Fig. 2: The E3 ligase function of SMURF1 controls necroptosis by suppressing necrosome formation.

A HT-29 cells were transduced with lentivirus to generate stable SMURF1-knockdown (KD) cell lines. The expression levels of RIPK1, RIPK3, MLKL, and SMURF1 in each cell line were analyzed by western blotting. B HT-29 cells depleted of SMURF1 using the indicated shRNAs were treated with T/Bi/Z in the presence or absence of 0.1 μM GSK'963 for 2.5 h. Flow cytometry was performed by counting 10,000 cells based on FSC (Forwarder Scatter) and SSC (Side Scatter), and calculating the percentage of cells in the Annexin V-FITC positive region. This experiment was independently repeated 3 times. Data are presented as means ± standard deviations (SDs), n = 3. Significance between groups was determined using two-sided Students’ test. C SMURF1 KD and control HT-29 cells were treated with T/Bi/Z to induce necroptosis. GSK'963 was used to confirm that this cell death is necroptosis. Cells were harvested at the indicated time points and analyzed by western blotting. D SMURF1 KD and control HT-29 cells treated with T/Bi/Z were lysed after 2 h. The necrosome was pulled down using an anti-RIPK3 antibody and protein-G agarose beads. The protein-bead conjugate was washed and analyzed by western blotting to assess the assembly of the RIPK1-RIPK3-MLKL complex. The asterisk indicates p-MLKL. E shSMURF1-infected cells, reconstituted with either SMURF1 WT or the E3 ligase-inactive form (C699A), were treated with T/Bi/Z in the presence or absence of 0.1 μM GSK'963 for 2 h. Flow cytometry was performed by counting 10,000 cells based on FSC (Forwarder Scatter) and SSC (Side Scatter), and calculating the percentage of cells in the Annexin V-FITC positive region. This experiment was independently repeated three times. Data are presented as means ± standard deviations (SDs), n  =  3. Significance between groups was determined using two-sided Students’ test. F shSMURF1-infected cells, reconstituted with either SMURF1 WT or the E3 ligase-inactive form (C699A), were treated with T/Bi/Z to induce necroptosis. GSK'963 was used to confirm that this cell death is necroptosis. Cells were harvested at the indicated time points and analyzed by western blotting. The asterisk indicates p-RIPK3. G shSMURF1-infected cells, reconstituted with either SMURF1 WT or the E3 ligase-inactive form (C699A), were treated with T/Bi/Z and lysed after 2 h. The necrosome was pulled down using an anti-RIPK3 antibody and protein-G agarose beads. The protein-bead conjugate was washed and analyzed by western blotting to examine the assembly of the RIPK1-RIPK3-MLKL complex.