Figure 5 ( A ) Effect of HDAC6 over - expression or CAMDI co - expression on the level of acetylated α - tubulin in the centrosome . HeLa cells co - transfected with Centrin2 - EGFP with / without HDAC6 - HA and FLAG - CAMDI were immunostained with anti - EGFP ( green ) and anti - acetylated α - tubulin antibodies ( red ) . Scale bar , 1 μm . ( B ) Quantification of intensity of acetylated α - tubulin at centrosome . n = 25 , 30 , 30 cells . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc test . Data are presented as mean ± SEM . ( C ) Effects of HDAC6 overexpression or CAMDI co - expression on the centrosomal localization of γ - tubulin . HeLa cells co - transfected with Centrin2 - EGFP with / without HDAC6 - HA and FLAG - CAMDI were immunostained with anti - EGFP ( green ) and anti - γ - tubulin antibodies ( red ) . DNA was stained with Hoechst 33258 . Scale bar , 1 μm . ( D ) Quantification of intensity of γ - tubulin at centrosome . n = 39 , 16 , 17 cells . * , p < 0 . 05 , * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc test . Data are presented as mean ± SEM . ( E ) CAMDI stabilizes centrosomal γ - tubulin through HDAC6 inhibition . Centrosomal fraction was isolated from cell lysates of SH - SY5Y cells expressing each construct , and γ - tubulin level was assessed by immunoblot analysis . n = 3 independent experiments . * * , p < 0 . 01 , One - way ANOVA followed with Bonferroni ' s post hoc test . Data are presented as mean ± SEM . ( F ) Effects of CAMDI knockdown on the centrosomal localization of γ - tubulin in vivo . Coronal sections through the somatosensory cortex of E17 . 5 were analyzed following in utero electroporation of EGFP and CAMDI - sh plasmid at E14 . 5 . The sections were immunostained with anti - EGFP ( green ) and anti - γ - tubulin antibodies ( red ) . Representative images of EGFP at Cortical Plateneuron expressing CAMDI - sh stained for endogenous γ - tubulin and with Hoechst ( blue ) . Z - stack number 6 indicates centrosome of EGFP - positive CAMDI - sh - expressing neuron . Z - stack number 19 indicates centrosome of a nearby non - electroporated neuron . Quantification of γ - tubulin intensity . EGFP ( - ) = 12 cells , EGFP ( + ) ( CAMDI - sh ) = 12 cells . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc test . Data are presented as mean ± SEM . Scale bar , 5 μm . ( G ) Reduced γ - tubulin in the centrosome fraction of CAMDI - KO brain . The centrosomal fraction was isolated from brain lysates of WT and CAMDI - KO mice and γ - tubulin level was assessed by immunoblot analysis . n = 3 independent experiments . * * , p < 0 . 01 , One - way ANOVA followed with Bonferroni ' s post hoc test . Data are presented as mean ± SEM .

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

Figure 5(A) Effect of HDAC6 over-expression or CAMDI co-expression on the level of acetylated α-tubulin in the centrosome. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-acetylated α-tubulin antibodies (red). Scale bar, 1 μm.(B) Quantification of intensity of acetylated α-tubulin at centrosome. n = 25, 30, 30 cells. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.(C) Effects of HDAC6 overexpression or CAMDI co-expression on the centrosomal localization of γ-tubulin. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). DNA was stained with Hoechst 33258. Scale bar, 1 μm.(D) Quantification of intensity of γ-tubulin at centrosome. n = 39, 16, 17 cells. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.(E) CAMDI stabilizes centrosomal γ-tubulin through HDAC6 inhibition. Centrosomal fraction was isolated from cell lysates of SH-SY5Y cells expressing each construct, and γ-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, One-way ANOVA followed with Bonferroni's post hoc test. Data are presented as mean ± SEM.(F) Effects of CAMDI knockdown on the centrosomal localization of γ-tubulin in vivo.Coronal sections through the somatosensory cortex of E17.5 were analyzed following in utero electroporation of EGFP and CAMDI-sh plasmid at E14.5. The sections were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). Representative images of EGFP at Cortical Plateneuron expressing CAMDI-sh stained for endogenous γ-tubulin and with Hoechst (blue). Z-stack number 6 indicates centrosome of EGFP-positive CAMDI-sh-expressing neuron. Z-stack number 19 indicates centrosome of a nearby non-electroporated neuron. Quantification of γ-tubulin intensity. EGFP (-) = 12 cells, EGFP (+) (CAMDI-sh) = 12 cells. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM. Scale bar, 5 μm.(G) Reduced γ-tubulin in the centrosome fraction of CAMDI-KO brain. The centrosomal fraction was isolated from brain lysates of WT and CAMDI-KO mice and γ-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, One-way ANOVA followed with Bonferroni's post hoc test. Data are presented as mean ± SEM.


Figure 6 ( A ) Rescue of a delayed cortical migration in CAMDI - KO mice by Tubastatin A . Immunohistochemical analysis of Cux1 expression in the somatosensory cortex at P2 after intraperitoneal injections of Tubastatin A into pregnant CAMDI - KO mice from E12 . 5 - 17 . 5 . Scale bar , 100 μm . ( B ) Quantification of the number of Cux1 - positive neurons . Note the abnormal distribution of neurons in deep cortical layers of CAMDI - KO mice . n = 3 mice / genotype ( WT ( vehicle ) = 3 , 628 cells , KO ( vehicle ) = 2 , 816 cells , WT ( Tubastatin A ) = 1 , 847 cells , KO ( Tubastatin A ) = 2 , 364 cells ) . * , p < 0 . 05 , * * , p < 0 . 01 , Two - way ANOVA followed by Tukey HSD test . Data are presented as mean ± SEM . ( C ) Enhanced α - tubulin acetylation at P0 by Tubastatin A . Ac - tubulin level was assessed by immunoblot analysis . ( D ) Quantification of the α - tubulin acetylation . n = 3 independent experiments . * , p < 0 . 05 , Two - way ANOVA followed by Scheffe ' s post - hoc test . Data are presented as mean ± SEM .

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

Figure 6(A) Rescue of a delayed cortical migration in CAMDI-KO mice by Tubastatin A. Immunohistochemical analysis of Cux1 expression in the somatosensory cortex at P2 after intraperitoneal injections of Tubastatin A into pregnant CAMDI-KO mice from E12.5-17.5. Scale bar, 100 μm.(B) Quantification of the number of Cux1-positive neurons. Note the abnormal distribution of neurons in deep cortical layers of CAMDI-KO mice. n = 3 mice/genotype (WT (vehicle) = 3,628 cells, KO (vehicle) = 2,816 cells, WT (Tubastatin A) = 1,847 cells, KO (Tubastatin A) = 2,364 cells). *, p<0.05, **, p<0.01, Two-way ANOVA followed by Tukey HSD test. Data are presented as mean ± SEM.(C) Enhanced α-tubulin acetylation at P0 by Tubastatin A. Ac-tubulin level was assessed by immunoblot analysis.(D) Quantification of the α-tubulin acetylation. n=3 independent experiments. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.


Figure 7 ( A ) Representative traces from control ( WT ) and CAMDI - KO ( KO ) mice in the open field test at P21 . Sample traces of locomotor activity were shown . ( B ) Distance traveled for 10 min . n = 13 for WT ( vehicle ) mice , n = 13 for KO ( vehicle ) mice , n = 5 for WT ( Tubastatin A ) mice , n = 16 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc test ( main effect of genotype F ( 1 , 43 ) = 6 . 25 , main effect of drug F ( 1 , 43 ) = 2 . 14 , interaction F ( 1 , 43 ) = 12 . 68 ) . Data are presented as mean ± SEM . ( C ) Repetitive jumping behavior . n = 13 for WT ( vehicle ) mice , n = 27 for KO ( vehicle ) mice , n = 5 for WT ( Tubastatin A ) mice , n = 18 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc test ( main effect of genotype F ( 1 , 59 ) = 5 . 04 , main effect of drug F ( 1 , 59 ) = 10 . 13 , interaction F ( 1 , 59 ) = 4 . 98 ) . Data are presented as mean ± SEM . ( D ) Grooming time in small open field test . n = 13 for WT ( vehicle ) mice , n = 13 for KO ( vehicle ) mice , n = 5 for WT ( Tubastatin A ) mice , n = 16 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc test ( main effect of genotype F ( 1 , 43 ) = 11 . 18 , main effect of drug F ( 1 , 43 ) = 7 . 23 , interaction F ( 1 , 43 ) = 9 . 34 ) . Data are presented as mean ± SEM . ( E ) Distance traveled for 15 min . n = 10 for adult WT ( Vehicle ) mice , n = 5 for KO ( Vehicle ) mice , n = 6 for WT ( Tubastatin A ) mice , n = 11 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , Two - way ANOVA followed by Scheffe ' s post - hoc test ( main effect of genotype F ( 1 , 28 ) = 1 . 73 , main effect of drug F ( 1 , 28 ) = 3 . 96 , interaction F ( 1 , 28 ) = 11 . 80 ) . Data are presented as mean ± SEM . ( F ) Light - dark test . Time in latency to the light chamber ( latency ) . n = 10 for adult WT ( Vehicle ) mice , n = 5 for KO ( Vehicle ) mice , n = 6 for WT ( Tubastatin A ) mice , n = 11 for KO ( Tubastatin A ) mice . * , p < 0 . 05 , Two - way ANOVA followed by Scheffe ' s post - hoc test ( main effect of genotype F ( 1 , 28 ) = 2 . 34 , main effect of drug F ( 1 , 28 ) = 3 . 82 , interaction F ( 1 , 28 ) = 10 . 64 ) . Data are presented as mean ± SEM .

