Figure 2.

Overexpression of ARNT/HIF‐1β contributes to drug resistance in MM. A, ARNT/HIF‐1β expression was analyzed by qPCR in a cohort of patients with newly diagnosed MM (n = 40) and healthy donors (HD, n = 5). P = .005 for MM patients vs healthy donors (left). IHC staining for HIF‐1β as well as FISH with a probe targeting MCL1 to detect 1q21 gain were performed using bone marrow biopsies obtained undergoing routine diagnostic procedures. Representative microscopic images were shown (right). B, ARNT/HIF‐1β expression in drug‐naive RPMI8226 vs revlimid‐resistant (RR) and bortezomib‐resistant (DR) cells was assessed by qPCR (lower panel) and Western blot analysis (upper panel), respectively. C, MM cells (eg, H929 carrying 1q21 gain, as well as RPMI8226 and U266 without 1q21 gain) were exposed to bortezomib (Btz, 3‐5 nmol/L), for 24 h, after which Western blot analysis was performed to monitor expression of HIF‐1β. D, ARP‐1 cells, a human MM cell line, were transiently transfected with ARNT1.3 or empty vector (EV). Overexpression (OE) of HIF‐1β was determined by Western blot analysis (upper panel). In parallel, cells were stained for HIF‐1β by immunofluorescence (IF) and counterstained by DAPI (lower panel). Red, HIF‐1β; blue, DAPI for nucleus; bottom, merged images, indicating nuclear localization of HIF‐1β. E, ARP‐1 cells with ectopic overexpression of HIF‐1β and EV control were cultured for 4 d, and cell number was counted every 24 h (*P < .05 and **P < .01 for OE vs EV, ns = not significant). F, Alternatively, cells were exposed to a series of concentrations of Btz (0.25‐16 nmol/L) for 24 h, after which the CCK‐8 assay was performed to determine cell viability (*P < .05 and **P < .01 for OE vs EV). For E and F, values represent mean ± SD for at least 3 independent experiments performed in triplicate, respectively