Fig. 6.

RGMb inhibits necroptosis in TKPTS, HT-29, and HK-2 cells. (A) Necroptosis in TKPTS cells. Cells were treated with DMSO (Ctrl), TNF-α (100 ng/mL), CHX (2 μg/mL), zVAD-fmk (25 μM), and/or Nec-1 (50 μM) as indicated (TC, TCZ, or TCZN) for 24 h. Cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay. (B) RGMb inhibits TCZ-induced necroptosis but not TC-induced apoptosis in TKPTS cells. Cells were transfected with increasing amounts of Rgmb cDNA. Forty-eight hours after transfection, cells were treated with DMSO, TC (TNF-α and CHX), or TCZ (TNF-α, CHX, and zVAD-fmk) for 24 h before cell viability was measured. (C) RGMb reduces the release of HMGB1 from TKPTS cells in the presence of TCZ. Cells were transfected with or without Rgmb cDNA (300 ng/mL). Forty-eight hours after transfection, cells were treated with DMSO (lanes 1 and 2) or TCZ (lanes 3–8) for 24 h. Supernatants were collected and concentrated. Cells were lysed. Western blotting was performed for HMGB1 in supernatants and HMGB1 and RGMb in whole-cell lysates. β-actin was used as the loading control for cell lysates. (D) Necroptosis in HT-29 cells. Cells were treated with DMSO (Ctrl), TNF-α (30 ng/mL), the Smac mimetic Birinapant (50 nM), zVAD-fmk (20 μM), and/or Nec-1 (50 μM) as indicated (TS, TSZ, or TSZN) for 24 h. MTT assay was performed to determine cell viability. (E) RGMb inhibits TSZ-induced necroptosis but not TS-induced apoptosis in HT-29 cells. Cells were transfected with or without Rgmb cDNA (300 ng/mL). Forty-eight hours after transfection, cells were treated with DMSO, TS, or TSZ for 8 h before the cells were subjected to MTT assay. (F) RGMb dose-dependently inhibits necroptosis in HT-29 cells. Cells were transfected with increasing amounts of Flag-Rgmb plasmid. Forty-eight hours after transfection, cells were treated with DMSO or TSZ for 8 h before the cells were subjected to MTT assay. (G) CaOx crystals induce necroptosis in HK-2 cells. Cells were incubated with 1 mg/mL CaOx crystals in the absence or presence of Nec-1 for 24 h before cell viability was measured by MTT assay. (H) RGMb inhibits CaOx-induced necroptosis in HK-2 cells. Cells were transfected with increasing amounts of Rgmb plasmid. Twenty-four hours after transfection, cells were incubated with or without CaOx crystals for 24 h before cell viability was measured. (I) RGMb-TM inhibits TSZ-induced necroptosis. Cells were transfected with increasing amounts of Rgmb-TM cDNA (100, 200, and 400 ng/mL) or fixed amount of Rgmb cDNA (300 ng/mL). Forty-eight hours after transfection, cells were treated with DMSO or TSZ for 6 h before the cells were subjected to MTT assay. n = 6 for A, B, D, F, and I; n = 3 for C; n = 10 for E and G; n = 8 for H. *P < 0.05; **P < 0.01; ***P < 0.001. In B, ** indicates the differences between the doses 150 or 300 ng/mL with 0, and # indicates the difference between the doses 150 and 300 ng/mL.