Fig. 7: EFHD2 interacts with Cofilin and inhibits Cofilin phosphorylation to block TNFR1 internalization.

a Silver staining of the gel separating proteins immunoprecipitated by FLAG beads from lysates prepared from HCT-116 cells transfected with FLAG-EFHD2 treated with 50 ng/ml TNF for the indicated time. b Co-IP analysis of the interaction between FLAG-EFHD2 and Cofilin in HCT-116 cells treated with TNF for the indicated time by western blot. The quantification of Cofilin/EFHD2 ratio by ImageJ was shown below. c Western blot analysis and quantification of the p-Cofilin in wild-type and EFHD2 interfered HCT-116 cells treated with TNF for the indicated time. d Flow cytometric analysis (left) and proportions (right) of PI^- caspase-3/7 active cells from HCT-116 cells treated with TS for 4 hours, without or with 20 μM or 50 μM BMS-3 pretreatment (TSB). e Flow cytometric analysis and proportions of PI^- caspase-3/7 active cells from WT and EFHD2^-/- HT-29 cells treated with TS for the indicated time, without or with 20 μM BMS-3 pretreatment. f Flow cytometric analysis and proportions of cell-surface TNFR1-positive cells from WT and EFHD2^-/- HT-29 cells treated with TS for 30 minutes, without or with 20 μM BMS-3 pretreatment. n = 3 (b–f). g Representative images of immunostaining for EFHD2 on intestinal biopsy samples from UC patients that were responsive (n = 5 patients) and non-responsive (n = 6 patients) to infliximab treatment. Histologic scores of EFHD2 staining were using a 3-point quantification scale. Scale bar, 300 μm. h Model depicting how deficiency of EFHD2 promotes TNFR1 internalization and intestinal inflammation due to excessive apoptosis. Data are representative of three independent experiments; error bars show means ± s.d. P values were determined by unpaired two-tailed t-test in b, d–g. Two-way ANOVA analysis with Sidak’s multiple comparisons test in c. Source data are provided as a Source Data file.