Hypoxia increases IL-32 expression in an HIF1α-dependent manner. (A) BM-derived CD138⁺ MM cells were cultured for 24 hours in normoxia (20% oxygen) or hypoxia (2% oxygen) as indicated, and IL-32, HIF1α, and β-actin were detected by immunoblotting in total cellular lysates. (B) MM cell lines were cultured for 48 hours under normoxia or hypoxia before collection of cells and cell culture supernatant for EV isolation. Total cell lysates and isolated EVs were immunoblotted as indicated. (C) IL-32 mRNA expression was determined by quantitative PCR in JJN3 cells cultured in hypoxia for the indicated time before reoxygenation as indicated. (D) JJN3 cells were transfected with scrambled control siRNA (siCtr) or HIF1α siRNA (siHIF1α), and expression of IL-32 and HIF1α was examined by immunoblotting after 24 hours. (E) Density of IL-32 and HIF1α bands was quantified by Image Studio Software and normalized to levels of β-actin. Data presented are mean + standard error of the mean from 4 independent experiments. (F) RNA sequencing data were downloaded from the CoMMPass IA8 release and analyzed using GSEA software v2.2.3 to identify functionally related groups of genes with statistically significant enrichment. The figure shows the enrichment plot for the hypoxia-related gene set for patients with IL32 levels in the upper 15th percentile. (G) The same RNA sequencing data in panel F were also analyzed to determine the correlation between relative IL32 mRNA expression vs relative hypoxic gene signature expression in MM as defined previously.²¹  Relative IL32 gene expression significantly correlated with the hypoxic signature (Spearman ρ = 0.4; P < .0001). ***P < .001.