NEDD8 trimers bind to GFP - PARP - 1 . NEDP1 U2OS KO were transfected with GFP or with GFP - PARP - 1 24 hours later lysates were collected . Immunoprecipitation was then performed with GFP - Trap , bound were resolved by SDS - PAGE processed for Western blot with indicated antibodies . GFP - PARP - 1 but not GFP alone can co - immunoprecipitate NEDD8 trimers from NEDP1 KO .

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NEDD8 trimers bind to GFP-PARP-1. NEDP1 U2OS KO cells were transfected with GFP or with GFP-PARP-1 and 24 hours later cell lysates were collected. Immunoprecipitation was then performed with GFP-Trap, and bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies. GFP-PARP-1 but not GFP alone can co-immunoprecipitate NEDD8 trimers from NEDP1 KO cells.


NEDD8 trimers form in WT U2OS after treatment with H2O2 co - immunoprecipitate with endogenous PARP - 1 but not with immunoglobulin control ( IGG ) . WT U2OS were left untreated of treated with H2O2 ( 600 μM ) for 30 min were harvested for immunoprecipitation Western blot

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NEDD8 trimers form in WT U2OS cells after treatment with H2O2 and co-immunoprecipitate with endogenous PARP-1 but not with immunoglobulin control (IGG). WT U2OS cells were left untreated of treated with H2O2 (600 μM) for 30 min and were harvested for immunoprecipitation and Western blot analysis


NEDD8 trimers bind to GFP - PARP - 1 . WT or NEDP1 U2OS KO were transfected with GFP or with full - GFP - PARP - 1 24 hours later treated as indicated with H2O2 ( 600 μM ) for 30 min before lysates were collected . Immunoprecipitation was then performed with GFP - Trap , bound were resolved by SDS - PAGE processed for Western blot with indicated antibodies . GFP - PARP - 1 but not GFP alone can co - immunoprecipitate NEDD8 trimers from WT only after H2O2 treatment or from both treated or untreated NEDP1 KO .

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NEDD8 trimers bind to GFP-PARP-1. WT or NEDP1 U2OS KO cells were transfected with GFP or with full-length GFP-PARP-1 and 24 hours later treated as indicated with H2O2 (600 μM) for 30 min before cell lysates were collected. Immunoprecipitation was then performed with GFP-Trap, and bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies. GFP-PARP-1 but not GFP alone can co-immunoprecipitate NEDD8 trimers from WT cells only after H2O2 treatment or from both treated or untreated NEDP1 KO cells.


NEDD8 trimers bind to - binding domain of PARP - 1 ( Zn1 - 3 ) but not to its automodification catalytic domain ( CAT ) . NEDP1 KO U2OS were transfected with GFP , GFP - PARP - 1 , GFP fused to - binding domain of PARP - 1 ( AA 1 - 336 ) or with GFP fused to PARP - 1 automodification catalytic domain ( AA 336 - 1014 ) . 24 hours post transfection , lysates were collected immunoprecipitation with GFP - Trap was performed . Bound were eluted resolved by SDS - PAGE , followed by Western blot with indicated antibodies .

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NEDD8 trimers bind to the DNA-binding domain of PARP-1 (Zn1-3) but not to its automodification and catalytic domain (CAT). NEDP1 KO U2OS cells were transfected with GFP, GFP-PARP-1, GFP fused to the DNA-binding domain of PARP-1 (AA 1-336) or with GFP fused to the PARP-1 automodification and catalytic domain (AA 336-1014). 24 hours post transfection, cell lysates were collected and immunoprecipitation with GFP-Trap was performed. Bound proteins were eluted and resolved by SDS-PAGE analysis, followed by Western blot analysis with the indicated antibodies.


NEDD8 trimers specifically bind to second zinc finger of PARP - 1 . NEDP1 KO U2OS were transfected with GFP , GFP - PARP - 1 , or with GFP fused to Zn1 domain ( AA 1 - 96 ) , Zn2 domain ( AA 97 - 215 ) , Zn3 domain ( AA 216 - 336 ) or Zn1 + 2 domains ( AA 1 - 215 ) of PARP - 1 . 24 hours post transfection , lysates were collected immunoprecipitation with GFP - Trap was performed . Bound were eluted resolved by SDS - PAGE , followed by Western blot with indicated antibodies .

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NEDD8 trimers specifically bind to the second zinc finger of PARP-1. NEDP1 KO U2OS cells were transfected with GFP, GFP-PARP-1, or with GFP fused to the Zn1 domain (AA 1-96), the Zn2 domain (AA 97-215), the Zn3 domain (AA 216-336) or the Zn1+2 domains (AA 1-215) of PARP-1. 24 hours post transfection, cell lysates were collected and immunoprecipitation with GFP-Trap was performed. Bound proteins were eluted and resolved by SDS-PAGE analysis, followed by Western blot analysis with the indicated antibodies.


2D gel electrophoresis of lysate from WT followed by Western blot with α - NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point ( pI ) of 6 .

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2D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α-NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point (pI) of 6.59


2D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST - Zn1 + 2 followed by Western blot with α - NEDD8 antibody . Some of 25 - kDa NEDD8 band migrates to same isoelectric point as unconjugated NEDD8 ( denoted by triangle ) , indicating species is most likely an unanchored NEDD8 trimer . However , majority of NEDD8 trimer migrates to multiple spots of lower pI ( indicated in brackets ) , which suggests that NEDD8 trimers undergo a post - translational modification that either adds a negative charge or blocks a positive charge on NEDD8 trimer .

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2D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST-Zn1+2 followed by Western blot analysis with α-NEDD8 antibody. Some of the 25-kDa NEDD8 band migrates to the same isoelectric point as unconjugated NEDD8 (denoted by white triangle), indicating the species is most likely an unanchored NEDD8 trimer. However, the majority of NEDD8 trimer migrates to multiple spots of lower pI (indicated in the brackets), which suggests that NEDD8 trimers undergo a post-translational modification that either adds a negative charge or blocks a positive charge on the NEDD8 trimer.


Overexpression of HDAC1 or HDAC2 decreases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS were transfected with GFP or GFP fused to first two zinc finger domains of PARP - 1 ( GFP - Zn1 + 2 ) . were also co - transfected , as indicated , with empty FLAG vector , HDAC1 - FLAG , or HDAC2 - FLAG . 24 hours post transfection , were harvested immunoprecipitation was performed on lysates with GFP - Trap . Immunoprecipitated were resolved by SDS - PAGE followed by Western blot with indicated antibodies .

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Overexpression of HDAC1 or HDAC2 decreases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). Cells were also co-transfected, as indicated, with empty FLAG vector, HDAC1-FLAG, or HDAC2-FLAG. 24 hours post transfection, cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.


HDAC inhibition increases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS were transfected with GFP or GFP fused to first two zinc finger domains of PARP - 1 ( GFP - Zn1 + 2 ) . 24 hours later , were left untreated or treated with HDAC inhibitor sodium butyrate ( NaB ) ( 10 mM ) for 4 hours . were harvested immunoprecipitation was performed on lysates with GFP - Trap . Immunoprecipitated were resolved by SDS - PAGE followed by Western blot with indicated antibodies .

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HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). 24 hours later, cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.


HDAC inhibition increases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS were left untreated or treated with HDAC inhibitor sodium butyrate ( NaB ) ( 10 mM ) for 4 hours . were harvested lysates were incubated with recombinant Zn1 - GFP or Zn2 - GFP ( 25 nM ) for 1 hour followed by immunoprecipitation with GFP - Trap . Immunoprecipitated were resolved by SDS - PAGE followed by Western blot with indicated antibodies .

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HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and lysates were incubated with recombinant Zn1-GFP or Zn2-GFP (25 nM) for 1 hour followed by immunoprecipitation with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.


Overexpression of HDAC1 or HDAC2 in NEDP1 KO can rescue induction of PAR polymer following H2O2 treatment . U2OS WT or NEDP1 KO were transfected with indicated FLAG vectors . 48 post - transfection , were treated with H2O2 ( 600 μM ) for indicated amount of time harvested directly in sample loading buffer . Lysates were resolved by SDS - PAGE processed for Western blot with indicated antibodies .

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Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment. U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors. 48 post-transfection cells, were treated with H2O2 (600 μM) for the indicated amount of time and harvested directly in sample loading buffer. Lysates were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.


In vitro de - neddylation by NEDP1 is sensitive to oxidation . Poly - NEDD8 reactions were prepared with NAE , UBE2M , NEDD8 as in Figure 1E . NEDD8 reactions were stopped by addition of NAE inhibitor MLN4924 ( 3 μM ) . NEDD8 reactions were incubated with GST - NEDP1 at 30 ° C for indicated amount of time under reducing ( 50 μM DTT ) or oxidizing conditions ( 50 μM DTT + 150 μM H2O2 ) . Reactions were stopped by addition of LDS sample loading buffer resolved by SDS - PAGE before processing for Western blot with indicated antibodies .

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In vitro de-neddylation by NEDP1 is sensitive to oxidation. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. NEDD8 reactions were stopped by the addition of NAE inhibitor MLN4924 (3 μM). NEDD8 reactions were incubated with GST-NEDP1 at 30°C for the indicated amount of time under reducing (50 μM DTT) or oxidizing conditions (50 μM DTT + 150 μM H2O2). Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.


