Fig. 3.

Expression of HIF-1α is associated with cell density-dependent culture conditions. A Total mRNA was extracted from low- or high-density cultured U251-MG cells and analyzed by next-generation sequencing (NGS). Genes highly expressed in high-density cultured cells are indicated as OEG-high, target gene sets of transcription factors (x) are indicated as TF(x)_targets, and the intersection of the OEG-high gene set and each TF(x)_target is designated as the TF(x)_ratio. Each TF(x)_ratio of 199 transcription factors was calculated. B The top 15 ranked transcription factors are displayed with p values. C Expression levels of HIF-1α encoded by HIF1A gene, cyclin D1 encoded by CCND1 gene, and p65 (NF-κB p65 subunit) encoded by RELA gene in low- or high-density cultured U251-MG, U373-MG, LN215-MG, and Panc-1 cells were evaluated by immunoblot assays (L indicates low-density culture; H indicates high-density culture). GAPDH was used as the loading control. Relative pixel intensities were measured using ImageJ software. The data are representative of three individual experiments