Figure 7 ( A - B ) CANX co - immunoprecipitates with T6BP in model and professional APCs . Endogenous T6BP was immunoprecipitated from HeLa - CIITA , B ( DG - 75 ) and dendritic - like ( KG - 1 , DC ) cells . The input , the flow through ( FT ) and the immunoprecipitation ( IP ) fractions were analyzed by Western blot using the indicated antibodies . Bottom panels , control IPs using the beads without antibody ( IP CTRL ) are presented for each IP . ( B ) As in ( A ) but using anti - CANX Ab for the IP . For A and B , one representative experiment out of three biological replicates is shown . ( C ) CANX interacts with T6BP using proximity ligation assay ( PLA ) . 48h post - transfection with the indicated siRNAs , HeLa - CIITA cells were fixed , stained with anti - T6BP and anti - CANX antibodies and proximity revealed using PLA ( Duolink ) . Nuclei were stained using DAPI . Top panel , two representative fields are shown . Bottom panel , quantitative analysis using ImageJ displaying the number of PLA per cell . The continuous line indicates the mean ( + / - SEM ) . PLA were quantified in at least 130 cells corresponding to three biological replicates . Scale bars : 10μm . CTRL : control ; Mann - Whitney ' s test ; * * * : p < 0 . 0003 . ( D ) Calnexin interacts with T6BP through its cytosolic tail . HeLa - CIITA cells were transfected with a plasmid encoding the GST - tagged transmembrane ( TM ) and cytosolic ( CytoTail ) domains of CANX ( GST - TM - CytoTail - CANX ) or the vector not encoding TM - CytoTail - CANX as control and immunoprecipitated with anti - GST antibodies . The input , FT , the wash and the IP fractions were analyzed by Western blot and revealed using the indicated antibodies . One representative experiment out of three biological replicates is shown . ( E ) Silencing of T6BP does not influence CANX expression levels while silencing of CANX reduces the level of CD74 expression . HeLa - CIITA cells transfected with the indicated siRNAs and samples analyzed , 48h post transfection , by Western blot with the indicated antibodies . These western blot results are representative of at least three biological replicates .

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

Figure 7(A-B) CANX co-immunoprecipitates with T6BP in model and professional APCs. Endogenous T6BP was immunoprecipitated from HeLa-CIITA, B (DG-75) and dendritic-like (KG-1, DC) cells. The input, the flow through (FT) and the immunoprecipitation (IP) fractions were analyzed by Western blot using the indicated antibodies. Bottom panels, control IPs using the beads without antibody (IP CTRL) are presented for each IP. (B) As in (A) but using anti-CANX Ab for the IP. For A and B, one representative experiment out of three biological replicates is shown.(C) CANX interacts with T6BP using proximity ligation assay (PLA). 48h post-transfection with the indicated siRNAs, HeLa-CIITA cells were fixed, stained with anti-T6BP and anti-CANX antibodies and proximity revealed using PLA (Duolink). Nuclei were stained using DAPI. Top panel, two representative fields are shown. Bottom panel, quantitative analysis using ImageJ displaying the number of PLA per cell. The continuous line indicates the mean (+/-SEM). PLA were quantified in at least 130 cells corresponding to three biological replicates. Scale bars: 10μm. CTRL: control; Mann-Whitney's test; ***: p<0.0003.(D) Calnexin interacts with T6BP through its cytosolic tail. HeLa-CIITA cells were transfected with a plasmid encoding the GST-tagged transmembrane (TM) and cytosolic (CytoTail) domains of CANX (GST-TM-CytoTail-CANX) or the vector not encoding TM-CytoTail-CANX as control and immunoprecipitated with anti-GST antibodies. The input, FT, the wash and the IP fractions were analyzed by Western blot and revealed using the indicated antibodies. One representative experiment out of three biological replicates is shown.(E) Silencing of T6BP does not influence CANX expression levels while silencing of CANX reduces the level of CD74 expression. HeLa-CIITA cells transfected with the indicated siRNAs and samples analyzed, 48h post transfection, by Western blot with the indicated antibodies. These western blot results are representative of at least three biological replicates.


Figure 1A Liver biopsies showing no ( IS0 ) , moderate ( IS3 ) , and severe ( IS6 ) fibrosis stained by PSR . Size bar , 300 μm . B Immunohistochemistry ( brown staining ) for αSMA and SOX9 on consecutive sections from patients with no ( IS0 ) , moderate ( IS3 ) , and severe ( IS6 ) fibrosis . Boxed areas are shown to the right at higher magnification . Bile duct ( bd ) . SOX9 detected in HSCs with elongated nuclei ( hatched line ) next to scar and in hepatocytes with rounded nuclei ( arrows ) . Size bars , 200 µm . C Immunofluorescence for SOX9 ( red ) co - stained with CK19 , CK7 , α1AT , and αSMA ( green ) . Arrowheads show elongated nuclei in αSMA - positive HSCs . D Immunohistochemistry for SOX9 and EpCAM ( brown staining ) on consecutive sections from severe ( IS6 ) fibrosis and normal fetal liver at 18 weeks post - conception ( wpc ) . Dual immunofluorescence confirms SOX9 and EpCAM in the same cell in severe fibrosis . E , F , G Data for SOX9 index for the biliary , HSC / Hep , and total ( biliary + HSC / Hep ) populations . In worsening categories of liver fibrosis ( E ) . In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow - up biopsy ( F ) . The lines shows the best fit by ordinal logistic regression analysis . In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow - up biopsy ( G ) . E , F , G Data for SOX9 index for the biliary , HSC / Hep , and total ( biliary + HSC / Hep ) populations . In worsening categories of liver fibrosis ( E ) . In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow - up biopsy ( F ) . The lines shows the best fit by ordinal logistic regression analysis . In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow - up biopsy ( G ) . E , F , G Data for SOX9 index for the biliary , HSC / Hep , and total ( biliary + HSC / Hep ) populations . In worsening categories of liver fibrosis ( E ) . In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow - up biopsy ( F ) . The lines shows the best fit by ordinal logistic regression analysis . In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow - up biopsy ( G ) . I - L Plots of SOX9 index ( total , HSC / Hep , and biliary ) and NI score marking progressors in green and non - progressors in red . The solid line indicates the mean in each graph . The broken line in NI dot plot ( L ) indicates the threshold ( score = 8 ) below which all NI is categorized as ' mild ' .

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

Figure 1A Liver biopsies showing no (IS0), moderate (IS3), and severe (IS6) fibrosis stained by PSR. Size bar, 300 μm.B Immunohistochemistry (brown staining) for αSMA and SOX9 on consecutive sections from patients with no (IS0), moderate (IS3), and severe (IS6) fibrosis. Boxed areas are shown to the right at higher magnification. Bile duct (bd). SOX9 detected in HSCs with elongated nuclei (hatched line) next to scar and in hepatocytes with rounded nuclei (arrows). Size bars, 200 µm.C Immunofluorescence for SOX9 (red) co-stained with CK19, CK7, α1AT, and αSMA (green). Arrowheads show elongated nuclei in αSMA-positive HSCs.D Immunohistochemistry for SOX9 and EpCAM (brown staining) on consecutive sections from severe (IS6) fibrosis and normal fetal liver at 18 weeks post-conception (wpc). Dual immunofluorescence confirms SOX9 and EpCAM in the same cell in severe fibrosis.E, F, G Data for SOX9 index for the biliary, HSC/Hep, and total (biliary + HSC/Hep) populations. In worsening categories of liver fibrosis (E). In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow-up biopsy (F). The lines shows the best fit by ordinal logistic regression analysis. In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow-up biopsy (G).E, F, G Data for SOX9 index for the biliary, HSC/Hep, and total (biliary + HSC/Hep) populations. In worsening categories of liver fibrosis (E). In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow-up biopsy (F). The lines shows the best fit by ordinal logistic regression analysis. In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow-up biopsy (G).E, F, G Data for SOX9 index for the biliary, HSC/Hep, and total (biliary + HSC/Hep) populations. In worsening categories of liver fibrosis (E). In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow-up biopsy (F). The lines shows the best fit by ordinal logistic regression analysis. In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow-up biopsy (G).I-L Plots of SOX9 index (total, HSC/Hep, and biliary) and NI score marking progressors in green and non-progressors in red. The solid line indicates the mean in each graph. The broken line in NI dot plot (L) indicates the threshold (score = 8) below which all NI is categorized as 'mild'.