In vitro neddylation reactions with UBE2M are insensitive to H2O2 . Poly - NEDD8 reactions were prepared with NAE , UBE2M , NEDD8 as in Figure 1E . Before reactions were started , increasing concentrations of H2O2 ( as indicated ) were added to NEDD8 reactions . Subsequently reactions were incubated at 30 ° C for indicated amount of time . Reactions were stopped by addition of LDS sample loading buffer resolved by SDS - PAGE before processing for Western blot with indicated antibodies .

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In vitro neddylation reactions with UBE2M are insensitive to H2O2. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. Before reactions were started, increasing concentrations of H2O2 (as indicated) were added to the NEDD8 reactions. Subsequently the reactions were incubated at 30°C for the indicated amount of time. Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.


A Intracellular localization of total ( HA ) polymeric ATZ ( 2C1 ) in WT MEF mock - treated , Confocal Scanning Microscopy ( CLSM ) . B Same as A for MEF exposed to 50 nM BafA1 for 12 h . C Same as A , 4 h after BafA1 wash - out . D of ATZ - positive , LAMP1 - positive endolysosomes ( EL ) ( n = 13 , 10 , 11 , respectively ) . One - way ANOVA Dunnett ' s multiple comparisons , ns P > 0 . 05 , * * * * P < 0 . 0001 . Data information : Scale bars : 10 μm .

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A Intracellular localization of total (HA) and polymeric ATZ (2C1) in WT MEF mock-treated, Confocal Laser Scanning Microscopy (CLSM). B Same as A for MEF exposed to 50 nM BafA1 for 12 h. C Same as A, 4 h after BafA1 wash-out. D Quantification of ATZ-positive, LAMP1-positive endolysosomes (EL) (n=13, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. Data information: Scale bars: 10 μm.


E ATZ polymers immunoisolated from lysates of WT MEF mock treated ( lane 1 ) , incubated for 12 h with BafA1 ( lane 2 ) 4 h after BafA1 wash - out ( lane 3 ) . Immunoprecipitation ( IP ) of ATZ polymers with polymer - specific 2C1 antibody , transfer on PVDF , revealed with anti - HA antibody on western blot ( WB ) . F of E , n = 3 , mean ± . Unpaired two - t - , ns P > 0 . 05 , * P < 0 . 05 . Data information : Scale bars : 10 μm .

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E ATZ polymers immunoisolated from lysates of WT MEF mock treated (lane 1), incubated for 12 h with BafA1 (lane 2) and 4 h after BafA1 wash-out (lane 3). Immunoprecipitation (IP) of ATZ polymers with polymer-specific 2C1 antibody, transfer on PVDF membrane, revealed with anti-HA antibody on western blot (WB). F Quantification of E, n=3, mean ± SEM. Unpaired two-tailed t-test, ns P>0.05, * P<0.05. Data information: Scale bars: 10 μm.


G - K Same as B in WT MEF , in exposed to 20 mM CST in Cnx - , Crt - ERp57 - KO MEF . L of ATZ - positive EL ( n = 10 , 9 , 11 , 10 , 11 , respectively ) . One - way ANOVA Dunnett ' s multiple comparisons , ns P > 0 . 05 , * * * * P < 0 . 0001 . Data information : Scale bars : 10 μm .

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G-K Same as B in WT MEF, in cells exposed to 20 mM CST and in Cnx-, Crt- and ERp57-KO MEF. L Quantification of ATZ-positive EL (n=10, 9, 11, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. Data information: Scale bars: 10 μm.


A HEK293 transfected with empty vector ( lanes 1 , 6 ) , ATZ - HA ( 2 , 7 ) , FAM134B - V5 ( 3 8 ) , FAM134B - V5 ATZ - HA ( 4 , 9 ) , or FAM134BLIR - V5 ATZ - HA ( 5 , 10 ) , incubated for 12 h with 50 nM BafA1 then treated with cross - linker DSP before lysis as described in Methods . Lanes 1 - 5 , WB of total extract ( TCE ) ; lanes 6 - 10 , WB of antiV5 immunoprecipites to isolate complexes containing ectopically expressed FAM134B or FAM134BLIR . membranes were probed with antiV5 ( upper panels ) , antiCNX , antiHA antiLC3 antibodies . B of LC3 co - precipitating with FAM134B - V5 ( A , lanes 8 9 ) . Mean ± , n = 3 , unpaired two - t - , * P < 0 . 05 .

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A HEK293 cells transfected with empty vector (lanes 1, 6), ATZ-HA (2, 7), FAM134B-V5 (3 and 8), FAM134B-V5 and ATZ-HA (4, 9), or FAM134BLIR-V5 and ATZ-HA (5, 10), incubated for 12 h with 50 nM BafA1 and then treated with the cross-linker DSP before lysis as described in Methods. Lanes 1-5, WB of the total cell extract (TCE); lanes 6-10, WB of antiV5 immunoprecipites to isolate complexes containing ectopically expressed FAM134B or FAM134BLIR. The membranes were probed with antiV5 (upper panels), antiCNX, antiHA and antiLC3 antibodies. B Quantification of LC3 co-precipitating with FAM134B-V5 (A, lanes 8 and 9). Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05.


WT MEF . Scale bar : 10 μm . D Ectopic expression of FAM134BLIR in WT MEF inhibits ATZ delivery to endolysosomes . Scale bar : 10 μm .

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WT MEF. Scale bar: 10 μm. D Ectopic expression of FAM134BLIR in WT MEF inhibits ATZ delivery to endolysosomes. Scale bar: 10 μm.


E , F Distribution of gold - labeled ATZ - HA by IEM in BafA1 treated WT MEF WT MEF overexpressing FAM134BLIR , respectively . EV , ER - derived vesicles , red arrowheads ; EL , endolysosome . G of ATZ - gold density of E , F ( n = 75 79 EL , respectively ) . Unpaired two - t - , * * * * P < 0 . 0001 .

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E, F Distribution of gold-labeled ATZ-HA by IEM in BafA1 treated WT MEF and WT MEF overexpressing FAM134BLIR, respectively. EV, ER-derived vesicles, red arrowheads; EL, endolysosome. G Quantification of ATZ-gold density of E, F (n=75 and 79 EL, respectively). Unpaired two-tailed t-test, **** P <0.0001.


H Max projection of same as in D after deconvolution . Insets show orthogonal section of select regions . Scale bar : 10 μm .

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H Max projection of the same cell as in D after deconvolution. Insets show orthogonal section of select regions. Scale bar: 10 μm.


I Decay of ATZ polymers ( CHX chase , upper panel ) immunoisolated with polymer - specific 2C1 antibody ( visualized with antiHA in WB ) in HEK293 mock - transfected ( lanes 1 - 4 ) or expressing FAM134BLIR - V5 ( lanes 5 - 8 ) . Middle panel , expression of FAM134BLIR - V5 assessed by WB ; lower panel , loading control . ( J ) of ( I ) ( Mean ± , n = 3 , unpaired two - t - , * P < 0 . 05 ) .

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I Decay of ATZ polymers (CHX chase, upper panel) immunoisolated with the polymer-specific 2C1 antibody (visualized with antiHA in WB) in HEK293 cells mock-transfected (lanes 1-4) or expressing FAM134BLIR-V5 (lanes 5-8). Middle panel, expression of FAM134BLIR-V5 assessed by WB; lower panel, loading control. (J) Quantification of (I) (Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05).


K Flow cytometry of ATZ - HA polymer in MEFs mock treated , exposed to BafA1 , co - expressing FAM134LIR . MFI : mean ( Mean ± , n = 5 , unpaired two - t - , ns P > 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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K Flow cytometry analysis of ATZ-HA polymer levels in MEFs mock treated, exposed to BafA1, and co-expressing FAM134LIR. MFI: mean fluorescence intensity (Mean ± SEM, n=5, unpaired two-tailed t-test, ns P>0.05, ** P<0.01, *** P <0.001).


A WB showing KO efficiency for CRISPR134B MEF .

A WB analysis showing KO efficiency for CRISPR134B MEF.


WB showing KO efficiency for CRISPR62 MEF .

WB analysis showing KO efficiency for CRISPR62 MEF.


C - E CRISPR WT MEF , CRISPR134B MEF , CRISPR62 MEF , respectively . Scale bars : 10 μm . F of C - E ( n = 10 , 8 , 7 , respectively ) . One - way ANOVA Dunnett ' s multiple comparisons , ns P > 0 . 05 , * * * * P < 0 . 0001 .

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C-E CRISPR WT MEF, CRISPR134B MEF, CRISPR62 MEF, respectively. Scale bars: 10 μm. F Quantification of C-E (n=10, 8, 7 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.


A - H Intracellular localization of LAMP1 - positive EL , total ( HA ) polymeric ( 2C1 ) ATZ in WT MEF exposed to BafA1 for 12 h ( A ) ; in Atg4BKO MEF ( B ) ; in Atg7KO MEF ( C ) ; in Fip200KO MEF ( D ) ; in Ulk1 / 2 double - KO MEF ( E ) ; in Atg13KO MEF ( F ) ; in Atg9KO MEF ( G ) ; in RubiconKO MEF ( H ) . Scale bars : 10 μm . I Quantifications of A - H ( n = 10 , 9 , 9 , 10 , 7 , 15 , 9 , 12 ) . One - way ANOVA Dunnett ' s multiple comparisons , ns P > 0 . 05 , * * * * P < 0 . 0001 .

