Figure 4.

Hypoxia influence on several processes upon melanoma cells cultivation under low oxygen condition (1% O₂, 5%CO₂ and 94% N₂). (A) Western blot assay of hypoxia-inducible factor 1-alpha (HIF1α), hypoxia-inducible factor 2-alpha (HIF2α), phosphorylated extracellular signal-regulated kinase (p-ERK) and extracellular signal-regulated kinases (ERK) protein expressions in HEK293T, A375, MNT1 and SKMEL28 cells. HEK293T, A375, MNT1 and SKMEL28 cells were kept in normoxia and hypoxia (1% O2) for 24 hours, at 37°C. Cells were harvested in RIPA buffer and specific proteins were identified. Expression levels of HIF1α and HIF2α proteins were normalized to calnexin (n=3). (B) Western blotting of autophagy related proteins (LC3 and p62). Quantitative analysis of LC3-II to LC3-I protein ratio, (n=3). (C) The same experiment as in (A) was performed for cathepsin D protein expression. Densitometric quantification of cathepsin D protein bands was assessed using calnexin as internal control. Data are represented as mean ± SEM (two-way ANOVA with Sidak multiple comparisons test; **p < 0.01, *p < 0.05). (D) Box plot of log2 transformed LFQ intensity values of cathepsin D in normoxia and hypoxia, as emerge from mass spectrometry data results.