Figure 2A Immunohistochemistry for SOX9 ( brown ) in wild - type ( WT ) mice following CCl4 or BDL induced fibrosis . SOX9 positive bile ducts ( bd ) and hepatocytes ( arrowheads ) indicated . B ( Right panel ) Immunofluorescence for SOX9 ( green ) and HNF4α ( red ) in WT fibrotic mice . Arrowheads ( orange / yellow staining ) indicate SOX9 + / HNF4α + hepatocytes and star ( * ; red ) indicates SOX - / HNF4α + hepatocytes . ( Left panel ) Immunofluorescence for SOX9 ( green nucleus ) and CK19 ( red cytoplasm ) in WT fibrotic mice . Arrowheads ( green ) indicate SOX9 + hepatocytes and star ( * ) indicates SOX - hepatocytes identified by DAPI staining in Appendix Figure S1 . C Co - localization by immunohistochemistry ( top panel ) and in situ hybridization ( lower panel ) for SOX9 ( red ) and αSMA ( brown ) in wild - type ( WT ) mice following CCl4 or BDL induced fibrosis . D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control . E Immunohistochemistry for SOX9 ( brown ) in control ( Sox9fl / fl ; RosaCre - ) or Sox9 - null ( Sox9fl / fl ; RosaCre + ) mice following fibrosis . Bile ducts ( bd ) and hepatocytes ( arrowheads ) indicated . SOX9 is absent , even in bile ducts in Sox9 - null liver . Size bar , 25 μm ( A , B and E ) , 10 μm ( C ) .

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Figure 2A Immunohistochemistry for SOX9 (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis. SOX9 positive bile ducts (bd) and hepatocytes (arrowheads) indicated.B (Right panel) Immunofluorescence for SOX9 (green) and HNF4α (red) in WT fibrotic mice. Arrowheads (orange/yellow staining) indicate SOX9+/HNF4α+ hepatocytes and star (*; red) indicates SOX-/ HNF4α+ hepatocytes. (Left panel) Immunofluorescence for SOX9 (green nucleus) and CK19 (red cytoplasm) in WT fibrotic mice. Arrowheads (green) indicate SOX9+ hepatocytes and star (*) indicates SOX- hepatocytes identified by DAPI staining in Appendix Figure S1.C Co-localization by immunohistochemistry (top panel) and in situ hybridization (lower panel) for SOX9 (red) and αSMA (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis.D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control.E Immunohistochemistry for SOX9 (brown) in control (Sox9fl/fl;RosaCre-) or Sox9-null (Sox9fl/fl;RosaCre+) mice following fibrosis. Bile ducts (bd) and hepatocytes (arrowheads) indicated. SOX9 is absent, even in bile ducts in Sox9-null liver. Size bar, 25 μm (A, B and E), 10 μm (C).


Figure 3 A - J Representative images and quantification shown for olive oil treated ( n = 6 ) or chronic CCl4 ( n = 5 Sox9fl / fl ; RosaCreER - ; n = 8 Sox9fl / fl ; RosaCreER + ) induced fibrosis or following sham operation ( n = 5 ) or BDL ( n = 7 Sox9fl / fl ; RosaCreER - ; n = 5 Sox9fl / fl ; RosaCreER + ) ) induced fibrosis . A Picrosirius red ( PSR ) staining ( collagen deposition in red ) counterstained with fast green ( top row ) and immunohistochemistry for α - SMA ( brown staining bottom row ; activated HSC / myofibroblast marker ) in olive oil treated ( left ) or chronic CCl4 induced fibrosis ( right ) in control and Sox9 - null mice . Size bar = 200 μm . B , C Quantification of surface area covered by the PSR staining or α - SMA in control ( Cnt ) and Sox9 - null ( Null ) in ( A ) . D PSR staining ( red ; top row ) and immunohistochemistry for α - SMA ( brown ; middle row ) and CK19 ( brown ; bottom row ) in control and Sox9 - null mice following sham operation ( left ) or BDL induced fibrosis ( right ) . Size bar = 500 μm . E , F Quantification of surface area covered by the PSR staining or α - SMA in ( D ) . G , H Liver function is improved in Sox9 - null mice following CCl4 ( G ) or BDL ( H ) induced fibrosis compared to control mice . Reduction in serum alanine aminotransferase ( ALT ; G , H ) and bilirubin ( H ) down to levels shown in non - fibrotic mice ( olive oil treated groups ; Oil ) . G , H Liver function is improved in Sox9 - null mice following CCl4 ( G ) or BDL ( H ) induced fibrosis compared to control mice . Reduction in serum alanine aminotransferase ( ALT ; G , H ) and bilirubin ( H ) down to levels shown in non - fibrotic mice ( olive oil treated groups ; Oil ) . I Sox9 - loss improved severity of fibrosis compared to control mice indicated by quantification of bridging fibrosis in PSR sections following CCl4 . Olive oil treated mice had no bridging fibrosis , in line with a non - fibrotic liver histology in ( A ) . J Ductal hyperplasia as quantified by the surface area covered by CK19 positive ducts in ( D ) is reduced in Sox9 - null mice compared to control following BDL . All mice were treated with tamoxifen ( Tam ) which did not induce ectopic expression of SOX9 in non - fibrotic livers ( Figure 2A , E and Appendix Figure S3 ) . All experiments are n ≥ 5 as indicated . Two - tailed unpaired t - test was used for statistical analysis . Data in bar charts show means ± s . e . m . P values indicated .

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

Figure 3 A-J Representative images and quantification shown for olive oil treated (n=6) or chronic CCl4 (n=5 Sox9fl/fl;RosaCreER-; n=8 Sox9fl/fl;RosaCreER+) induced fibrosis or following sham operation (n=5) or BDL (n=7 Sox9fl/fl;RosaCreER-; n=5 Sox9fl/fl;RosaCreER+)) induced fibrosis. A Picrosirius red (PSR) staining (collagen deposition in red) counterstained with fast green (top row) and immunohistochemistry for α-SMA (brown staining bottom row; activated HSC / myofibroblast marker) in olive oil treated (left) or chronic CCl4 induced fibrosis (right) in control and Sox9-null mice. Size bar = 200 μm. B, C Quantification of surface area covered by the PSR staining or α-SMA in control (Cnt) and Sox9-null (Null) in (A).D PSR staining (red; top row) and immunohistochemistry for α-SMA (brown; middle row) and CK19 (brown; bottom row) in control and Sox9-null mice following sham operation (left) or BDL induced fibrosis (right). Size bar = 500 μm.E, F Quantification of surface area covered by the PSR staining or α-SMA in (D).G, H Liver function is improved in Sox9-null mice following CCl4 (G) or BDL (H) induced fibrosis compared to control mice. Reduction in serum alanine aminotransferase (ALT; G, H) and bilirubin (H) down to levels shown in non-fibrotic mice (olive oil treated groups; Oil).G, H Liver function is improved in Sox9-null mice following CCl4 (G) or BDL (H) induced fibrosis compared to control mice. Reduction in serum alanine aminotransferase (ALT; G, H) and bilirubin (H) down to levels shown in non-fibrotic mice (olive oil treated groups; Oil).I Sox9-loss improved severity of fibrosis compared to control mice indicated by quantification of bridging fibrosis in PSR sections following CCl4. Olive oil treated mice had no bridging fibrosis, in line with a non-fibrotic liver histology in (A).J Ductal hyperplasia as quantified by the surface area covered by CK19 positive ducts in (D) is reduced in Sox9-null mice compared to control following BDL. All mice were treated with tamoxifen (Tam) which did not induce ectopic expression of SOX9 in non-fibrotic livers (Figure 2A, E and Appendix Figure S3). All experiments are n≥5 as indicated. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated.