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 O O O O O O O O O O O O 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 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 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 7(A) Representative traces from control (WT) and CAMDI-KO (KO) mice in the open field test at P21. Sample traces of locomotor activity were shown.(B) Distance traveled for 10 min. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 43)=6.25, main effect of drug F(1, 43)=2.14, interaction F(1, 43)=12.68). Data are presented as mean ± SEM.(C) Repetitive jumping behavior. n = 13 for WT (vehicle) mice, n = 27 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 18 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 59)=5.04, main effect of drug F(1, 59)=10.13, interaction F(1, 59)=4.98). Data are presented as mean ± SEM.(D) Grooming time in small open field test. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 43)=11.18, main effect of drug F(1, 43)=7.23, interaction F(1, 43)=9.34). Data are presented as mean ± SEM.(E) Distance traveled for 15 min. n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. **, p<0.01, Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 28)=1.73, main effect of drug F(1, 28)=3.96, interaction F(1, 28)=11.80). Data are presented as mean ± SEM.(F) Light-dark test. Time in latency to the light chamber (latency). n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 28)=2.34, main effect of drug F(1, 28)=3.82, interaction F(1, 28)=10.64). Data are presented as mean ± SEM.


Figure 1 ( B ) Assessment of exogenous DDX60 expression . HEK293T cells transfected with DDX60 wild type ( wt ) , or DDX60 mutants and analyzed by western blot for DDX60 , ß - actin and GAPDH ( loading controls ) , and RFP ( reporter ) . DDX60 and RFP quantification relative to GAPDH from one representative blot are shown below . ( C ) HCV antiviral assays with DDX60 wt or mutant panel . Huh - 7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase ( Fluc , negative , and transfection control ) , IRF1 ( positive antiviral control ) , DDX60 wt , or DDX60 mutants and infected with HCV - Ypet , a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein consisting of C , E1 , E2 , p7 , NS2 , NS3 , 4A , 4B , NS5A , and NS5B is driven by EMCV IRES . Data information : For ( C ) percent of Ypet + cells in RFP + cells is scaled to one replicate of Fluc control . Data shows mean ± SD for at least n = 3 biological replicates ; ns = not significant , * p < 0 . 05 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against Fluc . ( D ) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs . Huh - 7 cells transfected as in ( C ) and infected with either bicistronic HCV - Ypet ( left ) or monocistronic HCV J6 / JFH - 5AB - YPet . Ypet reporter in monocistronic HCV is placed in between NS5A and NS5B . Data information : For ( D ) , percent of Ypet + cells in RFP + cells is scaled to one replicate of Fluc control . Data shows mean ± SD for at least n = 3 biological replicates ; ns = not significant , * p < 0 . 05 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against Fluc .

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

Figure 1(B) Assessment of exogenous DDX60 expression. HEK293T cells transfected with DDX60 wild type (wt), or DDX60 mutants and analyzed by western blot for DDX60, ß-actin and GAPDH (loading controls), and RFP (reporter). DDX60 and RFP quantification relative to GAPDH from one representative blot are shown below.(C) HCV antiviral assays with DDX60 wt or mutant panel. Huh-7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase (Fluc, negative, and transfection control), IRF1 (positive antiviral control), DDX60 wt, or DDX60 mutants and infected with HCV-Ypet, a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein consisting of C, E1, E2, p7, NS2, NS3, 4A, 4B, NS5A, and NS5B is driven by EMCV IRES. Data information: For (C) percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.(D) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs. Huh-7 cells transfected as in (C) and infected with either bicistronic HCV-Ypet (left) or monocistronic HCV J6/JFH-5AB-YPet. Ypet reporter in monocistronic HCV is placed in between NS5A and NS5B. Data information: For (D), percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.


Figure 2 ( A ) Plasmid - based dual luciferase bicistronic reporter assays . HEK293T cells co - transfected with dual luciferase bicistronic reporter plasmid ( Renilla luciferase ( Rluc ) translationally driven by a 5 ' cap , and Firefly luciferase ( Fluc ) translationally driven by different IRESs as indicated ) and GFP plasmid ( negative control ) or increasing amounts of IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A mutant . Total amount of DNA transfected was kept constant by supplementing transfection mixes with GFP plasmid . Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells ( left ) and ratio of IRES Fluc units over 5 ' cap Rluc units ( middle ) . Diagram to the right of ratios plot depicts expected ratios of IRES - driven Fluc units to 5 ' cap - driven Rluc units given either : equal translation of 5 ' cap - driven Rluc and IRES - driven Fluc ( top ) , greater translation of IRES - driven Fluc ( middle ) , or greater translation of 5 ' cap - driven Rluc ( bottom ) . Data information : Data shows mean ± SD for at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against GFP . ( B ) RNA - based monocistronic luciferase reporter assays . HEK293T cells transfected with GFP ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A and subsequently transfected with in vitro transcribed 5 ' cap or different IRES driven Fluc mRNA constructs as indicated . Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells . Data information : Data shows mean ± SD for at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against GFP .

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

Figure 2(A) Plasmid-based dual luciferase bicistronic reporter assays. HEK293T cells co-transfected with dual luciferase bicistronic reporter plasmid (Renilla luciferase (Rluc) translationally driven by a 5' cap, and Firefly luciferase (Fluc) translationally driven by different IRESs as indicated) and GFP plasmid (negative control) or increasing amounts of IRF1 (positive control), DDX60 wt, or DDX60 E890A mutant. Total amount of DNA transfected was kept constant by supplementing transfection mixes with GFP plasmid. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells (left) and ratio of IRES Fluc units over 5' cap Rluc units (middle). Diagram to the right of ratios plot depicts expected ratios of IRES-driven Fluc units to 5' cap-driven Rluc units given either: equal translation of 5' cap-driven Rluc and IRES-driven Fluc (top), greater translation of IRES-driven Fluc (middle), or greater translation of 5' cap-driven Rluc (bottom). Data information: Data shows mean ± SD for at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against GFP.(B) RNA-based monocistronic luciferase reporter assays. HEK293T cells transfected with GFP (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and subsequently transfected with in vitro transcribed 5' cap or different IRES driven Fluc mRNA constructs as indicated. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells. Data information: Data shows mean ± SD for at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against GFP.


Figure 3 ( A - E ) ( A , B , E ) Multi - cycle infection assays with type I and type II IRES containing viruses . HeLa cells stably expressing Firefly luciferase ( Fluc ) ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A and infected with ( A ) encephalomyocarditis virus , ( B ) poliovirus , or ( E ) a chimeric poliovirus with the poliovirus IRES replaced with the IRES of EMCV ( EMCV - IRES - PV ) at MOI 0 . 001 . Supernatants were collected 24 hours post infection ( hpi ) ( EMCV ) or 48 hpi ( poliovirus and EMCV - IRES - PV ) and titers determined via plaque assay on HeLa cells . ( C , D ) Single - cycle infection assays with type I and type II IRES containing viruses . BHK - J cells stably expressing empty vector ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A and infected with ( C ) foot and mouth disease virus , or ( D ) bovine enterovirus - 1 at MOI 1 . Supernatants were collected 5 hours post infection ( hpi ) and titers determined via plaque assay on BHK - 21 clone 13 cells . Data information : Data shows mean ± SEM percent infectious titers relative to Fluc from at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA and Dunnett ' s multiple comparison test against DDX60 E890A .

