs transfected with DNase II expression vector or control were analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 . ( G ) CSCs treated with chloroquine ( 50 μM ) were analyzed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

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 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 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 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 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 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

Figure 5(A) CSCs and non-CSCs were analyzed for mitochondrial metabolism using seahorse assay. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method (A, **p < 0.01, ***p < 0.001.(B) CSCs were treated with 2-DG (10 mM) or Malonate (10 mM), and assayed for sphere formation plus tumorgenicity. Mean±SEM from 6 patients. Data information: ANOVA test with Turkey's method (B). **p < 0.01, ***p < 0.001.(C) CSCs treated with DAPT (10 μM) were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.(D) CSCs transfected with Notch1 shRNA or control shRNA were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.(E) CSCs treated with EtBr (50 ng/ml) were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.(F) CSCs transfected with DNase II expression vector or control were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.(G) CSCs treated with chloroquine (50 μM) were analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


Figure 6 ( A ) CSCs stimulated with or without CpG ODN ( 10 μg / ml ) were analyzed for expression of phosphor - AMPKα . Representative and mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( B ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) in the presence or absence of DAPT ( 10 μM ) , and analyzed for phosphor - AMPKα . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( C ) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN ( 10 μg / ml ) and analyzed for phosphor - AMPKα . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( D ) CSCs were treated with DAPT ( 10 μM ) in the presence or absence of A769662 ( 10 μM ) , and analysed for mitochondrial OCR . Ctrl represents CSCs without any treatment . Mean ± SEM from 6 patients . Data information : ANOVA with Turkey method ( D ) , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( E ) CSCs were stimulated with or without CpG ODN ( 10 μg / ml ) and analyzed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( F ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) in the presence or absence of Compound C ( 10 μM ) and analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( G ) CSCs were transfected with 2 different AMPKα shRNA or the control , stimulated with CpG ODN ( 10 μg / ml ) , and analysed for mitochondrial OCR . Mean ± SEM from 6 patients . Data information : Paired t - test with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 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 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 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 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 6(A) CSCs stimulated with or without CpG ODN (10 μg/ml) were analyzed for expression of phosphor-AMPKα. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.(B) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.(C) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN (10 μg/ml) and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.(D) CSCs were treated with DAPT (10 μM) in the presence or absence of A769662 (10 μM), and analysed for mitochondrial OCR. Ctrl represents CSCs without any treatment. Mean±SEM from 6 patients. Data information: ANOVA with Turkey method (D), **p < 0.01, ***p < 0.001, ****p < 0.0001.(E) CSCs were stimulated with or without CpG ODN (10 μg/ml) and analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.(F) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of Compound C (10 μM) and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.(G) CSCs were transfected with 2 different AMPKα shRNA or the control, stimulated with CpG ODN (10 μg/ml), and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.