Figure 4A - C Representative images shown for olive oil treated ( n = 5 ) or chronic CCl4 ( n = 5 ) induced fibrosis or following sham operation ( n = 6 Sox9fl / fl ; AlbCre - ; n = 5 Sox9fl / fl ; AlbCre + ) or BDL ( n = 5 Sox9fl / fl ; AlbCre - ; n = 8 Sox9fl / fl ; AlbCre + ) induced fibrosis . SOX9 immunohistochemistry ( brown ; A ) , in situ hybridization for Sox9 ( brown ) and α - Sma ( red ) and collagen deposition by PSR staining ( red ; B ) in control ( Sox9fl / fl ; AlbCre - ) or Sox9 - null ( Sox9fl / fl ; AlbCre + ) mice following fibrosis . Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl / fl ; AlbCre + mice ( A ) . A - C Representative images shown for olive oil treated ( n = 5 ) or chronic CCl4 ( n = 5 ) induced fibrosis or following sham operation ( n = 6 Sox9fl / fl ; AlbCre - ; n = 5 Sox9fl / fl ; AlbCre + ) or BDL ( n = 5 Sox9fl / fl ; AlbCre - ; n = 8 Sox9fl / fl ; AlbCre + ) induced fibrosis . SOX9 immunohistochemistry ( brown ; A ) , in situ hybridization for Sox9 ( brown ) and α - Sma ( red ) and collagen deposition by PSR staining ( red ; B ) in control ( Sox9fl / fl ; AlbCre - ) or Sox9 - null ( Sox9fl / fl ; AlbCre + ) mice following fibrosis . Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl / fl ; AlbCre + mice ( A ) . A - C Representative images shown for olive oil treated ( n = 5 ) or chronic CCl4 ( n = 5 ) induced fibrosis or following sham operation ( n = 6 Sox9fl / fl ; AlbCre - ; n = 5 Sox9fl / fl ; AlbCre + ) or BDL ( n = 5 Sox9fl / fl ; AlbCre - ; n = 8 Sox9fl / fl ; AlbCre + ) induced fibrosis . SOX9 immunohistochemistry ( brown ; A ) , in situ hybridization for Sox9 ( brown ) and α - Sma ( red ) and collagen deposition by PSR staining ( red ; B ) in control ( Sox9fl / fl ; AlbCre - ) or Sox9 - null ( Sox9fl / fl ; AlbCre + ) mice following fibrosis . Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl / fl ; AlbCre + mice ( A ) . D , E . Quantification of surface area covered by the PSR staining in ( C ) . D , E . Quantification of surface area covered by the PSR staining in ( C ) . F Expression of SOX9 protein by immunohistochemistry in quiescent ( Q ) and activated ( A ) HSCs extracted from Sox9fl / fl ; AlbCre + mice . G Individual fluorescent channels showing localization and expression of α - SMA ( left panel ) , SOX9 ( middle panel ) and composite image for α - SMA ( green ) and SOX9 ( red ) in right panel in activated Sox9fl / fl ; AlbCre + HSCs ( genotyping shown in Appendix Figure S9 ) . Two - tailed unpaired t - test was used for statistical analysis . Data in bar charts show means ± s . e . m . Size bars = 100 μm ( A ) , 10 μm ( B ) 200 μm ( C ) , 25 μm ( G ) .

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

Figure 4A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A). A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A). A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A).D, E. Quantification of surface area covered by the PSR staining in (C).D, E. Quantification of surface area covered by the PSR staining in (C).F Expression of SOX9 protein by immunohistochemistry in quiescent (Q) and activated (A) HSCs extracted from Sox9fl/fl;AlbCre+ mice.G Individual fluorescent channels showing localization and expression of α-SMA (left panel), SOX9 (middle panel) and composite image for α-SMA (green) and SOX9 (red) in right panel in activated Sox9fl/fl;AlbCre+ HSCs (genotyping shown in Appendix Figure S9). Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. Size bars = 100 μm (A), 10 μm (B) 200 μm (C), 25 μm (G).


Figure 5A , B Flow cytometry of CD45 + CD11B + cells in control ( Sox9fl / fl ; RosaCre - ; A ) and Sox9 - null ( Sox9fl / fl ; RosaCre + ; B ) livers stained for Ly6C and MHCII ( far left ) to identify four populations of cells with differences in their expression of MerTK , CD64 , F4 / 80 and MHCII . C Graphical representation of Ly6C and F4 / 80 staining in macrophages from population 3 and 4 . D , E Graphical representation of cell numbers in the four Ly6C and MHCII cell populations in control and Sox9 - null livers . Data are representative of four independent experiments . F F4 / 80 immunohistochemistry ( brown staining ) in control and Sox9 - null mice livers following 4 - weeks CCl4 induced fibrosis . Size bar = 100μm . Two - tailed unpaired t - test was used for statistical analysis . Data in bar charts show means ± s . e . m . P values indicated . All experiments are n = 4 .

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 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 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

Figure 5A, B Flow cytometry of CD45+CD11B+ cells in control (Sox9fl/fl;RosaCre-; A) and Sox9-null (Sox9fl/fl;RosaCre+; B) livers stained for Ly6C and MHCII (far left) to identify four populations of cells with differences in their expression of MerTK, CD64, F4/80 and MHCII.C Graphical representation of Ly6C and F4/80 staining in macrophages from population 3 and 4.D, E Graphical representation of cell numbers in the four Ly6C and MHCII cell populations in control and Sox9-null livers. Data are representative of four independent experiments.F F4/80 immunohistochemistry (brown staining) in control and Sox9-null mice livers following 4-weeks CCl4 induced fibrosis. Size bar = 100μm. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated. All experiments are n=4.


Figure 6 A , B Immunofluorescence of activated mouse HSCs cultured on plastic ( A ) or hydrogels ( A , B ) . Individual fluorescent channels showing localization and expression of SOX9 ( left ) , YAP ( middle ) and composite image for SOX9 ( red ) and YAP ( green ) in right panel . ( B ) Individual and composite images for αSMA ( green ) and TAZ ( red ) or TEAD ( red ) . Size bar , 25μm . C Quantified decrease in SOX9 protein levels following inhibition of YAP using verteporfin ( VP ) in activated mouse HSCs expressed relative to DMSO control ( n = 3 ) . D Immunohistochemistry for SOX9 ( brown ) following CCl4 or BDL induced fibrosis in mice treated with DMSO ( CCl4 n = 4 , BDL n = 7 ) or verteporfin ( CCl4 n = 3 , BDL n = 5 ) . E , F quantification of SOX9 cell numbers in the HSC / Hep population . Size bar , 50 μm . E , F quantification of SOX9 cell numbers in the HSC / Hep population . Size bar , 50 μm .

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

Figure 6 A, B Immunofluorescence of activated mouse HSCs cultured on plastic (A) or hydrogels (A, B). Individual fluorescent channels showing localization and expression of SOX9 (left), YAP (middle) and composite image for SOX9 (red) and YAP (green) in right panel. (B) Individual and composite images for αSMA (green) and TAZ (red) or TEAD (red). Size bar, 25μm. C Quantified decrease in SOX9 protein levels following inhibition of YAP using verteporfin (VP) in activated mouse HSCs expressed relative to DMSO control (n=3).D Immunohistochemistry for SOX9 (brown) following CCl4 or BDL induced fibrosis in mice treated with DMSO (CCl4 n=4, BDL n=7) or verteporfin (CCl4 n=3, BDL n=5).E, F quantification of SOX9 cell numbers in the HSC/Hep population. Size bar, 50 μm.E, F quantification of SOX9 cell numbers in the HSC/Hep population. Size bar, 50 μm.


Figure 1C E139R , E143R , and the combination ER / ER mutant substitutions in UBE2S increase the amount of automodification , monitored by the presence of * Ub ( * denotes fluorescently labeled protein ) and using SDS - PAGE and fluorescent scanning . D ER / ER substitutions increase Ub chain formation in multiple UBE2S constructs ( WT , UBC only , and UBC - UBD fusion ) , monitored by Coomassie - staining of non - reduced SDS - PAGE gels .

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Figure 1C E139R, E143R, and the combination ER/ER mutant substitutions in UBE2S increase the amount of automodification, monitored by the presence of *Ub (* denotes fluorescently labeled protein) and using SDS-PAGE and fluorescent scanning.D ER/ER substitutions increase Ub chain formation in multiple UBE2S constructs (WT, UBC only, and UBC-UBD fusion), monitored by Coomassie-staining of non-reduced SDS-PAGE gels.


Figure 2E , F , G , H Pulse - chase diUb synthesis assays using different E2s , UBE2S ( E ) , UBE2C ( F ) , UBE2D2 ( G ) , or UBE2R2 ( H ) . DiUb formation was monitored by fluorescent scanning ( top ) , quantitated ( bottom ) , and assessed for statistical significance by either an unpaired Welch ' s t - test ( UBE2C and UBE2R2 ) or one - way ANOVA ( UBE2S and UBE2D2 ) ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates for all E2s ) . Error bars : SEM . Data normalized to the maximal WT diUb formation . # denotes a contaminant band .

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Figure 2E, F, G, H Pulse-chase diUb synthesis assays using different E2s, UBE2S (E), UBE2C (F), UBE2D2 (G), or UBE2R2 (H). DiUb formation was monitored by fluorescent scanning (top), quantitated (bottom), and assessed for statistical significance by either an unpaired Welch's t-test (UBE2C and UBE2R2) or one-way ANOVA (UBE2S and UBE2D2) (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates for all E2s). Error bars: SEM. Data normalized to the maximal WT diUb formation. # denotes a contaminant band.


