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. 2016 Mar 16;6:23135. doi: 10.1038/srep23135

Figure 4. GPE inhibits the expression of members of NOX system and HIF-1α in human PCa 22Rv1 cells.

Figure 4

(A) 22Rv1 cells were treated with GPE (1–10 μg/ml) under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h. Thereafter, cells were collected and analyzed for mRNA expression of NOX1, p47phox and HIF-1α by RT-PCR using gene specific primers as mentioned in ‘Materials and Methods’. β-actin served as an endogenous internal standard. (B) 22Rv1 cells were treated with GPE (1–5 μg/ml) under hypoxic (1% O2) condition for 24 h. Thereafter, cells were collected and cytosolic and membrane fractions were prepared and analyzed for levels of NOX1, NOX2, p67phox and p47phox by immunoblotting. β-actin and E-cadherin were used to check the purity of cytoplasmic and membrane fractions as well as for loading control. Densitometry data presented below the bands are ‘fold change’ as compared with control (DMSO treated) after normalization with respective loading control. (C) 22Rv1 cells were treated with DPI (20 μM) and GPE (10 and 20 μg/ml) under hypoxic (1% O2) condition for 6 h. Cells cultured under normoxic condition (21% O2) served as relevant control. Thereafter, cells were collected and total cell lysates were prepared and analyzed for HIF-1α, HIF-1β, HIF-2α and tubulin. Densitometry data presented below the bands are ‘fold change’ as compared with hypoxia control (DMSO treated) after normalization with respective loading control. (D) 22Rv1 cells were treated with GPE (10 and 20 μg/ml) under hypoxic (1% O2) condition for 6 h. Cells cultured under normoxic condition (21% O2) served as relevant control. Thereafter, cells were collected and nuclear/cytoplasmic fractions were prepared and analyzed for HIF-1α expression. Membranes were also probed for TBP and tubulin as loading control for nuclear and membrane fractions, respectively. Densitometry data presented below the bands are ‘fold change’ as compared with hypoxia control (DMSO treated) after normalization with respective loading control. Abbreviations: N: Normoxic; H: Hypoxic; GPE: Graviola pulp extract; DPI: Diphenyleneiodonium.