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A-H Intracellular localization of LAMP1-positive EL, total (HA) and polymeric (2C1) ATZ in WT MEF exposed to BafA1 for 12 h (A); in Atg4BKO MEF (B); in Atg7KO MEF (C); in Fip200KO MEF (D); in Ulk1/2 double-KO MEF (E); in Atg13KO MEF (F); in Atg9KO MEF (G); in RubiconKO MEF (H). Scale bars: 10 μm. I Quantifications of A-H (n=10, 9, 9, 10, 7, 15, 9, 12 cells). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.


A Distribution of gold - labeled ATZ - HA by IEM in WT MEF exposed to BafA1 ( on right is not transfected , as specificity control for gold labeling ) . ER , endoplasmic reticulum ; EV , ER - derived vesicle ; EL , endolysosomes . Scale bar : 1 μm . B Inset of A . Red asterisks show site of EV : EL membranes fusion

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A Distribution of gold-labeled ATZ-HA by IEM in WT MEF exposed to BafA1 (the cell on the right is not transfected, as specificity control for gold labeling). ER, endoplasmic reticulum; EV, ER-derived vesicle; EL, endolysosomes. Scale bar: 1 μm. B Inset of A. Red asterisks show site of EV:EL membranes fusion


C - E Distribution of gold - labeled ATZ - HA by IEM in WT MEF . ES , ER subdomains . Red asterisk shows site of EV : EL membranes fusion . Scale bar : 1 μm .

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C-E Distribution of gold-labeled ATZ-HA by IEM in WT MEF. ES, ER subdomains. Red asterisk shows site of EV:EL membranes fusion. Scale bar: 1 μm.


F 3D visualization by means of electron tomography of organelles containing gold labeled ATZ - HA ( visualized as dots ES , ER subdomains . Scale bar : 500 nm .

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F 3D visualization by means of electron tomography of organelles containing gold labeled ATZ-HA (visualized as white dots ES, ER subdomains. Scale bar: 500 nm.


G CLEM showing ATZ ( red ) , LAMP1 - positive EL ( cyan ) GFP - derived ( green ) in correspondence to heavily labeled vesicles , which were identified as autophagosomes for their double ( insets 1 - 3 ) . GFP - LC3 was also stained by immunogold . Scale bar : 1 μm .

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G CLEM showing ATZ (red), LAMP1-positive EL (cyan) and GFP-derived fluorescence (green) in correspondence to heavily labeled vesicles, which were identified as autophagosomes for their double membrane (insets 1-3). GFP-LC3 was also stained by immunogold. Scale bar: 1 μm.


A , B Triple of with LAMP1 ( cyan ) , HaloTag - ATZ ( red ) luminal ER marker sfGFP - KDEL ( green ) .

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A, B Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and the luminal ER marker sfGFP-KDEL (green).


Triple of with LAMP1 ( cyan ) , HaloTag - ATZ ( red ) with CNX as ER membranes marker .

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Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and with CNX as ER membranes marker.


Triple of with LAMP1 ( cyan ) , HaloTag - ATZ ( red ) with CLIMP63 as ER membranes marker .

Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and with CLIMP63 as ER membranes marker.


G of ER markers HaloTag - ATZ co - localization in time ( sfGFP - KDEL n = 10 , 10 , 12 , 13 , CNX n = 8 , 10 , 9 , 8 , CLIMP63 n = 8 , 10 , 6 , 10 for corresponding time points ) . Mean ± , unpaired two - t - , * * * * P < 0 . 0001 . Data information : Scale bars 10 μm .

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G Quantification of ER markers and HaloTag-ATZ co-localization in time (sfGFP-KDEL n=10, 10, 12, 13, CNX n=8, 10, 9, 8, CLIMP63 n=8, 10, 6, 10 cells for corresponding time points). Mean ± SEM, unpaired two-tailed t-test, **** P<0.0001. Data information: Scale bars 10 μm.


H of double - positive endolysosomes at 300 ' ( ATZ : sfGFP - KDEL n = 14 , ATZ : CNX n = 8 , ATZ : CLIMP63 n = 10 . One - way ANOVA Dunnett ' s multiple comparisons , * * P < 0 . 01 * * * * P < 0 . 0001 ) . Data information : Scale bars 10 μm .

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H Quantification of double-positive endolysosomes at 300' (ATZ:sfGFP-KDEL n=14, ATZ:CNX n=8, ATZ:CLIMP63 n=10 cells. One-way ANOVA and Dunnett's multiple comparisons test, ** P<0.01 **** P<0.0001). Data information: Scale bars 10 μm.


C - E CLSM of ATZ delivery to LAMP1 - positive endolysosomes in BafA1 - treated CRISPRWT MEF ( C ) ; in CRISPR17 MEF ( D ) ; in CRISPR8 MEF ( E ) . Scale bars : 10 μm .

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C-E CLSM analysis of ATZ delivery to LAMP1-positive endolysosomes in BafA1-treated CRISPRWT MEF (C); in CRISPR17 MEF(D); in CRISPR8 MEF (E). Scale bars: 10 μm.


( n = 10 , 10 , 11 , respectively ) . One - way ANOVA Dunnett ' s multiple comparisons , * * * * P < 0 . 0001 .

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Quantification (n=10, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, **** P<0.0001.


G - I Distribution of gold - labelled ATZ - HA by IEM in CRISPRWT MEF exposed to BafA1 ( G ) ; in CRISPR17 ( H ) ; in CRISPR8 . ( I ) . ER , endoplasmic reticulum ; EV , ER - derived vesicles ; EL , endolysosomes . Scale bars : 1 μm .

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G-I Distribution of gold-labelled ATZ-HA by IEM in CRISPRWT MEF exposed to BafA1 (G); in CRISPR17 (H); in CRISPR8. (I). ER, endoplasmic reticulum; EV, ER-derived vesicles; EL, endolysosomes. Scale bars: 1 μm.


GFP reconstitution in ATZ - HA transfected CRISPR17 MEF expressing FAM134B - T11 GFP1 - 10 - LC3 . Scale bar : 10 μm . K Same as J by CLEM . ATZ - HA in red , LAMP1 - positive endolysosomes in cyan . FAM134B : LC - induce GFP reconstitution in green . FAM134B in gold is shown . Scale bar : 1 μm .

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GFP reconstitution in ATZ-HA transfected CRISPR17 MEF expressing FAM134B-T11 and GFP1-10-LC3. Scale bar: 10 μm. K Same as J by CLEM. ATZ-HA staining in red, LAMP1-positive endolysosomes in cyan. FAM134B:LC-induce GFP reconstitution in green. FAM134B in gold is shown. Scale bar: 1 μm.


A . production of Sox1 - GFP + neural - lineage during 7 days of neural differentiation , as shown by microscopy . Scale bars , 100 µm .

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A. The production of Sox1-GFP+ neural-lineage cells during 7 days of neural differentiation, as shown by microscopy. Scale bars, 100 µm.


B . qRT - PCR of NDIME expression during 7 days of neural differentiation ( D3 , P = 0 . 068 ; D5 , P = 0 . 0049 ; D7 , P = 0 . 0003 ) .

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B. qRT-PCR analysis of NDIME expression during 7 days of neural differentiation (D3, P = 0.068; D5, P = 0.0049; D7, P = 0.0003).


C . Relative expression level of NDIME mRNA in Sox1 - GFP - population Sox1 - GFP + population at day 7 of neural differentiation ( P = 0 . 0118 ) .

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C. Relative expression level of NDIME mRNA in the Sox1-GFP- population and Sox1-GFP+ population at day 7 of neural differentiation (P = 0.0118).


D . mESCs were cultured in differentiation medium alone , or with 10 % FBS , at day 5 of neural differentiation as shown by microscopy . Scale bars , 100 µm . E . Temporal expression profile of NDIME by qRT - PCR . mESCs were cultured in differentiation medium alone or with 10 % FBS ( D3 , P = 0 . 074 ; D5 , P = 0 . 0048 ; D7 , P = 0 . 0034 ) .

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D. mESCs were cultured in differentiation medium alone, or with 10% FBS, at day 5 of neural differentiation as shown by microscopy. Scale bars, 100 µm. E. Temporal expression profile of NDIME by qRT-PCR. mESCs were cultured in differentiation medium alone or with 10% FBS (D3, P = 0.074; D5, P = 0.0048; D7, P = 0.0034).


D . mESCs were cultured in differentiation medium alone , or with 10 % FBS , at day 5 of neural differentiation as shown by microscopy . Scale bars , 100 µm .

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D. mESCs were cultured in differentiation medium alone, or with 10% FBS, at day 5 of neural differentiation as shown by microscopy. Scale bars, 100 µm.


F . Distribution of NDIME in cytoplasm nucleus at day 7 of neural differentiation . XIST GAPDH mRNA were considered as positive controls for fractions , respectively . Data are represented as mean , error bars represent in panels . n = 3 .

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F. Distribution of NDIME in the cytoplasm and nucleus at day 7 of neural differentiation. The XIST RNA and GAPDH mRNA were considered as the positive controls for the nuclear and cytosolic fractions, respectively. Data are represented as mean, and error bars represent SEM in all panels. n= 3.


G . Verification of efficiency of knockout of exon 3 of NDIME in mESCs via qRT - PCR .

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G. Verification of the efficiency of knockout of exon 3 of NDIME in mESCs via qRT-PCR.


Knockout of NDIME strongly inhibited neural differentiation , as shown by microscopy analyses during 7 days of neural differentiation . Scale bars , 100 µm .

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Knockout of NDIME strongly inhibited neural differentiation, as shown by microscopy analyses during 7 days of neural differentiation. Scale bars, 100 µm.


