Fig. 5: USP5 regulates TNFα-induced necroptosis through its deubiquitinase function.

A HT-29 cells were transduced with lentivirus to generate stable USP5-knockdown (KD) cell lines. The expression levels of RIPK1, RIPK3, MLKL, and USP5 in each cell line were analyzed by western blotting. B HT-29 cells depleted of USP5 using the indicated shRNAs were treated with T/Bi/Z in the presence or absence of 0.1 μM GSK'963 for 3.5 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 USP5 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 USP5 KD and control HT-29 cells treated with T/Bi/Z were lysed after 3.5 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. E shUSP5-infected cells, reconstituted with either USP5 WT or the deubiquitinase-defective mutant USP5 C335A, were treated with T/Bi/Z in the presence or absence of 0.1 μM GSK'963 for 3.5 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. F shUSP5-infected cells, reconstituted with either USP5 WT or the deubiquitinase-defective mutant USP5 C335A, 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 and p-MLKL. G shUSP5-infected cells, reconstituted with either USP5 WT or the deubiquitinase-defective mutant USP5 C335A, were treated with T/Bi/Z and lysed after 3.5 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.