Figure 3C The E139R substitution decreases the stability of UBE2S ~ Ub compared to WT , revealed by fluorescent scanning of a lysine discharge assay ( top ) and its quantification ( bottom ) . Statistical comparison was performed using one - way ANOVA ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , n = 4 independent , technical replicates ) . Error bars : SEM . Data normalized to the initial timepoint of each respective E2 ~ Ub band . D UBE2R2 R149E has a similar E2 ~ Ub stability as WT , monitored by fluorescent scanning ( top ) of the lysine discharge assay and its quantification through an unpaired Welch ' s t - test ( bottom ) ( n = 3 independent , technical replicates ) . Error bars : SEM . Data normalized to the initial timepoint of each respective E2 ~ Ub band . E Fluorescent scans ( top ) and quantification ( bottom ) of a * LRLRGG peptide transfer assay comparing UBE2S WT and ER / ER reveals no significant difference through an unpaired Welch ' s t - test ( n = 3 independent , technical replicates ) . Error bars : SEM . Data normalized to the maximal * LRLRGG - Ub formation by WT UBE2S . F UBE2R2 R149E is still defective in the transfer of the * LRLRGG peptide to an acceptor Ub compared to WT , probed by fluorescent scanning ( top ) and its quantitation ( bottom ) . Statistical comparison was performed using an unpaired Welch ' s t - test ( * * p ≤ 0 . 01 , n = 5 independent , technical replicates ) . Error bars : SEM . Data normalized to the maximal * LRLRGG - Ub formation by WT UBE2R2 .

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 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 O O O O O O O O O O O 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 3C The E139R substitution decreases the stability of UBE2S~Ub compared to WT, revealed by fluorescent scanning of a lysine discharge assay (top) and its quantification (bottom). Statistical comparison was performed using one-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, n=4 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band.D UBE2R2 R149E has a similar E2~Ub stability as WT, monitored by fluorescent scanning (top) of the lysine discharge assay and its quantification through an unpaired Welch's t-test (bottom) (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band.E Fluorescent scans (top) and quantification (bottom) of a *LRLRGG peptide transfer assay comparing UBE2S WT and ER/ER reveals no significant difference through an unpaired Welch's t-test (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2S.F UBE2R2 R149E is still defective in the transfer of the *LRLRGG peptide to an acceptor Ub compared to WT, probed by fluorescent scanning (top) and its quantitation (bottom). Statistical comparison was performed using an unpaired Welch's t-test (** p ≤ 0.01, n=5 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2R2.


Figure 4Role of the UBE2RHTH in diUb formation is determined from kinetic parameters ( G - H ) for mutants upon titrating the acceptor Ub in pulse - chase reactions following UBE2R2 ~ * Ub Apparent Km and Vmax were determined using the Michaelis - Menten equation ( n ≥ 3 independent , technical replicates ) . Error bars : SEM . Data normalized to the WT apparent Vmax . Role of the UBE2RHTH in diUb formation is determined from kinetic parameters for mutants upon titrating the acceptor Ub in pulse - chase reactions following UBE2R2 ~ * Ub monitored by fluorescent scanning of nonreducing SDS - PAGE

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

Figure 4Role of the UBE2RHTH in diUb formation is determined from kinetic parameters (G-H) for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub Apparent Km and Vmax were determined using the Michaelis-Menten equation (n≥3 independent, technical replicates). Error bars: SEM. Data normalized to the WT apparent Vmax.Role of the UBE2RHTH in diUb formation is determined from kinetic parameters for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub monitored by fluorescent scanning of nonreducing SDS-PAGE gels


Figure 5B Reducing the positive charge of * Ub reduces the autoubiquitination of the E139R - substituted UBE2S variant , monitored by fluorescent scanning of an SDS - PAGE gel . D Plot of 15N - labeled UBE2SUBC WT and ER / ER 1H linewidth changes in the presence of Ub revealing the ER / ER variant has a more dynamic active - site cysteine and HTH , but a more stabilized crossover helix , indicative of the " closed " state . Shown are the best - fit values and standard deviations ( SD ) estimated from the covariance matrix analysis of NMR spectra with n = 2048 measurement points for 16 ppm in the 1H dimension . F C118A substitutions decrease UBE2S autoubiquitination , measured by fluorescent scanning of SDS - PAGE gels , indicating the significance of the " closed " formation of the UBE2S ~ Ub for activity .

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

Figure 5B Reducing the positive charge of *Ub reduces the autoubiquitination of the E139R-substituted UBE2S variant, monitored by fluorescent scanning of an SDS-PAGE gel.D Plot of 15N-labeled UBE2SUBC WT and ER/ER 1H linewidth changes in the presence of Ub revealing the ER/ER variant has a more dynamic active-site cysteine and HTH, but a more stabilized crossover helix, indicative of the "closed" state. Shown are the best-fit values and standard deviations (SD) estimated from the covariance matrix analysis of NMR spectra with n = 2048 measurement points for 16 ppm in the 1H dimension.F C118A substitutions decrease UBE2S autoubiquitination, measured by fluorescent scanning of SDS-PAGE gels, indicating the significance of the "closed" formation of the UBE2S~Ub for activity.


Figure 6C Securin ubiquitination by APC / C and either UBE2C WT or K157E , resolved by SDS - PAGE and fluorescent scanning . Quantification of 4 or more ubiquitins added on the substrate ( right ) are calculated as a fraction of total ubiquitination and analyzed by an unpaired Welch ' s t - test ( * * * * p ≤ 0 . 0001 , n = 5 independent , technical replicates ) . Error bars : SEM . D Similar to C , Ub - securin ubiquitination by APC / C and either UBE2D2 WT or charge - swap variants . Quantification of 4 or more ubiquitins ( right ) added on the substrate are calculated as a fraction of total ubiquitination and analyzed by one - way ANOVA ( * * * * p ≤ 0 . 0001 , n = 4 independent , technical replicates ) . Error bars : SEM . F The recombinant UBE2S ER / ER variant increases Cyclin B degradation in G1 extracts compared to UBE2S wild - type ( WT ) , monitored by immunoblot . G1 extracts were prepared from HeLa S3 cells treated with either control firefly luciferase siRNA ( siFF ) or two UBE2S siRNAs ( siUBE2S ) .

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 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

Figure 6C Securin ubiquitination by APC/C and either UBE2C WT or K157E, resolved by SDS-PAGE and fluorescent scanning. Quantification of 4 or more ubiquitins added on the substrate (right) are calculated as a fraction of total ubiquitination and analyzed by an unpaired Welch's t-test (**** p ≤ 0.0001, n=5 independent, technical replicates). Error bars: SEM. D Similar to C, Ub-securin ubiquitination by APC/C and either UBE2D2 WT or charge-swap variants. Quantification of 4 or more ubiquitins (right) added on the substrate are calculated as a fraction of total ubiquitination and analyzed by one-way ANOVA (**** p ≤ 0.0001, n=4 independent, technical replicates). Error bars: SEM.F The recombinant UBE2S ER/ER variant increases Cyclin B degradation in G1 extracts compared to UBE2S wild-type (WT), monitored by immunoblot. G1 extracts were prepared from HeLa S3 cells treated with either control firefly luciferase siRNA (siFF) or two UBE2S siRNAs (siUBE2S).


Figure 7B Representative fluorescent scan of an SDS - PAGE gel ( left ) and its quantification ( right ) where the charged - swapped variants of the UBE2SHTH enhance substrate ubiquitination of APC / Cs harboring APC2 substitutions . Statistical significance was assessed by two - way ANOVA ( * p ≤ 0 . 05 , * * * p ≤ 0 . 001 , n = 3 independent , technical replicates ) . Error bars : SEM . Data normalized to the negative control substrate band from reactions lacking an E2 . C APC / C - UBE2S - dependent diUb synthesis was monitored by fluorescent scanning ( left ) , quantitated ( right ) , and assessed for statistical significance by two - way ANOVA ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates ) , demonstrating that the UBE2SHTH substitutions rescue the defects in diUb synthesis created by mutating APC2 . Error bars : SEM . Data normalized to WT APC / C - UBE2S diUb reactions . D APC / C Platform - dependent activation of UBE2S - mediated diUb synthesis was monitored by fluorescent scanning ( left ) , quantitated ( right ) , and assessed for statistical significance by two - way ANOVA ( * p ≤ 0 . 05 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates ) . Error bars : SEM . Data normalized to WT APC / C Platform - UBE2S diUb reactions . E Similar to B , the role of the " closed " conformation in UBE2S - dependent Ub chain elongation of the APC / C substrate Ub - CycB * , monitored by the incorporation of C118A - substituted UBE2S variants ( top ) . Quantification of ubiquitinated Ub - CycB * was assessed ( bottom ) and statistical significance by one - way ANOVA between C118A and WT , C118A / E139R , and C118A / E143R ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates ) . Error bars : SEM . Data normalized to WT UBE2S 4 + Ub additions .