I . Knockout of NDIME strongly inhibited neural differentiation , as shown by FACS analyses during 7 days of neural differentiation . Scale bars , 100 µm .

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I. Knockout of NDIME strongly inhibited neural differentiation, as shown by FACS analyses during 7 days of neural differentiation. Scale bars, 100 µm.


J . Knockout of NDIME inhibits expression of neural - lineage genes at day 5 of neural differentiation .

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J. Knockout of NDIME inhibits the expression of neural-lineage genes at day 5 of neural differentiation.


K . NESTIN GFP immunostaining of in ( H ) at day 5 of neural differentiation . Nuclei were stained with Hoechst 33342 ( blue ) . Scale bars , 100 µm .

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K. NESTIN and GFP immunostaining of the cells in (H) at day 5 of neural differentiation. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100 µm.


L . NESTIN GFP immunostaining of Ctrl , NDIME - / - , NDIME - / - + Rs26 - NDIME at day 5 of neural differentiation . Scale bars , 100 µm .

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L. NESTIN and GFP immunostaining of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation. Scale bars, 100 µm.


M . Expression of neural - lineage genes of Ctrl , NDIME - / - , NDIME - / - + Rs26 - NDIME at day 5 of neural differentiation .

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M. Expression of neural-lineage genes of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation.


N . Relative expression of Emx1 , Brn2 , Sox5 , Zeb2 mRNA in different differentiation stages of Ctrl NDIME - knockout ( compared with control ) .

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N. Relative expression of Emx1, Brn2, Sox5, and Zeb2 mRNA in different differentiation stages of Ctrl and NDIME-knockout cells (compared with control cells).


A , Expression of MEF2C ( D0 , P = 0 . 0020 ; D3 , P = 0 . 0025 ; D5 , P = 0 . 0003 ; D7 , P = 0 . 0142 ) of Ctrl NDIME - / - during 7 days of neural differentiation , following knockout of NDIME at day 5 of neural differentiation .

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A, Expression levels of MEF2C (D0, P = 0.0020; D3, P = 0.0025; D5, P = 0.0003; D7, P = 0.0142) of Ctrl and NDIME-/- cells during 7 days of neural differentiation, following knockout of NDIME at day 5 of neural differentiation.


B . ( D0 , P = 0 . 0020 ; D3 , P = 0 . 0025 ; D5 , P = 0 . 0003 ; D7 , P = 0 . 0142 ) of Ctrl NDIME - / - during 7 days of neural differentiation , level of MEF2C following knockout of NDIME at day 5 of neural differentiation .

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B. (D0, P = 0.0020; D3, P = 0.0025; D5, P = 0.0003; D7, P = 0.0142) of Ctrl and NDIME-/- cells during 7 days of neural differentiation, and protein level of MEF2C following knockout of NDIME at day 5 of neural differentiation.


C . Western blot of effects of MEF2C overexpression in NDIME - knockout at day 5 of neural differentiation .

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C. Western blot analysis of the effects of MEF2C overexpression in NDIME-knockout cells at day 5 of neural differentiation.


Overexpression of MEF2C in NDIME - knockout rescued neural differentiation defects at day 5 of neural differentiation , as shown by microscopy

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Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by microscopy


Overexpression of MEF2C in NDIME - knockout rescued neural differentiation defects at day 5 of neural differentiation , as shown by

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E . Overexpression of MEF2C in NDIME - knockout rescued neural differentiation defects at day 5 of neural differentiation , as shown by FACS

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E. Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by FACS


E . Overexpression of MEF2C in NDIME - knockout rescued neural differentiation defects at day 5 of neural differentiation , as shown by

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F . NESTIN GFP immunostaining of in ( D ) at day 5 of neural differentiation . Scale bars , 100 µm .

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F. NESTIN and GFP immunostaining of the cells in (D) at day 5 of neural differentiation. Scale bars, 100 µm.


G . Expression of neural - lineage genes in Ctrl , NDIME - / - , NDIME - / - + Mef2c during 7 days of neural differentiation .

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G. Expression of neural-lineage genes in Ctrl, NDIME-/-, and NDIME-/-+Mef2c cells during 7 days of neural differentiation.


Enrichment of H3K27me3 at Mef2c promoter in NDIME - knockout at day 5 of neural differentiation compared with control .

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Enrichment of H3K27me3 at the Mef2c promoter in the NDIME-knockout cells at day 5 of neural differentiation compared with control cells.


B . Enrichment of polymerase II at Mef2c promoter in NDIME - knockout at day 5 of neural differentiation compared with control .

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B. Enrichment of RNA polymerase II at the Mef2c promoter in the NDIME-knockout cells at day 5 of neural differentiation compared with control cells.


C . NDIME interacts with EZH2 ( P = 0 . 0043 ) SUZ12 ( P = 0 . 0003 ) , but not EED ( P = 0 . 6720 ) EZH1 ( P = 0 . 0951 ) compared with IgG , as determined by RIP using EZH2 , SUZ12 , EED , EZH1 antibodies at day 5 of neural differentiation . U1 as a negative control . presence of NDIME U1 was measured by qRT - PCR with or without reverse transcriptase ( RT ) .

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C. NDIME interacts with EZH2 (P = 0.0043) and SUZ12 (P = 0.0003), but not EED (P = 0.6720) and EZH1 (P = 0.0951) compared with IgG, as determined by RIP using EZH2, SUZ12, EED, and EZH1 antibodies at day 5 of neural differentiation. Nuclear RNA U1 as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).


NDIME following EZH2 - RIP ( P = 0 . 0028 ) in HEK293T compared with pcDNA3 . 1 . U1 was used as a negative control . presence of NDIME U1 was measured by qRT - PCR with or without reverse transcriptase ( RT ) .

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NDIME levels following EZH2-RIP (P = 0.0028) in HEK293T cells compared with pcDNA3.1. Nuclear RNA U1 was used as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).


E . NDIME following SUZ12 - RIP ( P = 0 . 0015 ) in HEK293T compared with pcDNA3 . 1 . U1 was used as a negative control . presence of NDIME U1 was measured by qRT - PCR with or without reverse transcriptase ( RT ) .

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E. NDIME levels following SUZ12-RIP (P = 0.0015) in HEK293T cells compared with pcDNA3.1. Nuclear RNA U1 was used as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).


F , Enrichment of EZH2 at Mef2c promoter in NDIME - knockout at day 5 of neural differentiation compared with control .

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F, Enrichment of EZH2 at the Mef2c promoter in NDIME-knockout cells at day 5 of neural differentiation compared with control cells.


G . Enrichment of SUZ12 at Mef2c promoter in NDIME - knockout at day 5 of neural differentiation compared with control .

G. Enrichment of SUZ12 at the Mef2c promoter in NDIME-knockout cells at day 5 of neural differentiation compared with control cells.


H . Expression of neural - lineage genes at day 5 of neural differentiation in NDIME - knockout after adding EZH2 inhibitor GSK126 ( 7 . 5 µM ) partially rescued neural differentiation defects , as shown by qRT - PCR ; wild - type NDIME - knockout treated with DMSO .

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H. Expression of neural-lineage genes at day 5 of neural differentiation in the NDIME-knockout cells after adding the EZH2 inhibitor GSK126 (7.5 µM) partially rescued neural differentiation defects, as shown by qRT-PCR; the wild-type and NDIME-knockout cells treated with DMSO.


B . RIP using EZH2 antibody for co - transfection of Flag - EZH2 NDIME exons 1 - 3 ( P = 0 . 0177 ) or exon 4 ( P = 0 . 4335 ) in HEK293T extracts compared with IgG .

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B. RIP analysis using EZH2 antibody for the co-transfection of Flag-EZH2 and NDIME exons 1-3 (P = 0.0177) or exon 4 (P = 0.4335) in HEK293T extracts compared with IgG.


C . Verification of efficiency of overexpression of exons 1 - 3 or exon 4 of NDIME in NDIME - knockout at day 5 of neural differentiation via qRT - PCR . Three different primers P1 , P2 , P3 were used to analyze NDIME or exons of NDIME expression in Ctrl , NDIME - / - , NDIME - / - + Rs26 - Exon123 , NDIME - / - + Rs26 - Exon4 ( compared with control ) . position of primers P1 , P2 , P3 are shown in schematic diagram .

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C. Verification of the efficiency of overexpression of exons 1-3 or exon 4 of NDIME in NDIME-knockout cells at day 5 of neural differentiation via qRT-PCR. Three different primers P1, P2, and P3 were used to analyze NDIME or exons of NDIME expression in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells (compared with control cells). The position of primers P1, P2, and P3 are shown in the schematic diagram.


FACS , in Ctrl , NDIME - / - , NDIME - / - + Rs26 - Exon123 , NDIME - / - + Rs26 - Exon4 at day 5 of neural differentiation .

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FACS , in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation.


E . , expression of neural - lineage genes , in Ctrl , NDIME - / - , NDIME - / - + Rs26 - Exon123 , NDIME - / - + Rs26 - Exon4 at day 5 of neural differentiation .

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E. , expression of neural-lineage genes , in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation.


F . Microscopy of control , NDIME - / - , NDIME - / - + Rs26 - Exon123 , NDIME - / - + Rs26 - Exon4 at day 5 of neural differentiation . Scale bars , 100 µm .

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F. Microscopy of the control, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation. Scale bars, 100 µm.