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

Figure 3(A-E) (A, B, E) Multi-cycle infection assays with type I and type II IRES containing viruses. HeLa cells stably expressing Firefly luciferase (Fluc) (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (A) encephalomyocarditis virus, (B) poliovirus, or (E) a chimeric poliovirus with the poliovirus IRES replaced with the IRES of EMCV (EMCV-IRES-PV) at MOI 0.001. Supernatants were collected 24 hours post infection (hpi) (EMCV) or 48 hpi (poliovirus and EMCV-IRES-PV) and titers determined via plaque assay on HeLa cells. (C, D) Single-cycle infection assays with type I and type II IRES containing viruses. BHK-J cells stably expressing empty vector (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (C) foot and mouth disease virus, or (D) bovine enterovirus-1 at MOI 1. Supernatants were collected 5 hours post infection (hpi) and titers determined via plaque assay on BHK-21 clone 13 cells. Data information: Data shows mean ± SEM percent infectious titers relative to Fluc from at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against DDX60 E890A.


Figure 4B ) HEK293T cells transfected with DDX60 wt or DDX60 E890A ( negative control ) and transfected with in vitro transcribed 5 ' cap - or IRES - driven Fluc mRNA constructs as indicated and lysed 16 hours later . ( B ) IRES - or cap - driven translation from the same samples assayed in parallel using luciferase assay . Data information : B ) Mean ± SD from at least n = 3 biological replicates ; * * * * FDR < 0 . 01 % ( p < 0 . 0001 ) using unpaired t - test with Welch correction and Benjamini and Yekutieli correction for multiple testing comparing DDX60 wt versus DDX60 E890A transfected cells . ns , non - significant . ( C , HEK293T cells stably expressing a type I IFN sensitive response element ( ISRE ) driven Fluc gene were transfected with increasing amounts of RIG - I , RIG - I in combination with DDX60 wt , or RIG - I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected . Transfected cells were then treated with either PBS ( negative control ) or transfected with LMW poly ( I : C ) . In parallel , untransfected cells were treated with either PBS ( mock ) , transfected with LMW poly ( I : C ) , or treated with IFN - ß ( positive control ) . Cells were subsequently used for a luciferase assay to assess ISRE activity ( C ) Data information : ( C ) Mean ± SD from at least n = 3 biological replicates ; * p < 0 . 05 using unpaired t - test with Welch ' s correction , ns , not significant using repeated measures one - way ANOVA with Geisser - Greenhouse correction comparing RIG - I transfected vs RIG - I + DDX60 wt or RIG - I + DDX60 E890A transfected cells . D ) HEK293T cells stably expressing a type I IFN sensitive response element ( ISRE ) driven Fluc gene were transfected with increasing amounts of RIG - I , RIG - I in combination with DDX60 wt , or RIG - I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected . western blot for analysis of DDX60 , RIG - I , ß - actin ( loading control ) , or GAPDH ( loading control ) protein products ( D ) . Quantification of DDX60 and RIG - I band intensities relative to ß - actin are shown below each respective lane in ( D ) . Data information : ( D ) representative western blot from ( C ) .

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 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 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

Figure 4B) HEK293T cells transfected with DDX60 wt or DDX60 E890A (negative control) and transfected with in vitro transcribed 5' cap- or IRES-driven Fluc mRNA constructs as indicated and lysed 16 hours later. (B) IRES- or cap-driven translation from the same samples assayed in parallel using luciferase assay. Data information: B) Mean ± SD from at least n=3 biological replicates; ****FDR < 0.01% (p<0.0001) using unpaired t-test with Welch correction and Benjamini and Yekutieli correction for multiple testing comparing DDX60 wt versus DDX60 E890A transfected cells. ns, non-significant.(C, HEK293T cells stably expressing a type I IFN sensitive response element (ISRE) driven Fluc gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60 wt, or RIG-I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected. Transfected cells were then treated with either PBS (negative control) or transfected with LMW poly(I:C). In parallel, untransfected cells were treated with either PBS (mock), transfected with LMW poly(I:C), or treated with IFN-ß (positive control). Cells were subsequently used for a luciferase assay to assess ISRE activity (C) Data information: (C) Mean ± SD from at least n=3 biological replicates; *p < 0.05 using unpaired t-test with Welch's correction, ns, not significant using repeated measures one-way ANOVA with Geisser-Greenhouse correction comparing RIG-I transfected vs RIG-I + DDX60 wt or RIG-I + DDX60 E890A transfected cells.D) HEK293T cells stably expressing a type I IFN sensitive response element (ISRE) driven Fluc gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60 wt, or RIG-I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected. western blot for analysis of DDX60, RIG-I, ß-actin (loading control), or GAPDH (loading control) protein products (D). Quantification of DDX60 and RIG-I band intensities relative to ß-actin are shown below each respective lane in (D). Data information: (D) representative western blot from (C).


Figure 5 ( B ) Effect of DDX60 on 5 ' cap ( left ) or EMCV IRES ( right ) driven Fluc mRNA polysomes . Data information : Mean percent ± SEM of Fluc mRNA of each fraction relative to Fluc mRNA in all fractions , from n = 3 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

Figure 5(B) Effect of DDX60 on 5' cap (left) or EMCV IRES (right) driven Fluc mRNA polysomes. Data information: Mean percent ± SEM of Fluc mRNA of each fraction relative to Fluc mRNA in all fractions, from n=3 biological replicates.


Figure 6 ( B ) Cells depicted in ( A ) were subjected to polysome profiling by lysing in the presence of cycloheximide and protease and phosphatase inhibitors , applying cell lysates to 15 % - 50 % sucrose gradients , and subjecting to ultracentrifugation . Centrifuged gradients were run through a fractionator and total RNA in each fraction was measured by UV absorbance ( 254 nm ) . ( C ) Representative results of RNA electrophoresis analysis of individual fractions . Shown are results from one replicate of DDX60 - transfected , EMCV - IRES - PV - infected cells . RNA was analyzed by Bioanalyzer to visualize 28S , and 18S ribosome subunit distribution . RIN , RNA integrity number . ( D ) Effect of DDX60 expression on parental poliovirus ( type I IRES , purple circles ) versus chimeric poliovirus ( type II IRES , red squares ) VP4 mRNA polysomes ( left ) and cellular GAPDH mRNA polysomes in the same samples . Data information : Mean percent ± SEM of poliovirus VP4 or GAPDH mRNA in each fraction relative to VP4 or GAPDH mRNA in all fractions from n = 3 replicate plates per condition .

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 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

Figure 6(B) Cells depicted in (A) were subjected to polysome profiling by lysing in the presence of cycloheximide and protease and phosphatase inhibitors, applying cell lysates to 15%-50% sucrose gradients, and subjecting to ultracentrifugation. Centrifuged gradients were run through a fractionator and total RNA in each fraction was measured by UV absorbance (254 nm).(C) Representative results of RNA electrophoresis analysis of individual fractions. Shown are results from one replicate of DDX60-transfected, EMCV-IRES-PV-infected cells. RNA was analyzed by Bioanalyzer to visualize 28S, and 18S ribosome subunit distribution. RIN, RNA integrity number.(D) Effect of DDX60 expression on parental poliovirus (type I IRES, purple circles) versus chimeric poliovirus (type II IRES, red squares) VP4 mRNA polysomes (left) and cellular GAPDH mRNA polysomes in the same samples. Data information: Mean percent ± SEM of poliovirus VP4 or GAPDH mRNA in each fraction relative to VP4 or GAPDH mRNA in all fractions from n=3 replicate plates per condition.


Figure 1A . Four full - thickness excisional wounds were produced on each of CD18 - / - or WT mice by 6 mm biopsy punches . Each wound received intradermal injection of 2 . 5x105 AT - MSCs or PBS control . Each wound region was digitally photographed at the indicated time points , and wound areas were analyzed using Adobe Photoshop . Depicted are representative macroscopic pictures of PBS injected or AT - MSCs injected WT or CD18 - / - wounds at days 0 , 3 , 5 , 7 and 9 post - wounding . B . Quantitative analysis of 20 wound areas per group , expressed as percentage of the initial wound size at day 0 . The line in each group represents the mean value of 20 wounds from 5 mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t - test with Welch ' s correction .

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

Figure 1A. Four full-thickness excisional wounds were produced on each of CD18-/- or WT mice by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or PBS control. Each wound region was digitally photographed at the indicated time points, and wound areas were analyzed using Adobe Photoshop. Depicted are representative macroscopic pictures of PBS injected or AT-MSCs injected WT or CD18-/- wounds at days 0, 3, 5, 7 and 9 post-wounding. B. Quantitative analysis of 20 wound areas per group, expressed as percentage of the initial wound size at day 0. The line in each group represents the mean value of 20 wounds from 5 mice. *p<0.05, **p<0.01, ***p<0.001 by Student's t-test with Welch's correction.


