Fig 2: Interference and overexpression efficiency of TRIM9.IEC-6 cells were treated with TRIM9 overexpression or interference lentiviruses. (A) Representative bar graphs showing that TRIM9 mRNA expression was significantly increased in the oeTRIM9 group and reduced in the shTRIM9-1, -2, -3 groups. (B) TRIM9 protein expression was significantly increased in the oeTRIM9 group and reduced in the shTRIM9-1, -2, -3 groups. ***P<0.001 vs. Vector; #P<0.05, ##P<0.01 vs. shNC. TRIM9, tripartite motif protein 9; oe, overexpression; sh, short hairpin RNA; NC, negative control.

Fig 3: TRIM9 directly interacts with DUSP6 in IEC-6 cells. (A) The protein expression levels of TRIM9, DUSP1, DUSP4, DUSP5, DUSP6 and DUSP10 in IEC-6 cells transfected with oeTRIM9 was detected. **P<0.01, ***P<0.001 vs. Vector. (B) Co-immunoprecipitation showed the interaction between TRIM9 and DUSP6, and TRIM9 and DUSP10. DUSP6 (C) mRNA and (D) protein expression levels in IEC-6 cells transfected with shTRIM9-1 and shTRIM9-2 or oeTRIM9 were detected. **P<0.01 vs. shNC; ##P<0.01 vs. Vector. (E) After combined treatment with oeTRIM9 and MG132 (proteasome inhibitor), DUSP6 protein expression in IEC-6 cells was detected. **P<0.01 vs. Vehicle + Vector; ##P<0.01 vs. Vehicle + oeTRIM9. (F) Following TRIM9 overexpression, the ubiquitination of DUSP6 was detected. TRIM9, tripartite motif protein 9; DUSP, dual-specificity phosphatase; oe, overexpression; sh, short hairpin RNA; NC, negative control.

Fig 4: Knockdown of TRIM9 partly inhibits the irinotecan-induced inhibition of cell proliferation, intestinal barrier function impairment and inflammatory factor expression levels. IEC-6 cells were treated with irinotecan combined with TRIM9 interference. (A) Cell proliferation at 0, 12, 24 and 48 h was detected using a Cell Counting Kit-8 assay. (B) TEER was measured in each group. (C) The fluorescence intensity of FITC was tested in each group. (D) Cell apoptosis was detected with a flow cytometer. (E) The levels of IL-1ß and TNF-a expression were detected with an ELISA. (F) The protein expression levels of TRIM9, ZO-1, Claudin-4 and p-P38/P38 were detected via western blotting. **P<0.01, ***P<0.001 vs. Vehicle; #P<0.05, ##P<0.01, ###P<0.001 vs. Irinotecan + shNC. TRIM9, tripartite motif protein 9; TEER, transepithelial electrical resistance; TNF-a, tumor necrosis factor-a; IL-1ß, interleukin-1ß; ZO-1, zona occludens protein 1; p-, phosphorylated; sh, short hairpin RNA; NC, negative control.

Fig 5: TRIM9 regulates cell proliferation and intestinal barrier function in IEC-6 cells likely through modulating DUSP6 expression. The overexpression efficiency of DUSP6 in IEC-6 cells was detected via (A) reverse transcription-quantitative PCR and (B) western blotting. **P<0.01, ***P<0.001 vs. Vector. IEC-6 cells were transfected with oeTRIM9 combined with oeDUSP6. (C) Cell proliferation at 0, 12, 24 and 48 h was detected using a CCK-8 assay. (D) Cell apoptosis was detected with a flow cytometer. (E) TEER was measured in each group. (F) The fluorescence intensity of FITC was tested in each group. (G) The levels of IL-1ß and TNF-a expression were detected with an ELISA. (H) The protein expression levels of ZO-1, DUSP6, Claudin-4 and p-P38/P38 were detected via western blotting. *P<0.05, **P<0.01, ***P<0.001 vs. Vector; #P<0.05, ##P<0.01, ###P<0.001 vs. oeTRIM9 + Vector. IEC-6 cells were treated with irinotecan combined with oeDUSP6. (I) Cell proliferation at 0, 12, 24 and 48 h was detected with a CCK-8. (J) TEER was measured in each group. (K) The fluorescence intensity of FITC was tested in each group. (L) Cell apoptosis was detected with a flow cytometer. **P<0.01, ***P<0.001 vs. Vector; #P<0.05 vs. irinotecan + Vector. TRIM9, tripartite motif protein 9; DUSP, dual-specificity phosphatase; oe, overexpression; CCK-8, Cell Counting Kit-8; TEER, transepithelial electrical resistance; TNF-a, tumor necrosis factor-a; IL-1ß, interleukin-1ß; ZO-1, zona occludens protein 1; p-, phosphorylated.