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 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 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

Figure 7B Representative fluorescent scan of an SDS-PAGE gel (left) and its quantification (right) where the charged-swapped variants of the UBE2SHTH enhance substrate ubiquitination of APC/Cs harboring APC2 substitutions. Statistical significance was assessed by two-way ANOVA (* p ≤ 0.05, *** p ≤ 0.001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to the negative control substrate band from reactions lacking an E2.C APC/C-UBE2S-dependent diUb synthesis was monitored by fluorescent scanning (left), quantitated (right), and assessed for statistical significance by two-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates), demonstrating that the UBE2SHTH substitutions rescue the defects in diUb synthesis created by mutating APC2. Error bars: SEM. Data normalized to WT APC/C-UBE2S diUb reactions. D APC/C Platform-dependent activation of UBE2S-mediated diUb synthesis was monitored by fluorescent scanning (left), quantitated (right), and assessed for statistical significance by two-way ANOVA (* p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT APC/C Platform-UBE2S diUb reactions.E Similar to B, the role of the "closed" conformation in UBE2S-dependent Ub chain elongation of the APC/C substrate Ub-CycB*, monitored by the incorporation of C118A-substituted UBE2S variants (top). Quantification of ubiquitinated Ub-CycB* was assessed (bottom) and statistical significance by one-way ANOVA between C118A and WT, C118A/E139R, and C118A/E143R (** p ≤ 0.01, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT UBE2S 4+ Ub additions.


Figure 1A Quantitative RT - PCR ( RT - qPCR ) results showing relative expression levels of mouse Pabpn1l in somatic tissues , oocytes , and preimplantation embryos . n = 3 biological replicates . Error bars indicate S . E . M . B Western blot results of PABPN1L , BTG4 , and phosphorylated ERK1 / 2 ( pERK1 / 2 ) in oocytes and embryos . Total protein from 400 oocytes or embryos is loaded in each lane . DNA damage - binding protein 1 ( DDB1 ) is blotted as a loading control . C Western blot results of PABPN1L in MII oocytes of wild type ( WT ) and Pabpn1l − / − females . DDB1 is blotted as a loading control . D Cumulative pup numbers of WT and Pabpn1l - / - female mice . n = 5 for each genotype . Error bars , S . E . M . * * * P < 0 . 001 by two - tailed student ' s t - test . E Quantification of preimplantation embryos derived from WT and Pabpn1l - / - females when WT embryos reached the corresponding stages . The number of analyzed embryos is indicated ( n ) . Error bars , S . E . M . * * * P < 0 . 001 by two - tailed Student ' s t - test . F Representative images of the embryos collected from the oviducts of mated WT and Pabpn1l - / - females at the indicated time points after hCG injection . Scale bar = 100 μm .

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

Figure 1A Quantitative RT-PCR (RT-qPCR) results showing relative expression levels of mouse Pabpn1l in somatic tissues, oocytes, and preimplantation embryos. n = 3 biological replicates. Error bars indicate S.E.M.B Western blot results of PABPN1L, BTG4, and phosphorylated ERK1/2 (pERK1/2) in oocytes and embryos. Total protein from 400 oocytes or embryos is loaded in each lane. DNA damage-binding protein 1 (DDB1) is blotted as a loading control.C Western blot results of PABPN1L in MII oocytes of wild type (WT) and Pabpn1l−/− females. DDB1 is blotted as a loading control.D Cumulative pup numbers of WT and Pabpn1l-/- female mice. n = 5 for each genotype. Error bars, S.E.M. ***P < 0.001 by two-tailed student's t-test.E Quantification of preimplantation embryos derived from WT and Pabpn1l-/- females when WT embryos reached the corresponding stages. The number of analyzed embryos is indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.F Representative images of the embryos collected from the oviducts of mated WT and Pabpn1l-/- females at the indicated time points after hCG injection. Scale bar = 100 μm.


Figure 2A Scatter plot comparing the transcripts of GV oocytes and zygotes from WT and Pabpn1l - / - females . Transcripts that increased or decreased by more than 3 - fold in Pabpn1l - deleted GV oocytes or zygotes are highlighted in red or blue , respectively . D RT - qPCR results for relative expression levels of the indicated maternal transcripts in oocytes and zygotes from WT and Pabpn1l − / − females . n = 3 biological replicates . Error bars , SEM . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 by two - tailed Student ' s t - test . E Poly ( A ) tail assay results showing poly ( A ) - tail length of indicated transcripts in WT and Pabpn1l - deleted oocytes and embryos . The poly ( A ) tails of Actin are used as an internal control . Each sample was prepared from 100 oocytes or embryos . Plots show the averaged relative signal intensity ( y - axis ) and length of the PCR products based on mobility ( x - axis ) .

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Figure 2A Scatter plot comparing the transcripts of GV oocytes and zygotes from WT and Pabpn1l-/- females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted GV oocytes or zygotes are highlighted in red or blue, respectively.D RT-qPCR results for relative expression levels of the indicated maternal transcripts in oocytes and zygotes from WT and Pabpn1l −/− females. n = 3 biological replicates. Error bars, SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test.E Poly(A) tail assay results showing poly(A)-tail length of indicated transcripts in WT and Pabpn1l-deleted oocytes and embryos. The poly(A) tails of Actin are used as an internal control. Each sample was prepared from 100 oocytes or embryos. Plots show the averaged relative signal intensity (y-axis) and length of the PCR products based on mobility (x-axis).


Figure 3A Scatter plot comparing the transcripts of 2 - cell embryos from WT and Pabpn1l - / - females . Transcripts that increased or decreased by more than 3 - fold in Pabpn1l - deleted oocytes or embryos are highlighted in red or blue , respectively . C 5 - Ethynyl uridine ( EU ) fluorescence ( green ) showing RNA transcription in WT and Pabpn1l ♀ − / ♂ + 2 - cell embryos . Some WT embryos were treated with α - amanitin as early as the zygote stage and cultured to the 2 - cell stage . The phosphorylated RNA polymerase II CTD repeat YSPTSPS ( pS2 ) ( red ) is co - stained to label the RNA polymerase II activity . Nuclei are labeled by DAPI ( blue ) . Scale bar = 20 μm . D Quantification of EU and pS2 signals in ( A ) . The numbers of analyzed embryos are indicated ( n ) . Error bars , S . E . M . * * * P < 0 . 001 by two - tailed Student ' s t - test . E Relative intensity of tdTomato signals in ( F ) . The numbers of analyzed embryos are indicated ( n ) . Error bars , S . E . M . * * * P < 0 . 001 by two - tailed Student ' s t - test . F Representative images of MuERV - L : : tdTomato relative to GFP signal in the same embryo when WT embryos reached the corresponding stages . Zygotes were injected with the MuERV - L : : tdTomato reporter plasmid and polyadenylated Gfp mRNA ( as a positive control of microinjection ) , then allowed to develop in vitro . Cultured embryos were imaged at 24 h and 40 h after hCG injection . DIC , differential interference contrast . Scale bar = 100 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 3A Scatter plot comparing the transcripts of 2-cell embryos from WT and Pabpn1l-/- females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted oocytes or embryos are highlighted in red or blue, respectively.C 5-Ethynyl uridine (EU) fluorescence (green) showing RNA transcription in WT and Pabpn1l♀−/♂+ 2-cell embryos. Some WT embryos were treated with α-amanitin as early as the zygote stage and cultured to the 2-cell stage. The phosphorylated RNA polymerase II CTD repeat YSPTSPS (pS2) (red) is co-stained to label the RNA polymerase II activity. Nuclei are labeled by DAPI (blue). Scale bar = 20 μm.D Quantification of EU and pS2 signals in (A). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.E Relative intensity of tdTomato signals in (F). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.F Representative images of MuERV-L::tdTomato relative to GFP signal in the same embryo when WT embryos reached the corresponding stages. Zygotes were injected with the MuERV-L::tdTomato reporter plasmid and polyadenylated Gfp mRNA (as a positive control of microinjection), then allowed to develop in vitro. Cultured embryos were imaged at 24 h and 40 h after hCG injection. DIC, differential interference contrast. Scale bar = 100 μm.