Figure 2A . AT - MSCs or PBS were intradermally injected to WT or CD18 - / - wounds and the wound tissues were harvested at days 2 , 5 , 7 post - wounding . Depicted are representative photographs of wound cryosections at days 2 and 5 post - wounding stained for human - specific β2M ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bars : 100 µm . B . Genomic DNA was isolated from the wound tissues , and the amount of human - specific Alu DNA was quantified by real time PCR , and then converted to the number of AT - MSCs based on a standard curve . Data are expressed as mean ± SEM , n = 3 at each time point , * p < 0 . 05 by two - tailed unpaired t - test . C . Total TGF - β1 concentrations in wound lysates were measured by TGF - β1 specific ELISA . Results are expressed as mean ± SEM , n = 3 at each time pointD . The expression of human TGF - β1 in cultured AT - MSCs and wound tissues of WT or CD18 - / - wounds received AT - MSCs injection was quantified by qPCR with primers specifically amplify human TGF - β1 , and normalized on the numbers of MSCs in each condition Data are expressed as mean ± SEM , n = 8 wounds ( 2 wounds x 4 mice ) per time

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

Figure 2A. AT-MSCs or PBS were intradermally injected to WT or CD18-/- wounds and the wound tissues were harvested at days 2, 5, 7 post-wounding. Depicted are representative photographs of wound cryosections at days 2 and 5 post-wounding stained for human-specific β2M (green). Nuclei were stained with DAPI (blue). Scale bars: 100 µm.B. Genomic DNA was isolated from the wound tissues, and the amount of human-specific Alu DNA was quantified by real time PCR, and then converted to the number of AT-MSCs based on a standard curve. Data are expressed as mean ± SEM, n = 3 at each time point, *p<0.05 by two-tailed unpaired t-test.C. Total TGF-β1 concentrations in wound lysates were measured by TGF-β1 specific ELISA. Results are expressed as mean ± SEM, n = 3 at each time pointD. The expression of human TGF-β1 in cultured AT-MSCs and wound tissues of WT or CD18-/- wounds received AT-MSCs injection was quantified by qPCR with primers specifically amplify human TGF-β1, and normalized on the numbers of MSCs in each condition Data are expressed as mean ± SEM, n = 8 wounds (2 wounds x 4 mice) per time point


Figure 3A . Cryosections of PBS or AT - MSCs injected CD18 - / - wounds at days 5 and 7 post - wounding were immunostained for α - SMA ( red ) . Nuclei were stained with DAPI ( blue ) . The dashed lines separate epidermis and wound bed . e , epidermis ; wb , wound bed . Scale bars : 100 µm . B . Quantification of α - SMA immunofluorescence shown in ( A ) C . Cryosections of AT - MSCs injected CD18 - / - wounds at day 5 post - wounding were co - immunostained for human β2M ( green ) and TGF - β1 ( red , upper panel ) or α - SMA ( red , lower panel ) .

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

Figure 3A. Cryosections of PBS or AT-MSCs injected CD18-/- wounds at days 5 and 7 post-wounding were immunostained for α-SMA (red). Nuclei were stained with DAPI (blue). The dashed lines separate epidermis and wound bed. e, epidermis; wb, wound bed. Scale bars: 100 µm. B. Quantification of α-SMA immunofluorescence shown in (A) C. Cryosections of AT-MSCs injected CD18-/- wounds at day 5 post-wounding were co-immunostained for human β2M (green) and TGF-β1 (red, upper panel) or α-SMA (red, lower panel).


Figure 4A - F . Representative photographs of immunofluorescent staining for α - SMA ( red ) and human β2M ( green ) on cultured human AT - MSCs ( A ) , murine primary dermal fibroblasts ( mFB ) ( B ) , mFB treated with 2 ng / ml recombinant human TGF - β1 ( C ) , and co - cultures of mFB with AT - MSCs ( D ) or TGF - β1 siRNA transfected AT - MSCs ( E ) or control siRNA transfected AT - MSCs ( F ) after 48 h culture . Nuclei were counterstained with DAPI ( blue ) . G . Quantification of α - SMA immunofluorescence shown in ( A - F ) H . Four full - thickness excisional wounds were produced on each of CD18 - / - mouse by 6 mm biopsy punches . Each wound received intradermal injection of 2 . 5x105 AT - MSCs or human dermal fibroblasts ( HDF ) or TGF - β1 siRNA transfected AT - MSCs or control siRNA transfected AT - MSCs or PBS . Each wound region was digitally photographed at days 0 , 3 , 5 , 7 and 10 post - wounding , and wound areas were analyzed using Adobe Photoshop . The depicted result is the quantitative analysis of all wound areas per group , expressed as percentage of the initial wound size at day 0 .

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 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

Figure 4A-F. Representative photographs of immunofluorescent staining for α-SMA (red) and human β2M (green) on cultured human AT-MSCs (A), murine primary dermal fibroblasts (mFB) (B), mFB treated with 2 ng/ml recombinant human TGF-β1 (C), and co-cultures of mFB with AT-MSCs (D) or TGF-β1 siRNA transfected AT-MSCs (E) or control siRNA transfected AT-MSCs (F) after 48 h culture. Nuclei were counterstained with DAPI (blue). G. Quantification of α-SMA immunofluorescence shown in (A-F)H. Four full-thickness excisional wounds were produced on each of CD18-/- mouse by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or human dermal fibroblasts (HDF) or TGF-β1 siRNA transfected AT-MSCs or control siRNA transfected AT-MSCs or PBS. Each wound region was digitally photographed at days 0, 3, 5, 7 and 10 post-wounding, and wound areas were analyzed using Adobe Photoshop. The depicted result is the quantitative analysis of all wound areas per group, expressed as percentage of the initial wound size at day 0.


Figure 5A - C . 2 . 5x105 of TGF - β1 siRNA or control siRNA transfected AT - MSCs were intradermally injected around each of CD18 - / - murine wound . PBS mock injection served as negative control . Wound tissue were harvested at day 2 , 5 , and 7 post - wounding for quantification of human TGF - β1 mRNA ( A ) at day 2 by qPCR , total TGF - β1 ( B ) and active TGF - β1 ( C ) protein at day 5 by ELISA . Expression of α - SMA ( D , E ) at day 5 and 7 by immunostaining on tissue sections . The dashed lines indicate the border of the wound bed and epidermis or eschar . e , epidermis ; es , eschar ; wb , wound bed . Quantification data are expressed as mean ± SEM , n = 3 wounds per groupExpression of CD31 ( F , G ) at day 5 and 7 by immunostaining on tissue sections . The dashed lines indicate the border of the wound bed and epidermis or eschar . e , epidermis ; es , eschar ; wb , wound bed . Quantification data are expressed as mean ± SEM , n = 3 wounds per

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 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 5A-C. 2.5x105 of TGF-β1 siRNA or control siRNA transfected AT-MSCs were intradermally injected around each of CD18-/- murine wound. PBS mock injection served as negative control. Wound tissue were harvested at day 2, 5, and 7 post-wounding for quantification of human TGF-β1 mRNA (A) at day 2 by qPCR, total TGF-β1 (B) and active TGF-β1 (C) protein at day 5 by ELISA.Expression of α-SMA (D, E) at day 5 and 7 by immunostaining on tissue sections. The dashed lines indicate the border of the wound bed and epidermis or eschar. e, epidermis; es, eschar; wb, wound bed. Quantification data are expressed as mean ± SEM, n = 3 wounds per groupExpression of CD31 (F, G) at day 5 and 7 by immunostaining on tissue sections. The dashed lines indicate the border of the wound bed and epidermis or eschar. e, epidermis; es, eschar; wb, wound bed. Quantification data are expressed as mean ± SEM, n = 3 wounds per group


Figure 6A . AT - MSCs were cultured in the labelling media containing [ 35S ] - Met / Cys and increasing concentrations of r . h . TGF - β1 at 0 , 0 . 1 , 0 . 3 , 1 , 3 and 10 ng / ml for overnight . Supernatants were harvested and incubated with TGF - β1 capture antibody . After washing the captured TGF - β1 was transferred to glass filter membrane , and the radioactivity was counted with a scintillation counter ( black bars ) . The supernatants were incubated with new TGF - β1 capture antibody , and the radioactivity of captured TGF - β1 was counted again ( white bars ) . The data is given as mean ± SD of 5 repeated measurements . CPM , count per minute . AT - MSCs ( B ) were cultured in media containing increasing concentrations of r . h . TGF - β1 at 0 , 0 . 1 , 0 . 3 , 1 , 3 and 10 ng / ml for 24 h , before protein lysates or total RNAs were harvested . Western blot analysis of Smad7 was performed with protein lysates and semi - quantitatively analyzed with densitometry . β - actin served as loading control . Data is given as mean ± SEM of three independent blots . HDFs ( C ) were cultured in media containing increasing concentrations of r . h . TGF - β1 at 0 , 0 . 1 , 0 . 3 , 1 , 3 and 10 ng / ml for 24 h , before protein lysates or total RNAs were harvested . Western blot analysis of Smad7 was performed with protein lysates and semi - quantitatively analyzed with densitometry . β - actin served as loading control . Data is given as mean ± SEM of three independent blots . * D . From total RNA , the mature miR - 21 and endogenous control RNU6B were converted to cDNA , and quantified by qPCR based TaqMan microRNA assays . The expression of miR - 21 was normalized on RNU6B . Data is given as mean ± SEM of three independent experiments .