G . NESTIN GFP immunostaining of in ( F ) at day 5 of neural differentiation . Scale bars , 100 µm .

G. NESTIN and GFP immunostaining of the cells in (F) at day 5 of neural differentiation. Scale bars, 100 µm.


H . Enrichment of H3K27me3 at Mef2c promoter of in ( F ) at day 5 of neural differentiation .

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H. Enrichment of H3K27me3 at the Mef2c promoter of the cells in (F) at day 5 of neural differentiation.


I . Western blot of expression of MEF2C in in ( F ) at day 5 of neural differentiation .

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I. Western blot analysis of expression of MEF2C in the cells in (F) at day 5 of neural differentiation.


A . Summary of DEGs in NDIME - knockout compared with control at day 5 of neural differentiation .

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A. Summary of the DEGs in the NDIME-knockout cells compared with control cells at day 5 of neural differentiation.


C . Summary of DEGs in MEF2C - knockdown compared with control at day 5 of neural differentiation .

C. Summary of the DEGs in the MEF2C-knockdown cells compared with control cells at day 5 of neural differentiation.


I . illustrating expression of selected neurectoderm genes mESCs - specific genes that were shown as log2 FPKM in Ctrl , NDIME - / - , shCtrl shMef2c mESCs at day 5 of neural differentiation . Each lane corresponds to an independent biological sample .

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I. Heatmap illustrating the expression of selected neurectoderm genes and mESCs-specific genes that were shown as log2 FPKM in Ctrl, NDIME-/- , shCtrl and shMef2c mESCs at day 5 of neural differentiation. Each lane corresponds to an independent biological sample.


J . qRT - PCR of representative co - upregulated co - downregulated genes in ( G ) at day 5 of neural differentiation .

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J. qRT-PCR analysis of representative co-upregulated and co-downregulated genes in (G) at day 5 of neural differentiation.


L . Relative expression of selectively down - regulated ASD risk genes from DEGs of NDIME - knockout compared with control at day 5 from DEGs of MEF2C - knockdown compared with control at day 5 .

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L. Relative expression of selectively down-regulated ASD risk genes from the DEGs of the NDIME-knockout cells compared with control cells at day 5 and from the DEGs of the MEF2C-knockdown cells compared with control cells at day 5.


A . qRT - PCR of NDIME ( P = 0 . 0051 ) MEF2C ( P = 0 . 0062 ) in VPA - treated mice saline - treated mice ; n = 9 per group .

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A. qRT-PCR analysis of NDIME (P = 0.0051) and MEF2C (P = 0.0062) in VPA-treated mice and saline-treated mice; n = 9 per group.


B . Enrichment of EZH2 at Mef2c promoter in VPA - treated mice saline - treated mice ( compared with saline - treated mice ) ; n = 9 per group .

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B. Enrichment of EZH2 at the Mef2c promoter in VPA-treated mice and saline-treated mice (compared with saline-treated mice); n = 9 per group.


C . Western blot of of MEF2C GAPDH in hippocampus of VPA - treated mice ( n = 9 ) saline - treated mice ( n = 10 ) .

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C. Western blot analysis of the protein levels of MEF2C and GAPDH in the hippocampus of VPA-treated mice (n = 9) and saline-treated mice (n = 10).


D . Immunofluorescence for MEF2C in hippocampus of VPA - treated mice saline - treated mice . Nuclei were stained with Hoechst 33342 ( blue ) ; n = 9 per group . Scale bars , 50 µm . E . Statistical of grayscale of MEF2C in ( C ) by ImageJ ( P = 0 . 0074 ) ; saline - treated mice ( n = 10 ) , VPA - treated mice ( n = 9 ) . F . Statistical of Mean MEF2C / unit in ( D ) by ImageJ ( P = 0 . 0096 ) ; n = 9 per group .

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D. Immunofluorescence images for MEF2C in the hippocampus of VPA-treated mice and saline-treated mice. Nuclei were stained with Hoechst 33342 (blue); n = 9 per group. Scale bars, 50 µm. E. Statistical analysis of the grayscale levels of MEF2C in (C) by ImageJ (P = 0.0074); saline-treated mice (n = 10), VPA-treated mice (n = 9). F. Statistical analysis of the Mean MEF2C staining intensity/unit area in (D) by ImageJ (P = 0.0096); n = 9 per group.


D . Immunofluorescence for MEF2C in hippocampus of VPA - treated mice saline - treated mice . Nuclei were stained with Hoechst 33342 ( blue ) ; n = 9 per group . Scale bars , 50 µm .

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D. Immunofluorescence images for MEF2C in the hippocampus of VPA-treated mice and saline-treated mice. Nuclei were stained with Hoechst 33342 (blue); n = 9 per group. Scale bars, 50 µm.


G . Immunofluorescence for GFP in hippocampus of saline - treated mice , VPA - treated mice , VPA - treated mice with viral - mediated expression of NDIME

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G. Immunofluorescence images for GFP in the hippocampus of saline-treated mice, VPA-treated mice, and VPA-treated mice with viral-mediated expression of NDIME


G . Immunofluorescence for GFP in hippocampus of saline - treated mice , VPA - treated mice , VPA - treated mice with viral - mediated expression of

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H . Immunofluorescence for MEF2C of mice in ( G ) ; n = 9 per group . Scale bars , 50 µm .

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H. Immunofluorescence images for MEF2C of mice in (G); n = 9 per group. Scale bars, 50 µm.


I - K . Adeno - associated overexpression of NDIME in dentate gyrus ( DG ) of dorsal hippocampus of VPA - treated mice rescued autism - like social deficits . Open field ( I ) , three - chamber ( J ) , elevated - plus maze ( EPM ) ( K ) . Data in ( I - K ) are represented as mean ± ; n = 9 per group . Asterisks indicate a difference between VPA + AAV - GFP Saline + AAV - GFP , whereas hash tags indicate a difference between VPA + AAV - NDIME - GFP VPA + AAV - GFP . # P < 0 . 05 , * * / # # P < 0 . 01 , * * * P < 0 . 001 ( one - way ANOVA with Bonferroni post - hoc ) . NS - non ‐ significant .

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I-K. Adeno-associated virus overexpression of NDIME in the dentate gyrus (DG) of the dorsal hippocampus of VPA-treated mice rescued autism-like social deficits. Open field test (I), three-chamber test (J), and elevated-plus maze (EPM) test (K). Data in (I-K) are represented as mean ± SEM; n = 9 per group. Asterisks indicate a difference between VPA+AAV-GFP and Saline+AAV-GFP, whereas hash tags indicate a difference between VPA+AAV-NDIME-GFP and VPA+AAV-GFP. #P < 0.05, **/##P < 0.01, ***P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant.


C ) Western Blot showing of PgP control ( α - TUBB ) in RPE - 1 parental Taxol resistant clones .

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C) Western Blot showing the levels of the PgP and control (α-TUBB) in RPE-1 parental and Taxol resistant clones.


E ) Representative smRNA - FISH of RPE - 1 Parental clones for ABCB1 gene DAPI . are projections of 0 , 5μm sections a total 5 μm in thickness . Scale bar , 15μm .

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E) Representative smRNA-FISH images of RPE-1 Parental and clones for the ABCB1 gene and DAPI. The images are projections of 0,5μm sections and a total 5 μm in thickness. Scale bar, 15μm.


A ) TLA for ABCB1 contacts in RPE - 1 parental Taxol resistant clone sg6C9 covering whole genome . Green arrow in sg6C9 shows a de novo interaction found between ABCB1 a region in chromosome 6 . B ) TLA for RPE - 1 Parental sg6C9 . Zoom in in region of chr6 with de novo interaction for sg6C9 . Image modified from IGV viewer .

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A) TLA analysis for ABCB1 contacts in RPE-1 parental and Taxol resistant clone sg6C9 covering the whole genome. Green arrow in sg6C9 shows a de novo interaction found between ABCB1 and a region in chromosome 6. B) TLA analysis for RPE-1 Parental and sg6C9. Zoom in in the region of chr6 with de novo interaction for sg6C9. Image modified from IGV viewer.


D ) PCR products using primers in C ( 1 ) over ABCB1 EEF1A1 regions in RPE - 1 Parental different Taxol resistant clones . E ) PCR products using primers in C ( 2 ) over specific break site in ABCB1 promoter in RPE - 1 Parental different Taxol resistant clones .

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D) PCR products using the primers in C(1) over the ABCB1 and EEF1A1 regions in RPE-1 Parental and the different Taxol resistant clones. E) PCR products using the primers in C(2) over the specific break site in the ABCB1 promoter in RPE-1 Parental and the different Taxol resistant clones.


G ) of RPE - 1 targeted with ABCB1 promoter sgRNAs found with vector integration . Percentages were calculated from data in Fig 1A . Each dot represents a CFA replicate experiment . Horizontal bars represent mean of lentiviral integratation per sgRNA .

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G) Percentage of RPE-1 cells targeted with the ABCB1 promoter sgRNAs found with vector integration. Percentages were calculated from data in Fig 1A. Each dot represents a CFA replicate experiment. Horizontal bars represent the mean of lentiviral integratation per sgRNA.


A ) H3K9me3 ChIP - sequencing tracks from q21 . 12 arm of chromosome 7 in RPE - 1 ( Parental Taxol - resistant clones ) . ABCB1 gene region is shown in pink . Each colored track shows H3k9me3 profile of a different Taxol - resistant clone .

