NOX4 promotes TRPM7-mediated ferroptosis in primary rat articular chondrocytes and C28/I2 cells. (A, B) Western blot analysis for NOX4 in erastin-induced RACs and C28/I2 cells treated with/without 2-APB (100 μM). Data are means ± SD, n = 3. (C) Western blot analysis for NOX4 in indicated TRPM7 KD C28/I2 cells treated with erastin (2.5 μM, 12 h). Data are means ± SD, n = 3. (D) Visualization of RACs cell viability treated with/without erastin (5 μM) and GKT137831 (20 μM). (E) Measurement of ΔΨm in RACs. (F) Immunofluorescence staining for ACSL4 in RACs. (G) Cell viability and LDH release of RACs (n = 3). (H) GSH, Fe2+, SOD and MDA were measured in RACs (n = 3). (I) Lipid ROS production was assessed by flow cytometry using C11-BODIPY in RACs (n = 3). Data are means ± SD. **P < 0.01 erastin versus control; #P < 0.05, ##P < 0.01 erastin + GKT137831 versus erastin. (J) Western blot analysis for ACSL4, NOX4, SLC7A11, FTH and GPX4 in RACs (n = 3). (K) Cell viability, LDH release and GSH of erastin-induced TRPM7 KD C28/I2 cells with NOX4 OE (n = 3–6). (L) Western blot analysis for ACSL4, SLC7A11, NOX4 and GPX4 in erastin-induced TRPM7 KD C28/I2 cells with NOX4 overexpression (n = 3). Data are means ± SD. **P < 0.01 Control KD + erastin versus Control KD; ##P < 0.01 TRPM7 KD + erastin versus Control KD + erastin, &&P < 0.01 TRPM7 KD + NOX4 OE + erastin versus TRPM7 KD + erastin.