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

Figure 6A. AT-MSCs were cultured in the labelling media containing [35S]-Met/Cys and increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for overnight. Supernatants were harvested and incubated with TGF-β1 capture antibody. After washing the captured TGF-β1 was transferred to glass filter membrane, and the radioactivity was counted with a scintillation counter (black bars). The supernatants were incubated with new TGF-β1 capture antibody, and the radioactivity of captured TGF-β1 was counted again (white bars). The data is given as mean ± SD of 5 repeated measurements. CPM, count per minute.AT-MSCs (B) were cultured in media containing increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for 24 h, before protein lysates or total RNAs were harvested. Western blot analysis of Smad7 was performed with protein lysates and semi-quantitatively analyzed with densitometry. β-actin served as loading control. Data is given as mean ± SEM of three independent blots.HDFs (C) were cultured in media containing increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for 24 h, before protein lysates or total RNAs were harvested. Western blot analysis of Smad7 was performed with protein lysates and semi-quantitatively analyzed with densitometry. β-actin served as loading control. Data is given as mean ± SEM of three independent blots. *D. From total RNA, the mature miR-21 and endogenous control RNU6B were converted to cDNA, and quantified by qPCR based TaqMan microRNA assays. The expression of miR-21 was normalized on RNU6B. Data is given as mean ± SEM of three independent experiments.


Figure 7A . AT - MSCs were pretreated with a TGFβRI inhibitor SB431542 at 10 µM , or control DMSO for 1 h , and subsequently exposed to r . h . TGF - β1 at indicated concentrations . The cells were cultured for another 24 h and harvested for miR - 21 assay . The expression of miR - 21 was normalized on RNU6B . Data is given as mean ± SEM of three independent experiments . B - D . AT - MSCs were transfected with 30 nM Smad7 - siRNAs or control siRNA . Untransfected AT - MSCs served as control . RNA and protein were isolated 2 days after transfection for Smad7 expression by qPCR ( B ) and western blot ( C ) . The remaining cells were treated with r . h . TGF - β1 at indicated concentrations for another 24 h . The expression of human TGF - β1 was analyzed by qPCR and normalized on human GAPDH ( D ) . Data is given as mean ± SEM of three independent experiments . E - G . AT - MSCs were transfected with 5 nM miR - 21 mimic or control mimic , and were exposed to r . h . TGF - β1 2 days after transfection at indicated concentrations for another 24 h . The expression miR - 21 was analyzed by qPCR - based miR - 21 assay normalized on RNU6B ( E ) , Samd7 protein level was shown by western blot ( F ) , and human TGF - β1 expression was monitored by qPCR normalized on human GAPDH ( G ) . Data is given as mean ± SEM of three independent experiments . H - J . AT - MSCs were transfected with 50 nM miR - 21 inhibitor or control inhibitor , and were exposed to r . h . TGF - β1 2 days after transfection at indicated concentrations for another 24 h , and subsequently subjected to the analysis of expression of miR - 21 ( H ) , Smad7 protein ( I ) and human TGF - β1 mRNA ( J ) . Data is given as mean ± SEM of three independent experiments . K - L . Full - thickness excisional wounds were produced on CD18 - / - mice by 6 mm biopsy punches . One day after wounding , each wound received intradermal injection of 2 . 5x105 AT - MSCs that had been transfected with 50 nM miR - 21 inhibitor or control inhibitor 2 days after transfection . CD18 - / - wounds received mock injection with PBS served as control . Each wound region was digitally photographed at days 0 , 3 , and 6 post - wounding , and expressed as percentage of the initial wound size at day 0 ( K ) . Wound tissues were harvested on day 6 post - wounding for quantification of total TGF - β1 protein by ELISA ( L ) . Data is given as mean ± SEM ; n = 5 wounds per group ; * , p < 0 . 05 ; * * , p < 0 . 01 , by one - way ANOVA with Tukey ' s test . MSC _ miR - 21 - IN , miR - 21 inhibitor transfected AT - MSCs ; MSC _ ctrl - IN , control inhibitor transfected AT - MSCs .

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

Figure 7A. AT-MSCs were pretreated with a TGFβRI inhibitor SB431542 at 10 µM, or control DMSO for 1 h, and subsequently exposed to r.h. TGF-β1 at indicated concentrations. The cells were cultured for another 24 h and harvested for miR-21 assay. The expression of miR-21 was normalized on RNU6B. Data is given as mean ± SEM of three independent experiments.B-D. AT-MSCs were transfected with 30 nM Smad7-siRNAs or control siRNA. Untransfected AT-MSCs served as control. RNA and protein were isolated 2 days after transfection for Smad7 expression by qPCR (B) and western blot (C). The remaining cells were treated with r.h. TGF-β1 at indicated concentrations for another 24 h. The expression of human TGF-β1 was analyzed by qPCR and normalized on human GAPDH (D). Data is given as mean ± SEM of three independent experiments.E-G. AT-MSCs were transfected with 5 nM miR-21 mimic or control mimic, and were exposed to r.h. TGF-β1 2 days after transfection at indicated concentrations for another 24 h. The expression miR-21 was analyzed by qPCR-based miR-21 assay normalized on RNU6B (E), Samd7 protein level was shown by western blot (F), and human TGF-β1 expression was monitored by qPCR normalized on human GAPDH (G). Data is given as mean ± SEM of three independent experiments.H-J. AT-MSCs were transfected with 50 nM miR-21 inhibitor or control inhibitor, and were exposed to r.h. TGF-β1 2 days after transfection at indicated concentrations for another 24 h, and subsequently subjected to the analysis of expression of miR-21 (H), Smad7 protein (I) and human TGF-β1 mRNA (J). Data is given as mean ± SEM of three independent experiments.K-L. Full-thickness excisional wounds were produced on CD18-/- mice by 6 mm biopsy punches. One day after wounding, each wound received intradermal injection of 2.5x105 AT-MSCs that had been transfected with 50 nM miR-21 inhibitor or control inhibitor 2 days after transfection. CD18-/- wounds received mock injection with PBS served as control. Each wound region was digitally photographed at days 0, 3, and 6 post-wounding, and expressed as percentage of the initial wound size at day 0 (K). Wound tissues were harvested on day 6 post-wounding for quantification of total TGF-β1 protein by ELISA (L). Data is given as mean ± SEM; n = 5 wounds per group; *, p<0.05; **, p<0.01, by one-way ANOVA with Tukey's test. MSC_miR-21-IN, miR-21 inhibitor transfected AT-MSCs; MSC_ctrl-IN, control inhibitor transfected AT-MSCs.


Figure 1 ( A ) Gene set enrichment analysis ( GSEA ) of differentially expressed genes in colorectal cancer from the GEO database ( GDS4382 ) . ES , enrichment score ; NES , normalized enrichment score . n = 3 biological samples . ( B ) Kaplan - Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset ( n = 590 samples , P = 0 . 016 , Log - rank ( Mantel - Cox ) test ) . " Normal " represents the patients with unaltered expression of iron homoeostasis - related genes , including LTF , CP , FTH1 , SLC30A1 and TFRC ; " Dysfunctional iron homoeostasis " represents the patients with altered expression of iron homoeostasis - related genes . ( C ) Immunoblot analysis of protein levels of IREB2 in colorectal adenocarcinoma and its matched adjacent normal tissues ( Left ) , the relative protein level of IREB2 was assessed by ImageJ software ( Right ) ( n = 8 human samples , * P = 0 . 0114 , two - tailed paired Student ' s t - test ) . ( D ) Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue , and was subsequently analyzed by anti - ubiquitin antibody for the assessment of ubiquitination . ( E ) Mass spectrum analysis of IREB2 - associated proteins . Mock or IREB2 - FLAG was transfected into HEK293T cells and FLAG - tagged proteins were enriched by anti - FLAG M2 beads and incubated with lysates from normal mice colon tissues . Five matched peptides corresponding to OTUD1 were shown on the right panel . ( F ) In vivo ubiquitination assay of IREB2 . HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti - FLAG antibody followed by western blot analysis . ( G ) Half - life analysis of IREB2 in wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells . Cells were treated with 100 μg / ml cycloheximide ( CHX ) and collected at the indicated times for western blot analysis ( Up ) , the relative protein level of IREB2 was assessed by ImageJ software ( Down ) ( n = 3 biological replicates , mean ± s . e . m . , * * P = 0 . 0049 , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) . ( H ) Flow cytometric analysis of TFRC expression in mock and OTUD1 expressing CT26 cells with AFC ( 50 μM ) and Hemin ( 100 μM ) treatment . MFI , mean fluorescence intensity . Control uses isotype - matched control antibody ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0010 , * * * P = 0 . 0008 , two - tailed unpaired Student ' s t - test ) . ( I ) Flow cytometric analysis of TFRC expression in wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells with DFO ( 100 μM ) treatment . MFI , mean fluorescence intensity . ( n = 4 biological replicates , mean ± s . e . m . , * * * P = 0 . 0001 , two - tailed unpaired Student ' s t - test ) .