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A) H3K9me3 ChIP-sequencing tracks from the q21.12 arm of chromosome 7 in RPE-1 cells (Parental and Taxol-resistant clones). The ABCB1 gene region is shown in pink. Each colored track shows the H3k9me3 profile of a different Taxol-resistant clone.


B ) H3K9me3 ChIP - qPCR in ABCB1 promoter region . Bar graph show primer pairs amplifying sgRNA # 6 break spanning this region ( + / - base pairs ) for each Taxol - resistant clone compared to parental . Bar plots show mean of H3K9me3 relative enrichment . Each dot represents a technical replicate ( n = 3 ) .

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B) H3K9me3 ChIP-qPCR in the ABCB1 promoter region. Bar graph show primer pairs amplifying the sgRNA#6 break and spanning this region (+/- base pairs) for each Taxol-resistant clone compared to the parental. Bar plots show the mean of H3K9me3 relative enrichment. Each dot represents a technical replicate (n=3).


VERO AT2 were infected with WT alpha ( α ) SARS - CoV - 2 live variants , heat inactivated α variant at MOI 0 . 1 . ( B ) were lysed 48h post - infection viral as well as cycle associated expression was analysed by western blot . N , nucleocapsid .

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VERO AT2 cells were infected with WT and alpha (α) SARS-CoV-2 live virus variants, and heat inactivated α variant at MOI 0.1. (B) Cells were lysed 48h post-infection and viral protein as well as cell cycle associated protein expression was analysed by western blot. N, nucleocapsid.


VERO AT2 were infected with WT alpha ( α ) SARS - CoV - 2 live variants , heat inactivated α variant at MOI 0 . 1 . ( C ) VERO AT2 were fixed 24h post - infection stained for viral cyclins . Arrowheads highlight un - infected cyclin D / A localization . Arrowheads : cyclin in uninfected . Scale bars : 20μm . ( D ) VERO AT2 . of cyclin A2 , D1 D3 relocalization from nucleus after infection . Uninfected ( - ) SARS - CoV - 2 infected were identified by negative / positive nucleocapsid or Spike . between cytoplasm of cyclins was measured using ImageJ Harmony ( PerkinElmer ) . At least 50 were counted . Bars indicate mean with SD . Statistical was performed using two - sided unpaired Student ' s t - ; ns , non - significant ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

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VERO AT2 cells were infected with WT and alpha (α) SARS-CoV-2 live virus variants, and heat inactivated α variant at MOI 0.1. (C) VERO AT2 Cells were fixed 24h post-infection and stained for viral proteins and cyclins. Arrowheads highlight un-infected cells and cyclin D/A nuclear localization. Arrowheads: Nuclear cyclin staining in uninfected cells. Scale bars: 20μm. (D) VERO AT2 cells. Quantification of cyclin A2, D1 and D3 relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid or Spike staining. Ratio between nuclear and cytoplasm staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells were counted. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ns, non-significant; ∗∗∗∗p < 0.0001.


( E ) A549 AT2 were infected with WT , alpha ( α ) , delta ( ∆ ) SARS - CoV - 2 variants . were lysed 48h post - infection viral , cyclin expression was analysed by western blot . N , nucleocapsid .

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(E) A549 AT2 cells were infected with WT, alpha (α), and delta (∆) SARS-CoV-2 variants. Cells were lysed 48h post-infection and viral, cyclin proteins expression was analysed by western blot. N, nucleocapsid.


( F ) A549 AT2 . of cyclin A2 , D1 D3 relocalization from nucleus after infection . Uninfected ( - ) SARS - CoV - 2 infected were identified by negative / positive nucleocapsid or Spike . between cytoplasm ( N / C ) of cyclins was measured using ImageJ Harmony ( PerkinElmer ) . At least 50 were counted . Bars indicate mean with SD . Statistical was performed using two - sided unpaired Student ' s t - ; ns , non - significant ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

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(F) A549 AT2 cells. Quantification of cyclin A2, D1 and D3 relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid or Spike staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells were counted. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ns, non-significant; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


A549 AT2 were infected with Delta SARS - CoV - 2 variant . Proteasome inhibitor Bortezomib ( BZ , 1μM ) was added to 18h post - infection . were lysed 24h post - addition of inhibitor cyclins viral were detected by western blot . ( N ) nucleocapsid . Densitometry of western blots for D - cyclins ( normalized to actin ) in A549 AT2 . Plots are average of 2 ( cyclin D1 ) , 3 ( cyclin D3 ) biological replicates . Bars indicate mean with SD . Statistical was performed using ordinary two way ANOVA ; ns , non - significant ; ∗ p < 0 . 1 .

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A549 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1μM) was added to cells 18h post-infection. Cells were lysed 24h post-addition of inhibitor and cyclins and viral proteins were detected by western blot. (N) nucleocapsid. Densitometry analysis of western blots for D-cyclins (normalized to actin) in A549 AT2 cells. Plots are average of 2 (cyclin D1), 3 (cyclin D3) biological replicates. Bars indicate mean with SD. Statistical analysis was performed using ordinary two way ANOVA; ns, non-significant; ∗p < 0.1.


A459 AT2 were infected with Delta SARS - CoV - 2 variant . Proteasome inhibitor Bortezomib ( BZ , 1μM ) was added to 8h post - infection . were fixed stained 24h post - addition of inhibitor . ( N ) nucleocapsid . Scale bars : 40μm . Contour lines represent outlines of infected .

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A459 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1μM) was added to cells 8h post-infection. Cells were fixed and stained 24h post-addition of inhibitor. (N) nucleocapsid.Scale bars: 40μm. Contour lines represent outlines of infected cells.


of D3 cyclin relocalization from nucleus after infection . Uninfected ( - ) SARS - CoV - 2 infected were identified by negative / positive nucleocapsid . between cytoplasm ( N / C ) of cyclins was measured using ImageJ Harmony ( PerkinElmer ) . At least 50 have been counted . Bars indicate mean with SD . Statistical was performed using ordinary two - way ANOVA ; ∗ ∗ ∗ p < 0 . 001 .

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Quantification of D3 cyclin relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells have been counted. Bars indicate mean with SD. Statistical analysis was performed using ordinary two-way ANOVA; ∗∗∗p < 0.001.


( A , D A - cyclins were depleted using siRNA in A549 AT2 . ( A , Representative example of western blot from uninfected lysates collected at 72h post - knockdown .

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(A, D and A-cyclins were depleted using siRNA in A549 AT2 cells. (A, Representative example of western blot from uninfected cell lysates collected at 72h post-knockdown.


B ) D A - cyclins were depleted using siRNA in A549 AT2 . were infected 18h later with Delta ( ∆ ) , Alpha ( α ) or wild type ( WT ) SARS - CoV - 2 variants at MOI 0 . 001 , 0 . 1 , 0 . 1 respectively . were washed 4h post - infection new media were added . Supernatants were collected 48h later for TCID50 ( B ) . ( B , titres in culture supernatants were determined as TCID50 Data information : Graphs represent average of n = 3 ( B , Statistical was performed using one - way ANOVA with Dunnett ' s multiple comparisons . NT , non - targeting control . ns , non - significant ; ∗ p < 0 . 1 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD .

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B) D and A-cyclins were depleted using siRNA in A549 AT2 cells. Cells were infected 18h later with Delta (∆), Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.001, 0.1, 0.1 respectively. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for TCID50 (B). (B, Virus titres in cell culture supernatants were determined as TCID50 Data information: Graphs represent average of n=3 (B, Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.


( C , D A - cyclins were depleted using siRNA in VERO AT2 . C ) Representative example of western blot from uninfected lysates collected at 72h post - knockdown .

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(C, D and A-cyclins were depleted using siRNA in VERO AT2 cells. C) Representative example of western blot from uninfected cell lysates collected at 72h post-knockdown.


D ) D A - cyclins were depleted using siRNA in VERO AT2 . were infected 18h later with Alpha ( α ) or wild type ( WT ) SARS - CoV - 2 variants at MOI 0 . 1 . were washed 4h post - infection new media added . Supernatants were collected at 24h 48h later . D ) titres in culture supernatants were determined as TCID50 in VERO AT2 . Data information : Graphs represent average of n = 3 D ) ; Statistical was performed using one - way ANOVA with Dunnett ' s multiple comparisons . NT , non - targeting control . ns , non - significant ; ∗ p < 0 . 1 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD .

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D) D and A-cyclins were depleted using siRNA in VERO AT2 cells. Cells were infected 18h later with Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.1. Cells were washed 4h post-infection and new media added. Supernatants and cells were collected at 24h and 48h later. D) Virus titres in cell culture supernatants were determined as TCID50 in VERO AT2 cells. Data information: Graphs represent average of n=3 D); Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.


Cyclin D3 was depleted using siRNA in Calu3 . were infected 48h later with Delta SARS - CoV - 2 at MOI 0 . 01 . were washed 4h post - infection new media were added . Supernatants were collected 48h later for western blot TCID50 . Uninfected lysates were used for western blot . Data information : Graphs represent average of n = 4 ( E ) biological replicates . Statistical was performed using one - way ANOVA with Dunnett ' s multiple comparisons . NT , non - targeting control . ns , non - significant ; ∗ p < 0 . 1 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD .

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Cyclin D3 was depleted using siRNA in Calu3 cells. Cells were infected 48h later with Delta SARS-CoV-2 at MOI 0.01. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for western blot and TCID50. Uninfected cell lysates were used for western blot. Data information: Graphs represent average of n=4 (E) biological replicates. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.


