Fig. 6. Analysis of the modulation on NRP1 protein levels by an in vitro hypoxic microenvironment.

Hypoxia (Hx) was induced by incubating HCC cell lines with 100 µM CoCl₂ for the corresponding period of times. Protein expression of NRP1 was analyzed by (a) Western blot and (b) ICC. Bar graphs from (b) represent the NRP1 CTCF ratio. Magnification 63× and scale bar 10 µm. *P < 0.05, **P < 0.01, ***P < 0.001 vs normoxia (Nx). c NRP1 expression was also determined by Western blot in normoxia (Nx) and after hypoxia induction and/or autophagy inhibition by treatment with 100 nM bafilomycin A1 (Baf). *P < 0.05, **P < 0.01, ***P < 0.001 vs Nx; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 Hx+Baf vs Hx for each time point. d Autophagy was evaluated through analysis of autolysosome cell content by acridine orange staining and fluorescence microscopy. Magnification 40×, scale bar 25 µm. Bar graphs represent the quantification of red/green CTCF ratio (n = 5). ***P < 0.001 vs Hx for each time point. e Protein levels of p62/SQSTM1 and LC3 (LC3-I and LC3-II) were analyzed by Western blot. LC3 turnover assay was performed to determine the autophagic flux index. *P < 0.05, **P < 0.01, ***P < 0.001 vs Nx; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 Hx+Baf vs Hx for each time point. Data from (a), (b), (c) and (e) are represented as mean values of arbitrary units (a.u.) ± SD (n = 3)^, showing a representative immunoblot.