Figure 5.

NLGP targets HIF1α upstream signaling cascade. (A) Responsible factors for regulating HIF1α at protein level was determined by a time kinetics experiment. B16Mel cells were exposed to hypoxia w/wo NLGP treatment for various time points (0 min, 15 min, 30 min, 1 h, 4 h, and 6 h) and expression pAKT(Thr) (A), pAKT(Ser) (B), pERK (C), and pSTAT3 (D) was checked by flow cytometry. Illustrative histograms show percent positive cells for respective upstream signaling molecules (n = 6). Mean Fluorescence Intensity (MFI) measuring the mean level of phosphorylation is indicated in each histogram with a bar diagram showing mean + SD of n = 4 samples. (E) mRNA expression of hif1α gene after blocking with inhibitor PD was checked w/wo NLGP treatment, keeping β-actin as loading control. (F) Representative data and bar diagram showing mean ± SD (n = 6) of HIF1α expression by RT-PCR are presented. (G) B16Mel cells were either treated with NLGP or Stat3 siRNA or both in presence of hypoxia. mRNA expression by RT-PCR was checked for hif1α gene expression. Level of Stat3 was also checked to see efficacy of the silencing experiment. (H) Representative bar diagram showing mean ± SD of relative expression (n = 6) for respective genes are presented.