Figure 4A RNA immunoprecipitation ( RIP ) results showing the interaction of BTG4 with indicated transcripts , in the presence or absence of PABPN1L ( full length , RRM - deleted , or R171A mutant ) . HeLa cells were co - transfected with plasmids expressing FLAG - tagged PABPN1L and HA - tagged BTG4 for 48 h before immunoprecipitating with an anti - HA antibody . RNAs recovered from the immunoprecipitants were subjected to RT - qPCR of the indicated transcripts . Fold change values of both input and IP samples were normalized by RIP results of HA - BTG4 and FLAG - PABPN1L co - expression groups . n = 3 biological replicates . Error bars , S . E . M . The P - value represents the two - tailed Student ' s t - test comparing the RIP results of BTG4 with the indicated transcripts in the presence of PABPN1L , * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Co - IP and western blot results showing interactions between PABPN1L and BTG4 . Lysates from HeLa cells expressing HA - BTG4 ( WT or mutants shown in ( B ) ) and FLAG - PABPN1L were immunoprecipitated with an anti - HA antibody . The immunoprecipitated proteins are detected by western blot with the indicated antibodies . D Co - IP and western blot results showing interactions between PABPN1L and BTG4 . Lysates from HeLa cells expressing HA - BTG4 ( WT or mutants shown in ( B ) ) and FLAG - PABPN1L were immunoprecipitated with an anti - HA antibody . The immunoprecipitated proteins are detected by western blot with the indicated antibodies . E Diagrams of mouse BTG2 and BTG4 constructs , and co - IP results showing that PABPN1L binds to BTG4 but not BTG2 . F Diagrams and co - IP results showing BTG4 binding to PABPN1L . Lysates from HeLa cells expressing HA - BTG4 and FLAG - PABPN1L ( full length and C - terminal deleted ) were immunoprecipitated with an anti - FLAG antibody . The immunoprecipitated proteins are detected by western blot with the indicated antibodies . G Diagrams of PABPN1 and PABPN1L constructs , and co - IP results showing that PABPN1L , but not PABPN1 , binds to BTG4 .

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 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 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 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

Figure 4A RNA immunoprecipitation (RIP) results showing the interaction of BTG4 with indicated transcripts, in the presence or absence of PABPN1L (full length, RRM-deleted, or R171A mutant). HeLa cells were co-transfected with plasmids expressing FLAG-tagged PABPN1L and HA-tagged BTG4 for 48 h before immunoprecipitating with an anti-HA antibody. RNAs recovered from the immunoprecipitants were subjected to RT-qPCR of the indicated transcripts. Fold change values of both input and IP samples were normalized by RIP results of HA-BTG4 and FLAG-PABPN1L co-expression groups. n = 3 biological replicates. Error bars, S.E.M. The P-value represents the two-tailed Student's t-test comparing the RIP results of BTG4 with the indicated transcripts in the presence of PABPN1L, *P < 0.05, **P < 0.01, and ***P < 0.001.Co-IP and western blot results showing interactions between PABPN1L and BTG4. Lysates from HeLa cells expressing HA-BTG4 (WT or mutants shown in (B)) and FLAG-PABPN1L were immunoprecipitated with an anti-HA antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.D Co-IP and western blot results showing interactions between PABPN1L and BTG4. Lysates from HeLa cells expressing HA-BTG4 (WT or mutants shown in (B)) and FLAG-PABPN1L were immunoprecipitated with an anti-HA antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.E Diagrams of mouse BTG2 and BTG4 constructs, and co-IP results showing that PABPN1L binds to BTG4 but not BTG2.F Diagrams and co-IP results showing BTG4 binding to PABPN1L. Lysates from HeLa cells expressing HA-BTG4 and FLAG-PABPN1L (full length and C-terminal deleted) were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.G Diagrams of PABPN1 and PABPN1L constructs, and co-IP results showing that PABPN1L, but not PABPN1, binds to BTG4.


Figure 5B RT - qPCR results showing the relative expression level changes ( MII / GV ) of indicated transcripts . Fully grown GV oocytes of Pabpn1l - / - mice were microinjected with mRNAs encoding PABPN1L or BTG4 and were released from meiotic arrest at 12 h after microinjection . Total RNA was extracted from 10 oocytes in each sample . Error bars , S . E . M . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 by two - tailed Student ' s t - test compared to the first column . ns : non - significant . C Western blot results showing the expression of exogenous PABPN1L ( full length , RRM - deleted , or R171A mutants ) and BTG4 in Pabpn1l - / - oocytes at the MII stage . The total protein from 100 oocytes is loaded in each lane . pERK1 / 2 is blotted to indicate the developmental stages . DDB1 is blotted as a loading control . At least three independent experiments were done with consistent results .

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 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 5B RT-qPCR results showing the relative expression level changes (MII/GV) of indicated transcripts. Fully grown GV oocytes of Pabpn1l-/- mice were microinjected with mRNAs encoding PABPN1L or BTG4 and were released from meiotic arrest at 12 h after microinjection. Total RNA was extracted from 10 oocytes in each sample. Error bars, S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test compared to the first column. ns: non-significant.C Western blot results showing the expression of exogenous PABPN1L (full length, RRM-deleted, or R171A mutants) and BTG4 in Pabpn1l-/- oocytes at the MII stage. The total protein from 100 oocytes is loaded in each lane. pERK1/2 is blotted to indicate the developmental stages. DDB1 is blotted as a loading control. At least three independent experiments were done with consistent results.


Figure 6Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly ( A ) variants ( n = 6 ) . The RNA substrate comprised 20 non - poly ( A ) nucleotides followed by a 10 / 20 / 30 / 60 - adenosine poly ( A ) tail . PABPN1L - bound substrates were prepared with 2 / 1 / 0 . 5 / 0 . 2 / 0 . 1 RNA molecules per PABPN1L . The midpoint of the unfolding curve was determined as the melting temperature ( Tm ) . Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly ( A ) variants ( n = 6 ) . The RNA substrate comprised 20 non - poly ( A ) nucleotides followed by a 10 / 20 / 30 / 60 - adenosine poly ( A ) tail . PABPN1L - bound substrates were prepared with 2 / 1 / 0 . 5 / 0 . 2 / 0 . 1 RNA molecules per PABPN1L . The midpoint of the unfolding curve was determined as the melting temperature ( Tm ) . D RNA immunoprecipitation results showing the interaction of PABPN1L with indicated transcripts . Fully grown GV oocytes of WT mice were microinjected with mRNAs encoding PABPN1L ( WT , RRM - deleted , or R171A mutant ) , and were released from meiotic arrest at 12 h after microinjection . Total RNAs were extracted from 350 MI stage oocytes in each sample . RNAs recovered from the immunoprecipitants were subjected to RT - qPCR of the indicated transcripts . Fold change values of both Input and IP samples were normalized by RIP results of the FLAG - PABPN1L microinjection group . Error bars , S . E . M . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 by two - tailed Student ' s t - test comparing with RNA levels pulled - down by PABPN1L WT in the second column .

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 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 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

Figure 6Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly(A) variants (n = 6). The RNA substrate comprised 20 non-poly(A) nucleotides followed by a 10/20/30/60-adenosine poly(A) tail. PABPN1L-bound substrates were prepared with 2/1/0.5/0.2/0.1 RNA molecules per PABPN1L. The midpoint of the unfolding curve was determined as the melting temperature (Tm).Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly(A) variants (n = 6). The RNA substrate comprised 20 non-poly(A) nucleotides followed by a 10/20/30/60-adenosine poly(A) tail. PABPN1L-bound substrates were prepared with 2/1/0.5/0.2/0.1 RNA molecules per PABPN1L. The midpoint of the unfolding curve was determined as the melting temperature (Tm).D RNA immunoprecipitation results showing the interaction of PABPN1L with indicated transcripts. Fully grown GV oocytes of WT mice were microinjected with mRNAs encoding PABPN1L (WT, RRM-deleted, or R171A mutant), and were released from meiotic arrest at 12 h after microinjection. Total RNAs were extracted from 350 MI stage oocytes in each sample. RNAs recovered from the immunoprecipitants were subjected to RT-qPCR of the indicated transcripts. Fold change values of both Input and IP samples were normalized by RIP results of the FLAG-PABPN1L microinjection group. Error bars, S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test comparing with RNA levels pulled-down by PABPN1L WT in the second column.