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

Figure 1(A) Gene set enrichment analysis (GSEA) of differentially expressed genes in colorectal cancer from the GEO database (GDS4382). ES, enrichment score; NES, normalized enrichment score. n = 3 biological samples.(B) Kaplan-Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset (n = 590 samples, P = 0.016, Log-rank (Mantel-Cox) test). "Normal" represents the patients with unaltered expression of iron homoeostasis-related genes, including LTF, CP, FTH1, SLC30A1 and TFRC; "Dysfunctional iron homoeostasis" represents the patients with altered expression of iron homoeostasis-related genes. (C) Immunoblot analysis of protein levels of IREB2 in colorectal adenocarcinoma and its matched adjacent normal tissues (Left), the relative protein level of IREB2 was assessed by ImageJ software (Right) (n = 8 human samples, *P = 0.0114, two-tailed paired Student's t-test).  (D) Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue, and was subsequently analyzed by anti-ubiquitin antibody for the assessment of ubiquitination.  (E) Mass spectrum analysis of IREB2-associated proteins. Mock or IREB2-FLAG was transfected into HEK293T cells and FLAG-tagged proteins were enriched by anti-FLAG M2 beads and incubated with lysates from normal mice colon tissues. Five matched peptides corresponding to OTUD1 were shown on the right panel.  (F) In vivo ubiquitination assay of IREB2. HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti-FLAG antibody followed by western blot analysis.  (G) Half-life analysis of IREB2 in wild-type (WT) and OTUD1─/─ NCM460 cells. Cells were treated with 100 μg/ml cycloheximide (CHX) and collected at the indicated times for western blot analysis (Up), the relative protein level of IREB2 was assessed by ImageJ software (Down) (n = 3 biological replicates, mean ± s.e.m., **P = 0.0049, ***P < 0.0001, two-tailed unpaired Student's t-test).  (H) Flow cytometric analysis of TFRC expression in mock and OTUD1 expressing CT26 cells with AFC (50 μM) and Hemin (100 μM) treatment. MFI, mean fluorescence intensity. Control uses isotype-matched control antibody (n = 4 biological replicates, mean ± s.e.m., **P = 0.0010, ***P = 0.0008, two-tailed unpaired Student's t-test).  (I) Flow cytometric analysis of TFRC expression in wild-type (WT) and OTUD1─/─ NCM460 cells with DFO (100 μM) treatment. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., ***P = 0.0001, two-tailed unpaired Student's t-test).


Figure 2 ( A ) RT - qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues ( n = 101 human samples , * * * P < 0 . 001 , two - tailed paired Student ' s t - test ) ( B ) Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer ( n = 101 human samples , two - tailed Pearson correlation analysis , * * * * P < 0 . 0001 ) . ( C ) Immunoblot analysis of protein levels of OTUD1 and TFRC in colorectal adenocarcinoma and its matched adjacent normal tissues ( n = 4 human samples ) , the protein levels relative to GAPDH are marked . ( D ) Immunohistochemical staining of OTUD1 in colorectal adenocarcinoma and its matched adjacent normal tissue ( The scale bars represent 1000 μm ) . ( E ) Analysis of OTUD1 protein levels in colorectal adenocarcinoma patients with different clinical stages from tissue microarray CoI05 - 118e . ( F ) Representative immunohistochemistry staining pictures of OTUD1 protein levels in normal tissue and different colon diseases from tissue microarray CO809a ( The scale bars represent 1000 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 2 (A) RT-qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues (n = 101 human samples, ***P < 0.001, two-tailed paired Student's t-test)  (B) Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer (n = 101 human samples, two-tailed Pearson correlation analysis, ****P < 0.0001).  (C) Immunoblot analysis of protein levels of OTUD1 and TFRC in colorectal adenocarcinoma and its matched adjacent normal tissues (n = 4 human samples), the protein levels relative to GAPDH are marked.  (D) Immunohistochemical staining of OTUD1 in colorectal adenocarcinoma and its matched adjacent normal tissue (The scale bars represent 1000 μm).  (E) Analysis of OTUD1 protein levels in colorectal adenocarcinoma patients with different clinical stages from tissue microarray CoI05-118e. (F) Representative immunohistochemistry staining pictures of OTUD1 protein levels in normal tissue and different colon diseases from tissue microarray CO809a (The scale bars represent 1000 μm).


Figure 3 Intracellular iron concentration was measured in CT26 cells stably expressing mock , OTUD1 or OTUD1C320S ( n = 2 biological replicates ) Intracellular iron concentration was measured in wild - type ( WT ) or OTUD1 ─ / ─ NCM460 cells ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) ( C ) The protein levels of IREB2 , TFRC and OTUD1 were assessed by western blot in colon tissues from wild - type ( WT ) or Otud1 ─ / ─ mice . ( D ) Intracellular iron concentration was measured in colon tissues from wild - type ( WT ) or Otud1 ─ / ─ mice with supplementation of ferrous gluconate ( n = 3 biological replicates , mean ± s . e . m . , * * * P = 0 . 0007 , two - tailed unpaired Student ' s t - test ) . Mean corpuscular volume ( MCV ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * P = 0 . 0472 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times . mean corpuscular hemoglobin ( MCH ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0048 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times . mean corpuscular hemoglobin concentration ( MCHC ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0068 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times . , red boold cells ( RBCs ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0049 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times . hemoglobin ( HB ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0036 , * * * P = 0 . 0002 , two - tailed unpaired Student ' s t - test ) ( I ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Intracellular iron concentration was measured in CT26 cells stably expressing mock, OTUD1 or OTUD1C320S (n = 2 biological replicates)  Intracellular iron concentration was measured in wild-type (WT) or OTUD1─/─ NCM460 cells (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test)  (C) The protein levels of IREB2, TFRC and OTUD1 were assessed by western blot in colon tissues from wild-type (WT) or Otud1─/─ mice.  (D) Intracellular iron concentration was measured in colon tissues from wild-type (WT) or Otud1─/─ mice with supplementation of ferrous gluconate (n = 3 biological replicates, mean ± s.e.m., ***P = 0.0007, two-tailed unpaired Student's t-test).  Mean corpuscular volume (MCV) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0472, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  mean corpuscular hemoglobin (MCH) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0048, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  mean corpuscular hemoglobin concentration (MCHC) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0068, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  , red boold cells (RBCs) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0049, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  hemoglobin (HB) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0036, ***P = 0.0002, two-tailed unpaired Student's t-test) (I) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


Figure 4 ( A - B ) Macroscopic evaluation ( A ) and tumor volume ( mock , n = 7 biological replicates ; OTUD1 , n = 9 biological replicates ; OTUD1C320S , n = 9 biological replicates ; mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) ( B ) of mock , OTUD1 and OTUD1C320S expressing CT26 tumors in BALB / c mice . Macroscopic evaluation of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice . tumor volume ( n = 6 biological replicates , mean ± s . e . m . , * * P < 0 . 01 , * * * P = 0 . 0007 , two - tailed unpaired Student ' s t - test ) of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice . survival analysis ( n = 5 biological replicates , * * P = 0 . 0021 , Log - rank ( Mantel - Cox ) test ) of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice . ( F ) Endogenous IREB2 and TFRC protein levels in murine CT26 tumors were measured by anti - IREB2 antibody and anti - TFRC antibody . ( G ) Flow cytometric analysis of TFRC expression in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) and OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors . MFI , mean fluorescence intensity ( mean ± s . e . m . , * * P < 0 . 01 , two - tailed unpaired Student ' s t - test ) . ( H ) Flow cytometric analysis of TFRC expression in mock or OTUD1 expressing LLC tumors . MFI , mean fluorescence intensity . ( n = 5 biological replicates , mean ± s . e . m . , * * P = 0 . 0011 , two - tailed unpaired Student ' s t - test ) . ( I ) Iron concentration of mock , OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry ( n = 3 biological replicates , mean ± s . e . m . , * P = 0 . 0237 , * * P = 0 . 0098 , two - tailed unpaired Student ' s t - test ) . ( J ) Analysis of tumor infiltrating CD8 + T cells or CD4 + T cells in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) and OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors in BALB / c mice ( mean ± s . e . m . , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) . Flow cytometric analysis of CD8 + T cells , CD4 + T cells ( n = 6 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * * P = 0 . 0003 , two - tailed unpaired Student ' s t - test ) isolated from mock and OTUD1 overexpressing LLC tumors . PD - 1 expression in T cells ( n = 6 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) isolated from mock and OTUD1 overexpressing LLC tumors . MFI , mean fluorescence intensity .

