Effects of ING4 suppression and overexpression in HMCLs on IL-8 and OPN production and HIF-1α activity under hypoxic condition. HMCLs and fresh purified CD138⁺ MM cells obtained from 3 MM patients were incubated in the presence or absence of hypoxic conditions for 12 hours. ING4 mRNA expression was evaluated by real-time PCR. Graphs represent the mean plus or minus SD of −ΔCt value of 3 independent experiments (*P < .05) (A). RPMI-8226 was transfected by electroporation with 1 nmol smart pool double-stranded RNA oligonucleotides (siRNA) against ING4 or with a nonspecific control siRNA (Cy) and after 24 hours incubated in the presence or absence of hypoxic condition (1% O₂, 5% CO₂ atmosphere or CoCl₂ treatment) for 12 hours. Thereafter, ING4 and HIF-1α mRNA expression was evaluated by RT-PCR (B left panel). HIF-1α protein level was checked by Western blot in nuclear extracts (B right panel). HIF-1α activity was quantified in RPMI-8226–transfected cells by a transcriptional factor assay kit, as described in “NF-κB and HIF-1α activity.” Nuclear extracts of COS-7 treated with CoCl₂ in the presence or absence of wild-type (wt) or mutated (mt) competitor oligonucleotides were tested as control (Con). Graphs represent the mean plus or minus SD of HIF-1α activity normalized for 10 μg protein analyzed of 2 independent experiments performed in triplicate (OD = optical density) (C). The mRNA levels of ING4, NIP-3, OPN, and IL-8 in RPMI-8226–transfected cells incubated in hypoxic condition were quantified by real-time PCR. Graphs represent the mean plus or minus SD of −ΔCt value (D). Aliquots of conditioned media of RPMI-8226–transfected cells incubated in hypoxic condition were tested by ELISA. Graphs represent the mean plus or minus SD of IL-8 and OPN levels in 2 independent experiments measured in triplicate (*P < .05) (E). Both JJN3 and RPMI-8226 were infected by empty lentiviral vector, used as control, or ING4 overexpression vector, as described in “Cells and cell culture conditions; siRNA transfection.” p29ING4 protein was detected after 72 hours by Western blot analysis (F). RPMI-8226 cells overexpressing ING4 or the empty vector were incubated in the presence or absence of hypoxic condition for 12 hours and then evaluated for IL-8, OPN, and NIP-3 mRNA expression by real-time PCR. Graphs represent the mean plus or minus SD of −ΔCt value (G).