Knockdown of KDM3A leads myeloma cells to induce apoptosis under chronic hypoxia in MM. (A) qRT-PCR of KDM3A for U266 cell line stably transduced with shKDM3A#A, #B, #C, #D, and control scrambled shRNA (Scr). Bars represent mean ± 95% CI of 3 independent experiments. ***P < .001. Student t test was used to test for significance. (B) Western blot analysis of KDM3A for U266 cell line stably transduced with shKDM3A#A, #B, #C, #D, and control scrambled shRNA (Scr). (C) qRT-PCR of KDM3A for U266 cell line stably transduced with shKDM3A#C, #D, and control Scr and cultured in normoxia or hypoxia (1% O2) for 48 hours. Bars represent mean ± 95% CI of 3 independent experiments. ***P < .001. Student t test was used to test for significance. (D) Western blot analysis of KDM3A and IRF4 for U266 cell line stably transduced with shKDM3A#C, #D, and control Scr and cultured in normoxia or hypoxia (1% O2) for 48 hours. H, hypoxia; N, normoxia. (E) Cell count of U266 cells stably transduced with shKDM3A#C or control Scr and cultured in normoxia or hypoxia (1% O2; 24, 48, 72, and 96 hours). Cells (0.5 × 106) were cultured at 0 hours. Bars represent mean ± 95% CI of 3 independent experiments. **.001 ≤ P < .01. Student t test was used to test for significance. (F) Cell cycle analysis of U266 cells stably transduced with shKDM3A#C or control Scr and cultured in normoxia or hypoxia (1% O2; 48, 72, and 96 hours). X-axis: intensity of propidium iodide (PI). Y-axis, cell count. (G) SubG1 cells (%) of U266 cell line stably transduced with shKDM3A#C or control Scr and cultured in normoxia or hypoxia (1% O2; 48, 72, and 96 hours). Bars represent mean ± 95% CI of 3 independent experiments. **.001 ≤ P < .01; ***P < .001. Student t test was used to test for significance. (H) western blot analysis of PARP, cleaved PARP, Caspase 3, cleaved Caspase 3, Caspase 9, cleaved Caspase 9, and Bim for U266 cells stably transduced with shKDM3A#C or control scrambled shRNA (Scr) and cultured in hypoxia (1% O2; 0, 48, 72, 96 hours).
