Journal: Pigment cell & melanoma research
Article Title: Hypoxia-induced HIF1α targets in melanocytes reveal a molecular profile associated with poor melanoma prognosis
doi: 10.1111/pcmr.12579
Figure Lengend Snippet: (A) The migratory ability of melan-Ink4a-Arf−/− cells is significantly increased by 24 hr hypoxia exposure (***, P<0.0001). Representative images of migration wells for cells grown for 24 hr under normoxia (22% O2) and hypoxia (1% O2) are shown below the graph. (B) HIF1α protein is stabilized by 24 hr hypoxia, and HIF1α protein expression under hypoxia is eliminated by siHif1α treatment. Molecular weights: HIF1α=95 kDa, Tubulin = 50 kDa. (C) Hif1α mRNA levels are unchanged under hypoxia, consistent with HIF1α protein stabilization regulating signaling activity. Hif1α mRNA levels are significantly reduced by multiple siRNAs (**, P=0.0015). The mRNA expression levels of the known HIF1α target genes Kdm3a and Gapdh are significantly increased by 24 hr hypoxia (*, P<0.05), and are significantly downregulated with siHif1α KD under hypoxia, returning to near-normoxic levels (*, P<0.05). (D) 709 hypoxia-responsive genes were differentially expressed 1.5 fold under normoxia vs. hypoxia growth conditions. Ingenuity Pathway Analysis (IPA) predicted distinct upstream regulatory factors for upregulated and downregulated gene cohorts, listed at right. See Table S1 for the complete list of 709 hypoxia-responsive genes. (E) 712 HIF1α-dependent genes were differentially expressed 1.5-fold in both siHif1α KD RNAs as compared to non-silencing control. IPA predicted distinct upstream regulatory factors for upregulated and downregulated gene cohorts, listed at right. See Table S4 for the complete list of 712 HIF1α-dependent genes. Details of IPA analysis for predicted transcriptional regulators and downstream targets for the hypoxia-responsive genes and the HIF1α-dependent genes are in Tables S2 and S5, respectively. Norm = normoxia; Hpx = hypoxia; Ctr = control si; si1 and si6 = independent HIF1α-directed siRNA constructs.
Article Snippet: HIF1α dsRNA duplex oligonucleotides (HIF1α Dsi RNA duplex oligos) were as follows: for murine Hif1α , si6 - MMC.RNAI.N0104431.12.6, (GGACGAUGAACAUCAAGUCAGCAAC) and si1 -MMC.RNAI.N0104431.12.1 (AGACAAUAGCUUCGCAGAAUGCUCA); for Human HIF1α, si9- HSC.RNAi.N001530.12.9 (GCACUCAAUCAAGAAGUUGCAUUAA), si5- HSC.RNAi.N001530.12.5 (ACCAUAUAGAGAUACACAAAGUCGG) and si4 - HSC.RNAi.N001530.12.4 (GAUGGAAGCACUAGACAAAGUUCAC) (Integrated DNA technologies).
Techniques: Migration, Expressing, Activity Assay, Construct