A549 AT2 were depleted for D A2 cyclins ( A ) A representative western blot from lysates of uninfected knock - down .

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A549 AT2 cells were depleted for D and A2 cyclins (A) A representative western blot from lysates of uninfected knock-down cells.


( E ) A549 AT2 were transduced with VSV - G pseudotyped Fucci containing lentiviral particles VSV - G pseudotyped lentiviral particles containing WT cyclin D3 or T283A mutant cyclin D3 ( mutant not degraded by proteasome ) . were infected 24h later collected 48h post - infection for flow cytometry of early S western blot . n = 2 biological replicates ; Ordinary two - way ANOVA with Sidak ' s multiple comparisons : ns , non - significant ; ∗ ∗ p < 0 . 01 . Bars indicate mean with SD .

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(E) A549 AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and VSV-G pseudotyped lentiviral particles containing WT cyclin D3 or T283A mutant cyclin D3 (mutant not degraded by proteasome). Cells were infected 24h later and collected 48h post-infection for flow cytometry analysis of early S and western blot. n=2 biological replicates; Ordinary two-way ANOVA with Sidak's multiple comparisons test: ns, non-significant; ∗∗p < 0.01. Bars indicate mean with SD.


( F , G ) VERO AT2 were transduced with VSV - G pseudotyped Fucci containing lentiviral particles 18h later infected with Delta variant SARS - CoV - 2 . were fixed stained for D - cyclins 24h later . ( F ) Example of acquisition using automated microscopic platform . are identified for infection , expression of cyclin D3 , cycle ( Red / arrow = G1phase ; Green / arrowhead = S / G2 / M ; Red + Green / arrowhead = early S ) . Scale bar : 20μm . ( G ) of D - cyclins re - localization from nucleus to cytoplasm correlation with cycle phases using ImageJ Harmony ( PerkinElmer ) . At least 50 - 200 were analysed in each condition . Bars indicate mean with SD . Statistical was performed using two - sided unpaired Student ' s t - ; ∗ p < 0 . 1 .

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(F,G) VERO AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and 18h later infected with Delta variant SARS-CoV-2. Cells were fixed and stained for D-cyclins 24h later. (F) Example of acquisition using automated microscopic platform. Cells are identified for infection, expression of cyclin D3, and cell cycle (Red/arrow=G1phase; Green/arrowhead=S/G2/M; Red+Green/arrowhead=early S). Scale bar: 20μm. (G) Quantification of D-cyclins re-localization from nucleus to cytoplasm and correlation with cell cycle phases using ImageJ and Harmony (PerkinElmer). At least 50-200 cells were analysed in each condition. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ∗p < 0.1.


( A ) 293T were contransfected with HA - cyclin D3 Strep - tag - SARS - CoV - 2 E or nsp9 , control plasmid ( EV ) . Immunoprecipitation was performed using anti - HA antibody . immunoprecipitates were blotted with anti - Strep , anti - HA , cyclin D3 antibodies .

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(A) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E or nsp9, and control plasmid (EV). Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA, and cyclin D3 antibodies.


( B ) 293T were contransfected with HA - cyclin D3 Strep - tag - SARS - CoV - 2 E , M , both E M or nsp9 . Immunoprecipitation was performed using anti - HA antibody . immunoprecipitates were blotted with anti - Strep , anti - HA antibodies . WCL , whole lysate .

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(B) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E, M, both E and M or nsp9. Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA antibodies. WCL, whole cell lysate.


( C - G ) 293T GFP11 were transfected with Spike , / or with Envelope , , cyclin D3 . 24h post - transfection were seeded at a 1 : 1 with Vero - GFP10 of GFP + ve ( syncytia ) were determined 18h later . S , Spike ; E , Envelope ; M , ; D3 , cyclin D3 . ( C , D ) Representative of GFP + syncytia . Scale bars : 40μm . ( E - G ) of - to - fusion showing of GFP + ve to acquired total . ( E ) n = 4 biological replicates . ( F , G ) n = 4 biological replicates ; technical triplicates shown ; one - way ANOVA with Dunnett ' s multiple comparisons : ns , non - significant ; ∗ ∗ ∗ ∗ p < 0 . 0001 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ p < 0 . 01 . Bars indicate mean with SD .

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(C-G) 293T GFP11 cells were transfected with Spike, and/or with Envelope, Membrane, and cyclin D3. 24h post-transfection cells were seeded at a 1:1 ratio with Vero-GFP10 cells and percentage of GFP+ve area (syncytia) were determined 18h later. S, Spike; E, Envelope; M, Membrane; D3, cyclin D3. (C,D) Representative images of GFP+ syncytia. Scale bars: 40μm. (E-G) Quantification of cell-to-cell fusion showing percentage of the GFP+ve area to the acquired total cell area. (E) n=4 biological replicates. (F,G) n = 4 biological replicates; technical triplicates shown; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01 . Bars indicate mean with SD.


( I ) Representative example of western blot from purified SARS - CoV - 2 Like Particles ( VLPs ) whole lysates ( WCL ) . MCM2 was used as loading control . ( J ) of incorporation ( into VLPs ) or expression ( in WCL ) . n = 4 biological replicates ; one - way ANOVA with Dunnett ' s multiple comparisons : ns , non - significant ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD . N , nucleocapsid ; M , ; E , envelope ; S , spike .

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(I) Representative example of western blot from purified SARS-CoV-2 Virus Like Particles (VLPs) and whole cell lysates (WCL). MCM2 was used as loading control. (J) Quantification of protein incorporation (into VLPs) or expression (in WCL). n = 4 biological replicates; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗p < 0.001. Bars indicate mean with SD. N, nucleocapsid; M, membrane; E, envelope; S, spike.


( B - C ) Western blot ( B ) densitometric ( C ) of OTUD1 in aortas of control Ang II - induced mice . β - actin was used as loading control ( n = 5 biological replicates ) . ( D ) mRNA of OTUD1 in aortas of control Ang II - induced mice were determined by RT - qPCR . values were normalized to Rn18s ( n = 5 biological replicates ) .

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(B-C) Western blot analysis (B) and densitometric quantification (C) of OTUD1 in aortas of control and Ang II-induced mice. β-actin was used as the loading control (n=5 biological replicates). (D) mRNA levels of OTUD1 in aortas of control and Ang II-induced mice were determined by RT-qPCR. The values were normalized to Rn18s (n=5 biological replicates).


( E ) Representative immunofluorescence of CD31 ( green ) OTUD1 ( red ) of aortic sections ( scale bar = 25 μm ) . ( F ) Representative immunofluorescence of α - SMA ( green ) OTUD1 ( red ) of aortic sections ( scale bar = 25 μm ) .

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(E) Representative immunofluorescence staining of CD31 (green) and OTUD1 (red) of aortic sections (scale bar=25 μm). (F) Representative immunofluorescence staining of α-SMA (green) and OTUD1 (red) of aortic sections (scale bar=25 μm).


( G - H ) Time - course of OTUD1 induction in response to Ang II in HUVECs . HUVECs were exposed to 1 μM Ang II for indicated time . Western blot ( G ) densitometric ( H ) of OTUD1 were shown ( n = 3 biological replicates ) . GAPDH was used as loading control .

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(G-H) Time-course of OTUD1 induction in response to Ang II in HUVECs. HUVECs were exposed to 1 μM Ang II for indicated time. Western blot analysis (G) and densitometric quantification (H) of OTUD1 were shown (n = 3 biological replicates). GAPDH was used as the loading control.


( A ) Representative of H & E of aortic sections of mice from four groups ( scale bar = 25 μm ) . E , endothelium ; M , media ; A , adventitia . ( B ) of arterial wall thickness in panel A ; n = 5 biological replicates . ( C ) Representative of Masson ' s Trichrome of aortic sections of mice ( scale bar = 50 μm ) . ( D ) of collagen deposition in panel C ; n = 5 biological replicates .

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(A) Representative images of H&E staining of aortic sections of the mice from four groups (scale bar=25 μm). E, endothelium; M, media; A, adventitia. (B) The quantification of arterial wall thickness in panel A; n=5 biological replicates. (C) Representative images of Masson's Trichrome staining of aortic sections of the mice (scale bar=50 μm). (D) The quantification of collagen deposition in panel C; n=5 biological replicates.


( E - F ) Western blot ( E ) of VE - cadherin , Vimentin , Twist , Snail , OTUD1 densitometric ( F ) of VE - cadherin , Vimentin , Twist Snail in aortic tissues of mice . β - actin was used as loading control ( n = 5 biological replicates ) .

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(E-F) Western blot analysis (E) of VE-cadherin, Vimentin, Twist, Snail, and OTUD1 and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues of the mice. β-actin was used as the loading control (n=5 biological replicates).


( G - H ) mRNA of Cdh5 , Vim , Twist1 , Snai1 ( G ) , Tgfb1 Col1a1 ( H ) in aortic tissues of mice were examined by real - time qPCR . values were normalized to Rn18s ( n = 5 biological replicates ) .

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(G-H) mRNA levels of Cdh5, Vim, Twist1, Snai1 (G), Tgfb1 and Col1a1 (H) in aortic tissues of the mice were examined by real-time qPCR assay. The values were normalized to Rn18s (n=5 biological replicates).


( B ) HUVECs transfected with si - OTUD1 or NC were challenged with 1 μM Ang II for 24 h . Western blot of VE - cadherin , Vimentin , α - SMA , Twist Snail were performed . GAPDH was used as loading control .