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

Figure 4 (A-B) Macroscopic evaluation (A) and tumor volume (mock, n = 7 biological replicates; OTUD1, n = 9 biological replicates; OTUD1C320S, n = 9 biological replicates; mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) (B) of mock, OTUD1 and OTUD1C320S expressing CT26 tumors in BALB/c mice.  Macroscopic evaluation of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.  tumor volume (n=6 biological replicates, mean ± s.e.m., **P < 0.01, ***P = 0.0007, two-tailed unpaired Student's t-test) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.  survival analysis (n=5 biological replicates, **P = 0.0021, Log-rank (Mantel-Cox) test) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.  (F) Endogenous IREB2 and TFRC protein levels in murine CT26 tumors were measured by anti-IREB2 antibody and anti-TFRC antibody.  (G) Flow cytometric analysis of TFRC expression in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors. MFI, mean fluorescence intensity (mean ± s.e.m., **P < 0.01, two-tailed unpaired Student's t-test).  (H) Flow cytometric analysis of TFRC expression in mock or OTUD1 expressing LLC tumors. MFI, mean fluorescence intensity. (n = 5 biological replicates, mean ± s.e.m., **P = 0.0011, two-tailed unpaired Student's t-test).  (I) Iron concentration of mock, OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry (n = 3 biological replicates, mean ± s.e.m., *P = 0.0237, **P = 0.0098, two-tailed unpaired Student's t-test).  (J)Analysis of tumor infiltrating CD8+ T cells or CD4+ T cells in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors in BALB/c mice (mean ± s.e.m., **P < 0.01, ***P < 0.001, two-tailed unpaired Student's t-test).  Flow cytometric analysis of CD8+ T cells, CD4+ T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P = 0.0003, two-tailed unpaired Student's t-test) isolated from mock and OTUD1 overexpressing LLC tumors.  PD-1 expression in T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P < 0.0001, two-tailed unpaired Student's t-test) isolated from mock and OTUD1 overexpressing LLC tumors. MFI, mean fluorescence intensity.


Figure 5 A ) Quantitative proteomics study of wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells . The expression of TFRC and IREB2 were highlighted with red color . Intensity indicates label - free quantification ( LFQ ) intensity of proteins in mass spectrometry . ( B ) Wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining ( n = 3 biological replicates , mean ± s . e . m . , * P < 0 . 05 , * * P < 0 . 01 , two - tailed unpaired Student ' s t - test ) . ( C ) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer - 1 ( 10 μM ) , Z - VAD ( 25 μM ) and nec - 1 ( 20 μM ) . Cell viability was measured by PI staining n = 3 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * P = 0 . 0119 , * * P = 0 . 0015 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) . ( D ) CT26 cells stably expressing mock , OTUD1 or OTUD1C320S were treated with Erastin ( 10 μM ) and RSL3 ( 5 μM ) , and cell viability was measured by PI staining ( n = 3 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P < 0 . 01 , two - tailed unpaired Student ' s t - test ) . ( E ) Wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells were treated with Erastin ( 10 μM ) and RSL3 ( 5 μM ) for 24 hours and cell viability was measured by PI staining ( n = 3 biological replicates , mean ± s . e . m . , * * P = 0 . 0082 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) . F ) Intracellular ROS levels in mock , OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin ( 10 μM ) , AFC ( 50 μM ) or H2O2 ( 100 μM ) were detected by DCFDA staining . MFI , mean fluorescence intensity ( n = 2 biological replicates ) . ( G ) Intracellular ROS levels in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) or OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors ( mean ± s . e . m . , * P = 0 . 0189 , * * P = 0 . 0086 , two - tailed unpaired Student ' s t - test ) were detected by DCFDA staining . MFI , mean fluorescence intensity . ( I ) Tumor volume ( mock , n = 4 biological replicates ; mock + NP - VE , n = 6 biological replicates ; OTUD1 , n = 4 biological replicates ; OTUD1 + NP - VE , n = 6 biological replicates Flow cytometric analysis of DCFDA ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) of mock or OTUD1 expressing CT26 tumors treated with or without NP - VE . MFI , mean fluorescence intensity . Flow cytometric analysis of the percentage of tumor infiltrating CD8 + T cells ( n = 4 biological replicates , mean ± s . e . m . , * P = 0 . 0433 , * * * P = 0 . 0001 , two - tailed unpaired Student ' s t - test ) of mock or OTUD1 expressing CT26 tumors treated with or without NP - VE .

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

Figure 5 A) Quantitative proteomics study of wild-type (WT) and OTUD1─/─ NCM460 cells. The expression of TFRC and IREB2 were highlighted with red color. Intensity indicates label-free quantification (LFQ) intensity of proteins in mass spectrometry.  (B) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., *P < 0.05, **P < 0.01, two-tailed unpaired Student's t-test).  (C) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer-1 (10 μM), Z-VAD (25 μM) and nec-1 (20 μM). Cell viability was measured by PI staining n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0119, **P = 0.0015, ***P < 0.001, two-tailed unpaired Student's t-test).  (D) CT26 cells stably expressing mock, OTUD1 or OTUD1C320S were treated with Erastin (10 μM) and RSL3 (5 μM), and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P < 0.01, two-tailed unpaired Student's t-test).  (E) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with Erastin (10 μM) and RSL3 (5 μM) for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., **P = 0.0082, ***P < 0.001, two-tailed unpaired Student's t-test).  F) Intracellular ROS levels in mock, OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin (10 μM), AFC (50 μM) or H2O2 (100 μM) were detected by DCFDA staining. MFI, mean fluorescence intensity (n = 2 biological replicates).  (G) Intracellular ROS levels in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) or OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors (mean ± s.e.m., *P = 0.0189, **P = 0.0086, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity.  (I) Tumor volume (mock, n = 4 biological replicates; mock + NP-VE, n = 6 biological replicates; OTUD1, n = 4 biological replicates; OTUD1 + NP-VE, n = 6 biological replicates  Flow cytometric analysis of DCFDA (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE. MFI, mean fluorescence intensity.  Flow cytometric analysis of the percentage of tumor infiltrating CD8+ T cells (n = 4 biological replicates, mean ± s.e.m., *P = 0.0433, ***P = 0.0001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE.


Figure 6 CT26 cells stably expressing mock or OTUD1 were treated with 5 μM RSL3 , and medium was collected to measure ATP level ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) . wild - type ( WT ) or OTUD1 ─ / ─ NCM460 cells were treated with 5 μM RSL3 , and medium was collected to measure ATP level ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) . ( C ) Volcano plots of genes differentially expressed in tumor interstitial fluid ( TIF ) of mock and OTUD1 expressing CT26 tumors . Left , example of genes downregulated in OTUD1 overexpressing tumor interstitial fluid ; right , example of genes upregulated in OTUD1 overexpressing tumor interstitial fluid . ( D ) Immunoblot analysis of protein levels of HMGB1 , HSP70 and HSP90 in tumor interstitial fluid of mock , OTUD1 and OTUD1C320S expressing CT26 tumors . ( E ) ATP level in tumor interstitial fluid ( TIF ) of mock or OTUD1 overexpressing CT26 cells ( n = 5 mice , mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) . ( F and G ) BALB / c mice were immunized with 1 × 106 OTUD1 expressing or mock CT26 cells in the left flank and challenged with 2 × 106 wild - type CT26 cells in the right flank 10 days later . Macroscopic evaluation ( F ) and tumor volume ( n = 7 mice , mean ± s . e . m . , * * P = 0 . 0053 , two - tailed unpaired Student ' s t - test ) ( G ) of tumors in the right flank were shown .