Figure 7A Western blot results showing the levels of the indicated proteins in WT and Pabpn1l - / - oocytes . Total protein from 100 oocytes is loaded in each lane . DDB1 is blotted as a loading control . B HeLa cells were transfected with plasmids expressing HA - BTG4 and FLAG - PABPN1L for 12 h and then treated by cycloheximide ( CHX , 10 μM ) . Cells were harvested at the indicated time points for western blots . DDB1 is a loading control . C Co - IP and western blot results showing the interaction of βTrCP1 with BTG4 in the presence or absence of PABPN1L . D Co - IP and western blot results showing BTG4 polyubiquitination . HeLa cells transfected with plasmids encoding the indicated proteins were lysed and subjected to IP with an anti - HA affinity gel . Input cell lysates and precipitates are immunoblotted with the indicated antibodies . E Western blot results showing endogenous BTG4 levels in WT and Pabpn1l - / - oocytes at the MII stage . FLAG - PABPN1L was expressed in Pabpn1l - / - oocytes by mRNA microinjected at the GV stage . Total proteins from 100 oocytes are loaded in each lane . ERK1 / 2 is blotted as a loading control .

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

Figure 7A Western blot results showing the levels of the indicated proteins in WT and Pabpn1l-/- oocytes. Total protein from 100 oocytes is loaded in each lane. DDB1 is blotted as a loading control.B HeLa cells were transfected with plasmids expressing HA-BTG4 and FLAG-PABPN1L for 12 h and then treated by cycloheximide (CHX, 10 μM). Cells were harvested at the indicated time points for western blots. DDB1 is a loading control.C Co-IP and western blot results showing the interaction of βTrCP1 with BTG4 in the presence or absence of PABPN1L.D Co-IP and western blot results showing BTG4 polyubiquitination. HeLa cells transfected with plasmids encoding the indicated proteins were lysed and subjected to IP with an anti-HA affinity gel. Input cell lysates and precipitates are immunoblotted with the indicated antibodies.E Western blot results showing endogenous BTG4 levels in WT and Pabpn1l-/- oocytes at the MII stage. FLAG-PABPN1L was expressed in Pabpn1l-/- oocytes by mRNA microinjected at the GV stage. Total proteins from 100 oocytes are loaded in each lane. ERK1/2 is blotted as a loading control.


Figure 1 ( B ) Growth inhibition upon overexpression of HOS3 - NLS driven by the GAL1 promoter is suppressed by overexpression of the KAT Esa1 . 10 - fold serial dilutions of the indicated strains transformed with an empty vector or with the indicated plasmids , were spotted onto SC - Glu and SC - Gal medium ( to activate the GAL1 promoter ) and incubated at 25 ° C for 3 days . Note that HOS3 - NLS ( under the control of the native HOS3 promoter ) does not affect cell growth . ( C ) esa1 - ts and gcn5Δ esa1 - ts mutants have bud emergence defects . ( Top ) Cells of the indicated strains were arrested in G1 by treatment with ɑ - factor for 2 . 5 h at 25 ºC , shifted to 37 ºC for 1 h and released from the G1 arrest at 37 ºC . Cells were fixed at the indicated times and the presence of buds was assessed by microscopy . Mean and SEM are derived from n = number of independent experiments . At least 200 cells were scored for each strain and time point . ( Bottom ) Bright field images of the indicated strains 60 min after the ɑ - factor washout . Arrowheads point to cell buds . Scale bar , 4 µm . ( D ) Inactivation of ESA1 and GCN5 delays DNA replication . Cells of the indicated genotypes were synchronised as in panel C , and DNA content was evaluated by flow cytometry . Numbers indicate time in minutes after the release . This experiment was repeated three times with similar results ; one experiment is shown .

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

Figure 1(B) Growth inhibition upon overexpression of HOS3-NLS driven by the GAL1 promoter is suppressed by overexpression of the KAT Esa1. 10-fold serial dilutions of the indicated strains transformed with an empty vector or with the indicated plasmids, were spotted onto SC-Glu and SC-Gal medium (to activate the GAL1 promoter) and incubated at 25 °C for 3 days. Note that HOS3-NLS (under the control of the native HOS3 promoter) does not affect cell growth.(C) esa1-ts and gcn5Δ esa1-ts mutants have bud emergence defects. (Top) Cells of the indicated strains were arrested in G1 by treatment with ɑ-factor for 2.5 h at 25 ºC, shifted to 37 ºC for 1 h and released from the G1 arrest at 37 ºC. Cells were fixed at the indicated times and the presence of buds was assessed by microscopy. Mean and SEM are derived from n = number of independent experiments. At least 200 cells were scored for each strain and time point. (Bottom) Bright field images of the indicated strains 60 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.(D) Inactivation of ESA1 and GCN5 delays DNA replication. Cells of the indicated genotypes were synchronised as in panel C, and DNA content was evaluated by flow cytometry. Numbers indicate time in minutes after the release. This experiment was repeated three times with similar results; one experiment is shown.


Figure 2 ( A ) Overexpression of Esa1 and Gcn5 KATs leads to increased acetylation levels of the nuclear basket nucleoporin Nup60 . ( Top ) Nup60 - GFP was immunoprecipitated from extracts of the indicated strains , and its acetylation state probed with anti - AcLys antibodies . ( Bottom ) Total extracts probed with anti - HA antibodies to verify KAT overexpression . ( B ) nup60 - KN partially rescues the budding defect of esa1 - ts cells . ( Left ) Budding of cells of the indicated strains were determined as in Figure 1C . At least 200 cells were scored for each strain and time point . Data from 3 independent experiments is represented as mean and SEM ( esa1 - ts , esa1 - ts nup60 - KN ) . ( Right ) Bright field images of the indicated strains 40 min after the ɑ - factor washout . Arrowheads point to cell buds . Scale bar , 4 µm . ( C ) mRNA levels of CLN2 , CDC21 , SVS1 , and RNR1 were determined for cells of the indicated strains after G1 arrest and release at restrictive temperature , with samples collected at indicated times . Data from 3 independent experiments is represented as mean and SEM . ( D ) nup60 - KN mutation partially rescues the delay in synthesis of the G1 / S cyclin Cln2 in esa1 - ts cells . Cells of the indicated strains were processed as in ( B ) and the amount of Cln2 - HA protein at the indicated times was assessed by western blot . % of budded cells is indicated below for each corresponding strain and time point . Note the slow , inefficient budding in esa1 - ts mutants . In WT , the reduction of Cln2 at 60 min and its increase at 90 min reflects the start of a second cycle , which is absent in esa1 - ts cells . ( E ) nup60 - KN partially rescues the Whi5 export defect of esa1 - ts daughter cells . Composite of bright field and Whi5 - mCherry ( left ) and quantification of Whi5 nuclear export ( right ) in mother ( M ) and daughter ( D ) cells of the indicated strains . Whi5 export ( arrows ) is delayed in esa1 - ts mothers and daughters compared to WT ( p < 000 . 1 , Log - rank Mantel - Cox test ) ; esa1 - ts nup60 - KN advances Whi5 export relative to esa1 - ts in daughters ( p = 0 . 0105 ) , but not in mothers ( p > 0 . 05 ) . 3 z - confocal slices spaced 0 . 5 µm were acquired every 3 min ; maximum projections of selected timepoints are shown . Time is indicated in minutes ; t = 0 marks Whi5 nuclear import . Scale bar , 5 µm . n = number of cells , pooled from two independent experiments with similar results .

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

Figure 2(A) Overexpression of Esa1 and Gcn5 KATs leads to increased acetylation levels of the nuclear basket nucleoporin Nup60. (Top) Nup60-GFP was immunoprecipitated from extracts of the indicated strains, and its acetylation state probed with anti-AcLys antibodies. (Bottom) Total extracts probed with anti-HA antibodies to verify KAT overexpression.(B) nup60-KN partially rescues the budding defect of esa1-ts cells. (Left) Budding of cells of the indicated strains were determined as in Figure 1C. At least 200 cells were scored for each strain and time point. Data from 3 independent experiments is represented as mean and SEM (esa1-ts, esa1-ts nup60-KN). (Right) Bright field images of the indicated strains 40 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.(C) mRNA levels of CLN2, CDC21, SVS1, and RNR1 were determined for cells of the indicated strains after G1 arrest and release at restrictive temperature, with samples collected at indicated times. Data from 3 independent experiments is represented as mean and SEM.(D) nup60-KN mutation partially rescues the delay in synthesis of the G1/S cyclin Cln2 in esa1-ts cells. Cells of the indicated strains were processed as in (B) and the amount of Cln2-HA protein at the indicated times was assessed by western blot. % of budded cells is indicated below for each corresponding strain and time point. Note the slow, inefficient budding in esa1-ts mutants. In WT, the reduction of Cln2 at 60 min and its increase at 90 min reflects the start of a second cycle, which is absent in esa1-ts cells.(E) nup60-KN partially rescues the Whi5 export defect of esa1-ts daughter cells. Composite of bright field and Whi5-mCherry (left) and quantification of Whi5 nuclear export (right) in mother (M) and daughter (D) cells of the indicated strains. Whi5 export (arrows) is delayed in esa1-ts mothers and daughters compared to WT (p < 000.1, Log-rank Mantel-Cox test); esa1-ts nup60-KN advances Whi5 export relative to esa1-ts in daughters (p=0.0105), but not in mothers (p > 0.05). 3 z-confocal slices spaced 0.5 µm were acquired every 3 min; maximum projections of selected timepoints are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm. n = number of cells, pooled from two independent experiments with similar results.