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(B) HUVECs transfected with si-OTUD1 or NC were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.


( E ) HUVECs transfected with Flag - OTUD1 or EV were challenged with 1 μM Ang II for 24 h . Western blot of VE - cadherin , Vimentin , α - SMA , Twist Snail were performed . GAPDH was used as loading control .

(E) HUVECs transfected with Flag-OTUD1 or EV were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.


( C ) Co - immunoprecipitation of OTUD1 SMAD2 or SMAD3 in HUVECs transfected with Flag - OTUD1 . Exogenous Flag - OTUD1 was immunoprecipitated by anti - Flag antibody . IgG , immunoglobulin G .

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(C) Co-immunoprecipitation of OTUD1 and SMAD2 or SMAD3 in HUVECs transfected with Flag-OTUD1. Exogenous Flag-OTUD1 was immunoprecipitated by anti-Flag antibody. IgG, immunoglobulin G.


( G ) Immunoprecipitation of SMAD3 in 293T co - transfected with GFP - SMAD3 , HA - Ub Flag - OTUD1 then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody . ( H ) Immunoprecipitation of SMAD3 in 293T co - transfected with GFP - SMAD3 , HA - Ub , Flag - OTUD1 or Flag - OTUD1 ( C320S ) , then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody . ( I ) Immunoprecipitation of SMAD3 in 293T co - transfected with GFP - SMAD3 , Flag - OTUD1 , HA - Ub , HA - Ub - K48 ( K48 only ) , or HA - Ub - K63 ( K63 only ) , respectively , then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody .

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(G) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (H) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and Flag-OTUD1 or Flag-OTUD1(C320S), and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (I) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, Flag-OTUD1, and HA-Ub, HA-Ub-K48 (K48 only), or HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody.


( A - B ) 293T were transfected with various amount of Flag - OTUD1 . Western blot ( A ) densitometric ( B ) of SMAD3 . GAPDH was used as loading control ( n = 3 biological replicates ) .

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(A-B) 293T cells were transfected with various amount of Flag-OTUD1. Western blot analysis (A) and densitometric quantification (B) of SMAD3. GAPDH was used as the loading control (n=3 biological replicates).


( E - F ) Western blot ( E ) densitometric ( F ) of SMAD3 in aortic tissues of mice from four groups . β - actin was used as loading control ( n = 5 biological replicates ) .

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(E-F) Western blot analysis (E) and densitometric quantification (F) of SMAD3 in aortic tissues of the mice from four groups. β-actin was used as the loading control (n=5 biological replicates).


( G ) Co - immunoprecipitation of SMAD3 SMAD4 in 293T co - transfected with GFP - SMAD3 , HA - Ub , HA - Ub - K63 ( K63 only ) , respectively , then challenged with 10 μM MG132 for 6 h . Exogenous SMAD3 was immunoprecipitated by anti - GFP antibody .

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(G) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody.


( H ) Co - immunoprecipitation of SMAD3 SMAD4 in 293T co - transfected with GFP - SMAD3 Flag - OTUD1 then challenged with 10 μM MG132 for 6 h . Exogenous SMAD3 was immunoprecipitated by anti - GFP antibody . IgG , immunoglobulin G .

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(H) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3 and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody. IgG, immunoglobulin G.


( A - B ) Representative ( A ) of H & E of aortic sections ( scale bar = 25 μm ) of arterial wall thickness ( B ) ; n = 5 biological replicates . E , endothelium ; M , media ; A , adventitia . ( C - D ) Representative ( C ) of Masson ' s Trichrome of aortic sections ( scale bar = 25 μm ) of collagen deposition ( D ) ; n = 5 biological replicates .

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(A-B) Representative images (A) of H&E staining of aortic sections (scale bar=25 μm) and quantification of arterial wall thickness (B); n=5 biological replicates. E, endothelium; M, media; A, adventitia. (C-D) Representative images (C) of Masson's Trichrome staining of aortic sections (scale bar=25 μm) and quantification of collagen deposition (D); n=5 biological replicates.


( E - F ) Western blot ( E ) densitometric ( F ) of VE - cadherin , Vimentin , Twist Snail in aortic tissues . β - actin was used as loading control ( n = 5 biological replicates ) .

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(E-F) Western blot analysis (E) and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues. β-actin was used as the loading control (n=5 biological replicates).


Immunostaining for CCN1 ( green ) in adult brain coronal section . Scale bar : 50 μm . insert refers to same image with DAPI to show nuclei ( blue ) . LV , lateral ventricle .

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Immunostaining for CCN1 (green) in the adult brain coronal section. Scale bar: 50 μm. The insert refers to the same image with DAPI staining to show cell nuclei (blue). LV, lateral ventricle.


Adult V - SVZ whole - mounts stained for CCN1 ( ) S100β ( green ) . Scale bar : 10 μm .

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Adult V-SVZ whole-mounts stained for CCN1 (white) and S100β (green). Scale bar: 10 μm.


Adult V - SVZ whole - mounts stained for CCN1 ( ) , GFAP ( green ) VCAM1 ( magenta ) . Arrowhead points to a VCAM1 + B1 surrounded by CCN1 - expressing ependymal . Scale bar : 10 μm .

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Adult V-SVZ whole-mounts stained for CCN1 (white), GFAP (green) and VCAM1 (magenta). Arrowhead points to a VCAM1+ B1 cell surrounded by CCN1-expressing ependymal cells. Scale bar: 10 μm.


Confocal tracing a VCAM1 + GFAP + reveal CCN1 expression is absent in B1 . Yellow arrowheads point to apical side of a B1 in center of a NSC unit . arrowheads point to process of B1 . Scale bar : 10 μm .

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Confocal images tracing a VCAM1+GFAP+ cell reveal CCN1 expression is absent in B1 cells. Yellow arrowheads point to the apical side of a B1 cell in the center of a NSC unit. White arrowheads point to the process of the B1 cell. Scale bar: 10 μm.


V - SVZ whole - mount showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment . Scale bar : 10 μm .

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V-SVZ whole-mount staining showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment. Scale bar: 10 μm.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) GFAP ( red ) . Arrowheads point to B1 in pinwheel center . Scale bars : 500 μm ( left ) , 50 μm ( middle ) , 10 μm ( right ) .

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V-SVZ whole-mounts stained for γ-tubulin (cyan) and GFAP (red). Arrowheads point to B1 cells in the pinwheel center. Scale bars: 500 μm (left), 50 μm (middle), 10 μm (right).


Densities ( / mm2 ) of B1 in adult V - SVZ . n = 4 , * * * p = 0 . 0005 .

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Densities (cells/mm2) of B1 cells in the adult V-SVZ. n=4, ***p=0.0005.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) , GFAP ( red ) VCAM1 ( ) . Arrowheads point to VCAM1 + B1 in NSC units . Scale bars : 50 μm ( left ) , 10 μm ( right ) .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right).


Densities ( / mm2 ) of VCAM1 + B1 in adult V - SVZ . n = 4 , * * p = 0 . 002 . Densities ( units / mm2 ) of NSC units in adult V - SVZ . n = 4 , * * p = 0 . 0022 . Distribution of NSC units with different numbers of B1 . n = 4 , * * * * p < 0 . 0001 ( 1 B1 / unit ) , * * p = 0 . 0025 ( 2 B1 / unit ) , * * p = 0 . 0063 ( 3 B1 / unit ) , * p = 0 . 0385 ( 4 B1 / unit ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ. n=4, **p=0.002. Densities (units/mm2) of NSC units in the adult V-SVZ. n=4, **p=0.0022. Distribution of NSC units with different numbers of B1 cells. n=4, ****p<0.0001 (1 B1 cell/unit), **p=0.0025 (2 B1 cells/unit), **p=0.0063 (3 B1 cells/unit), *p=0.0385 (4 B1 cells/unit).


Densities ( / mm2 ) of B1 in aged V - SVZ . n = 3 , * * p = 0 . 0099 .

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

Densities (cells/mm2) of B1 cells in the aged V-SVZ. n=3, **p=0.0099.


Densities ( / mm2 ) of VCAM1 + B1 in aged V - SVZ . n = 3 , * * p = 0 . 0041 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

Densities (cells/mm2) of VCAM1+ B1 cells in the aged V-SVZ. n=3, **p=0.0041.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) , GFAP ( red ) VCAM1 ( ) in aged mice . Arrowheads point to VCAM1 + B1 in NSC units . Scale bars : 50 μm ( left ) , 10 μm ( right ) .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white) in aged mice. Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right).


Densities ( units / mm2 ) of NSC units in aged V - SVZ . n = 3 , * * p = 0 . 0093 . Average numbers of B1 in a NSC unit . n = 3 , n . s . not significant .

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

Densities (units/mm2) of NSC units in the aged V-SVZ. n=3, **p=0.0093. Average numbers of B1 cells in a NSC unit. n=3, n.s. not significant.


Densities ( / mm2 ) of GFAP + Ki67 + in V - SVZ whole - mounts 2 days after TAM treatment . n = 4 , * p = 0 . 0141 . V - SVZ whole - mounts stained for GFAP ( red ) Ki67 ( cyan ) . Arrowheads point to dividing GFAP + . Scale bar : 10 μm .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O 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-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 2 days after TAM treatment. n=4, *p=0.0141. V-SVZ whole-mounts stained for GFAP (red) and Ki67 (cyan). Arrowheads point to dividing GFAP+ cells. Scale bar: 10 μm.