Figure 3.

Hypoxia increases CAF activation level. (a-b) CAFs viability (a) and proliferation (b) are not affected by 48 hrs culture under hypoxic conditions (1% pO2). Viability and proliferation percentages (mean ± s.d. of three independent experiments) were normalized to “100” for normoxic conditions. (c-g) Expression of the CAF activation markers FAP, tested by RT-qPCR (c) and flow cytometry (f-g), and αSMA, evaluated by fluorescence microscopy (d) or western blot (e), is strongly increased in hypoxic condition. Vimentin expression was used as control. Results are expressed as the mean ± s.d. of three independent experiments, normalized to “1” in normoxic condition (c). A representative fluorescence microscopy image for CAF2 (D; scale bar: 10 μm) or western blot (E) and a representative flow cytometry histogram for CAF1 (f) together with the mean ± s.d. of three independent experiments (g) are shown. (h) Protein secretion level was measured in CAF culture supernatants (conditioned medium; CM) under normoxic and hypoxic conditions using SDS-Page and silver staining. The intensity level of each profile is indicated. A representative image (CAF2) is shown. P values (a-c, g) were determined by unpaired two-tailed student’s t-test. (NS: non-significant; *p < .05; ** p < .01). N: Normoxia; H: Hypoxia