Figure 3 ( A ) Composite of bright field and Whi5 - tdTomato ( BF / Whi5 ) and CLN2 - PP7 mRNA labelled with PCP - NLS - GFP ( CLN2 mRNA ) in mother ( M ) and daughter ( D ) cells of the indicated strains . Arrows indicate nuclear foci . Brighter GFP nucleoplasmic areas may correspond to the nucleolus . Insets show enhanced - contrast images to visualise cytoplasmic mRNA particles ( arrowheads ) . Numbers indicate minutes relative to Whi5 nuclear import . Maximum projections of whole - cell Z - stacks are shown for Whi5 and CLN2 mRNA except in the inset and in selected esa1 - ts images , where single Z - slices are shown for clarity . Scale bar : 4 µm ( inset : 1 µm ) . ( B ) Fraction of cells of the indicated strains with nuclear CLN2 - PP7 mRNA foci , aligned relative to Whi5 import . Symbols represent individual values ; lines were generated by smoothing of the nearest 3 neighbouring values ( 0th order polynomial ) . ( C ) Fraction of cells of the indicated strains with nuclear CLN2 - PP7 mRNA foci , aligned relative to Whi5 export . ( D ) Mean fluorescence intensity of nuclear CLN2 mRNA foci at the time of Whi5 export , normalised relative to the nuclear background . Data information boxes include 50 % of data points , lines represent the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; * * , p ≤ 0 . 01 ; n . s . , p > 0 . 05 , ordinary one - way ANOVA with Tukey multiple comparisons test . Adjusted p - values in ( D ) : WT vs . esa1 - ts p < 0 . 0001 , WT vs . esa1 - ts nup60 - KN p < 0 . 0001 , esa1 - ts vs . esa1 - ts nup60 - KN p = 0 . 9845 Foci were scored in individual Z - slices spanning the entire cell volume . n = number of cells , pooled from two independent experiments with similar results . ( E ) Number of cytoplasmic CLN2 - PP7 mRNA foci in early G1 ( 5 minutes after Whi5 import ) and at the G1 / S transition ( 5 min after Whi5 export ) . Data information : boxes include 50 % of data points , lines represent the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; * * , p ≤ 0 . 01 ; n . s . , p > 0 . 05 , ordinary one - way ANOVA with Tukey multiple comparisons test . Adjusted p - values in ( E ) : WT vs . esa1 - ts p < 0 . 0001 , esa1 - ts vs . esa1 - ts nup60 - KN p = 0 . 002 . Foci were scored in individual Z - slices spanning the entire cell volume . n = number of cells , pooled from two independent experiments with similar results . ( F ) The position of nuclear CLN2 - PP7 foci ( marked with arrowheads in the examples ) was scored relatively to the nuclear periphery ( visualised with PCP - NLS - GFP ) in the indicated strains . The fraction of non - perinuclear CLN2 - PP7 foci is significantly increased in esa1 - ts cells compared to the WT and rescued by nup60 - KN mutation ( two - sided Fisher ' s exact test p = 0 . 0209 and p = 0 . 0227 ) . Scale bar , 1 µm .

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

Figure 3(A) Composite of bright field and Whi5-tdTomato (BF/Whi5) and CLN2-PP7 mRNA labelled with PCP-NLS-GFP (CLN2 mRNA) in mother (M) and daughter (D) cells of the indicated strains. Arrows indicate nuclear foci. Brighter GFP nucleoplasmic areas may correspond to the nucleolus. Insets show enhanced-contrast images to visualise cytoplasmic mRNA particles (arrowheads). Numbers indicate minutes relative to Whi5 nuclear import. Maximum projections of whole-cell Z-stacks are shown for Whi5 and CLN2 mRNA except in the inset and in selected esa1-ts images, where single Z-slices are shown for clarity. Scale bar: 4 µm (inset: 1 µm).(B) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 import. Symbols represent individual values; lines were generated by smoothing of the nearest 3 neighbouring values (0th order polynomial). (C) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 export.(D) Mean fluorescence intensity of nuclear CLN2 mRNA foci at the time of Whi5 export, normalised relative to the nuclear background. Data information boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (D): WT vs. esa1-ts p < 0.0001, WT vs. esa1-ts nup60-KN p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.9845 Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.(E) Number of cytoplasmic CLN2-PP7 mRNA foci in early G1 (5 minutes after Whi5 import) and at the G1/S transition (5 min after Whi5 export). Data information: boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (E): WT vs. esa1-ts p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.002. Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.(F) The position of nuclear CLN2-PP7 foci (marked with arrowheads in the examples) was scored relatively to the nuclear periphery (visualised with PCP-NLS-GFP) in the indicated strains. The fraction of non-perinuclear CLN2-PP7 foci is significantly increased in esa1-ts cells compared to the WT and rescued by nup60-KN mutation (two-sided Fisher's exact test p = 0.0209 and p = 0.0227). Scale bar, 1 µm.


Figure 4 ( B ) Overexpression of MEX67 and MTR2 rescues the lethality of Hos3 - NLS overexpression . 10 - fold serial dilutions of the indicated strains transformed with the indicated plasmids spotted onto SC - Glu and SC - Gal medium and incubated at 25 ° C for 3 days . ( C ) A high - copy plasmid containing SAC3 rescues the lethality of Hos3 - NLS overexpression . Strains carrying a high copy plasmid containing the SAC3 ORF ( nucleotides 467 - 3905 ) , or the empty vector , were grown ( D ) Overexpression of Hos3 - NLS promotes nuclear accumulation of mRNA . ( Left ) Cultures of the indicated strains were treated with galactose overnight to induce HOS3 - NLS expression , cells were fixed , and FISH was performed using Cy3 - Oligo ( dT ) . ( Right ) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions . Data information : , arrows point to polyadenylated RNA in the nucleus , which was visualised by DAPI staining . Data from 3 independent biological replicates ( 7h30 ) is represented as mean and s . d . , and data from 2 independent biological replicates ( 0 and 3h30 ) as mean and range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . At least 200 cells were scored for each time - point and condition . Scale bar , 4 µm . ( E ) Inactivation of Esa1 impairs export of poly ( A ) RNA . ( Left ) Cultures of the indicated strains were incubated at 37 ºC . ( Right ) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions Data information : , arrows point to polyadenylated RNA in the nucleus , which was visualised by DAPI staining . Data from 3 independent biological replicates ( 7h30 ) is represented as mean and s . d . , and data from 2 independent biological replicates ( 0 and 3h30 ) as mean and range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . At least 200 cells were scored for each time - point and condition . Scale bar , 4 µm . ( F ) nup60 - KN mutation partially rescues the mRNA export defects of esa1 - ts . Cells of the indicated strains were processed Data information : arrows point to polyadenylated RNA in the nucleus , which was visualised by DAPI staining . Data from 3 independent biological replicates ( 7h30 ) is represented as mean and s . d . , and data from 2 independent biological replicates ( 0 and 3h30 ) as mean and range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . At least 200 cells were scored for each time - point and condition . Scale bar , 4 µm .

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 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 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 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

Figure 4(B) Overexpression of MEX67 and MTR2 rescues the lethality of Hos3-NLS overexpression. 10-fold serial dilutions of the indicated strains transformed with the indicated plasmids spotted onto SC-Glu and SC-Gal medium and incubated at 25 °C for 3 days.(C) A high-copy plasmid containing SAC3 rescues the lethality of Hos3-NLS overexpression. Strains carrying a high copy plasmid containing the SAC3 ORF (nucleotides 467-3905), or the empty vector, were grown(D) Overexpression of Hos3-NLS promotes nuclear accumulation of mRNA. (Left) Cultures of the indicated strains were treated with galactose overnight to induce HOS3-NLS expression, cells were fixed, and FISH was performed using Cy3-Oligo(dT). (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions. Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.(E) Inactivation of Esa1 impairs export of poly(A) RNA. (Left) Cultures of the indicated strains were incubated at 37 ºC. (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.(F) nup60-KN mutation partially rescues the mRNA export defects of esa1-ts. Cells of the indicated strains were processed Data information: arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.