Figure 6.

RNA-Seq analysis of MB knockdown cells after treatment with NO. MDA-MB-468 cells were transfected with Hs_MB_6 FlexiTube siRNA (KD) or control siRNA (WT) and subsequently challenged with 500 µM GNSO for 6 h. (A) Ingenuity pathway analysis (IPA) showed significantly enriched annotations classified into three categories: molecular and cellular function, physiological system development and function, and disease and disorders. (B) 1029 differentially expressed genes (DEGs) of the cancer network were compared to 587 potential HIF-1α target genes revealing 45 genes that were altered by a combination of MB KD and NO treatment. Of these, five genes were identified as genes of interest with a fold change >1.5 and p-value < 0.05. (C) Expression levels of the genes of interest (SLC2A1/GLUT-1, MKNK1, RAB8B, TMEFF1, and TREX2) were validated by qPCR, showing a significant downregulation for SLC2A1/GLUT-1, MKNK1, and RAB8B under MB KD conditions. As control, WT and MB KD cells were incubated without NO to show NO-mediated effects. Mean +/- SD, n = 4 in 2 independent experiments. *p < 0.05 as indicated.