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

Figure 6 CT26 cells stably expressing mock or OTUD1 were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test).  wild-type (WT) or OTUD1─/─ NCM460 cells were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test).  (C) Volcano plots of genes differentially expressed in tumor interstitial fluid (TIF) of mock and OTUD1 expressing CT26 tumors. Left, example of genes downregulated in OTUD1 overexpressing tumor interstitial fluid; right, example of genes upregulated in OTUD1 overexpressing tumor interstitial fluid.  (D) Immunoblot analysis of protein levels of HMGB1, HSP70 and HSP90 in tumor interstitial fluid of mock, OTUD1 and OTUD1C320S expressing CT26 tumors.  (E) ATP level in tumor interstitial fluid (TIF) of mock or OTUD1 overexpressing CT26 cells (n = 5 mice, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test).  (F and G) BALB/c mice were immunized with 1×106 OTUD1 expressing or mock CT26 cells in the left flank and challenged with 2×106 wild-type CT26 cells in the right flank 10 days later. Macroscopic evaluation (F) and tumor volume (n=7 mice, mean ± s.e.m., **P = 0.0053, two-tailed unpaired Student's t-test) (G) of tumors in the right flank were shown.


Figure 7 ( A ) Representative pictures of colon tumors in AOM / DSS - induced Colitis - Associated Cancer ( CAC ) model ( n = 6 biological replicates ) . ( B and C ) The tumor load ( B ) or tumor number ( C ) in the whole colon was measured ( n = 12 biological replicates , mean ± s . e . m . , * P = 0 . 0431 , * * P = 0 . 003 , two - tailed unpaired Student ' s t - test ) . ( D ) Representative H & E ( Hematoxylin - eosin ) staining pictures of colon tumors ( The scale bars represent 1000 μm ) . ( E ) Flow cytometric analysis of TFRC expression in intestinal epithelial cells ( IECs ) from wild - type ( WT ) and Otud1 ─ / ─ mice treated with AOM / DSS . MFI , mean fluorescence intensity . ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0062 , two - tailed unpaired Student ' s t - test ) . ( F ) GSEA of genes expressed in colon tissues from wild - type ( WT ) and Otud1 ─ / ─ mice treated with AOM / DSS ( n = 3 biological replicates ) . ES , enrichment score ; NES , normalized enrichment score . ( G ) Representative immunohistochemistry staining pictures of CD3 + T cells in colon tumors ( The scale bars represent 400 μm ) . ( H ) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells ( LPMC ) isolated from wild - type ( WT ) and Otud1 ─ / ─ mice treated with AOM / DSS ( n = 5 biological replicates , mean ± s . e . m . , * * P = 0 . 0025 , * * * P = 0 . 0005 , two - tailed unpaired Student ' s t - test ) . ( I ) Intracellular ROS levels in colon tissues from wild - type ( WT ) or Otud1 ─ / ─ mice treated with AOM / DSS ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0072 , two - tailed unpaired Student ' s t - test ) were detected by DCFDA staining . MFI , mean fluorescence intensity . ( J ) ATP level in tumor interstitial fluid ( TIF ) of mice treated with AOM / DSS ( n = 4 biological replicates , mean ± s . e . m . , * P = 0 . 0292 , two - tailed unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative pictures of colon tumors in AOM/DSS-induced Colitis-Associated Cancer (CAC) model (n = 6 biological replicates).  (B and C) The tumor load (B) or tumor number (C) in the whole colon was measured (n = 12 biological replicates, mean ± s.e.m., *P = 0.0431, **P = 0.003, two-tailed unpaired Student's t-test).  (D) Representative H&E (Hematoxylin-eosin) staining pictures of colon tumors (The scale bars represent 1000 μm).  (E) Flow cytometric analysis of TFRC expression in intestinal epithelial cells (IECs) from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., **P = 0.0062, two-tailed unpaired Student's t-test).  (F) GSEA of genes expressed in colon tissues from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 3 biological replicates). ES, enrichment score; NES, normalized enrichment score.  (G) Representative immunohistochemistry staining pictures of CD3+ T cells in colon tumors (The scale bars represent 400 μm ).  (H) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells (LPMC) isolated from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 5 biological replicates, mean ± s.e.m., **P = 0.0025, ***P = 0.0005, two-tailed unpaired Student's t-test).  (I) Intracellular ROS levels in colon tissues from wild-type (WT) or Otud1─/─ mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., **P = 0.0072, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity.  (J) ATP level in tumor interstitial fluid (TIF) of mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., *P = 0.0292, two-tailed unpaired Student's t-test).


Figure 1 ( b , c ) Immunoblots of recruitment reactions performed as described in ( a ) , with the indicated proteins omitted . In ( c ) , a mid reaction wash in high salt ( 0 . 5 M NaCl ) buffer ( HSW ) was included following DDK phosphorylation as indicated . ( d ) Recruitment reaction performed as in ( a ) , using wild type ( wt ) or a Cdc45 - binding mutant ( 3E1 ) of Sld3 . ( f ) Wild type or mutant FLAG - Sld3 was added to an S - phase protein extract and tested for interaction with Cdc45 . Immunoprecipitated proteins were analysed by immunoblot . ( h ) Reaction performed as in ( g ) . Nascent DNA was separated in a 0 . 7 % alkaline agarose gel in this and all subsequent replication reactions .

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

Figure 1(b, c) Immunoblots of recruitment reactions performed as described in (a), with the indicated proteins omitted. In (c), a mid reaction wash in high salt (0.5 M NaCl) buffer (HSW) was included following DDK phosphorylation as indicated.(d) Recruitment reaction performed as in (a), using wild type (wt) or a Cdc45-binding mutant (3E1) of Sld3.(f) Wild type or mutant FLAG-Sld3 was added to an S-phase protein extract and tested for interaction with Cdc45. Immunoprecipitated proteins were analysed by immunoblot.(h) Reaction performed as in (g). Nascent DNA was separated in a 0.7% alkaline agarose gel in this and all subsequent replication reactions.


Figure 2 ( a ) Reaction performed as in Fig . 1a , except Cdc45 was omitted . ( b ) Reaction performed as in Fig . 1g , with the indicated proteins omitted . ( e ) Reaction performed as in ( a ) , except Sld3 / 7 was incubated with Rad53 before addition to reactions . ATP was omitted from this pre - incubation as indicated . ( f ) Sld3 recruitment reaction performed as in ( a ) . Sld7 was omitted from this experiment .

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

Figure 2(a) Reaction performed as in Fig. 1a, except Cdc45 was omitted.(b) Reaction performed as in Fig. 1g, with the indicated proteins omitted.(e) Reaction performed as in (a), except Sld3/7 was incubated with Rad53 before addition to reactions. ATP was omitted from this pre-incubation as indicated.(f) Sld3 recruitment reaction performed as in (a). Sld7 was omitted from this experiment.


Figure 3 ( b ) Recruitment reaction performed as described in ( a ) . ( c ) Sld3 / 7 recruitment reaction conducted as in Fig . 2a . ( d ) Replication reaction performed as in Fig . 1g . ( e ) Representative tetrads from dissection of SLD3 + / sld3 - 6E heterozygotes .

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

Figure 3(b) Recruitment reaction performed as described in (a).(c) Sld3/7 recruitment reaction conducted as in Fig. 2a.(d) Replication reaction performed as in Fig. 1g.(e) Representative tetrads from dissection of SLD3+/sld3-6E heterozygotes.


Figure 4 ( b , c , D ) Immunoblot of reactions performed as described in ( a ) . In ( B ) , either reconstituted Sld3 / 7 complex ( lanes 6 and 7 ) or Sld3 alone ( lanes 8 and 9 ) was coupled to beads . In ( C ) , MCM was treated with Lambda Phosphatase after dissociation from DNA ( lanes 3 and 7 ) . ( b , c , D ) Immunoblot of reactions performed as described in ( a ) . In ( D ) , Sld7 was omitted . ( E ) 30 - residue phosphorylated Mcm6 peptides were bound to streptavidin - coated beads and used as bait to pulldown Sld3 . Peptide - coupled beads were dephosphorylated with Lambda Phosphatase as indicated . ( F ) Reaction conducted as in ( a ) , using wild type ( wt ) or the indicated S / T - A substitution mutant versions of Mcm4 and Mcm6 . Positions of mutations in Mcm6 are summarised in Fig . EV5c .

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 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 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 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

Figure 4(b, c, D) Immunoblot of reactions performed as described in (a). In (B), either reconstituted Sld3/7 complex (lanes 6 and 7) or Sld3 alone (lanes 8 and 9) was coupled to beads. In (C), MCM was treated with Lambda Phosphatase after dissociation from DNA (lanes 3 and 7).(b, c, D) Immunoblot of reactions performed as described in (a). In (D), Sld7 was omitted.(E) 30-residue phosphorylated Mcm6 peptides were bound to streptavidin-coated beads and used as bait to pulldown Sld3. Peptide-coupled beads were dephosphorylated with Lambda Phosphatase as indicated.(F) Reaction conducted as in (a), using wild type (wt) or the indicated S/T-A substitution mutant versions of Mcm4 and Mcm6. Positions of mutations in Mcm6 are summarised in Fig. EV5c.