Fig. 4.

MCF-7 ALDH1A1 regulates endothelial angiogenic features in VEGF dependent manner. a Viability of MCF-7 (Scr, ALDH1A1KD, ALDH1A1+) exposed to exogenous serum (10% FBS) or VEGF (2 and 20 ng/ml) at 72 h and evaluated by MTT assay. Data are reported as absorbance at 540 nm. ***p < 0.001 vs 0.1% FBS group. b MCF-7 were co-cultured with HUVEC for 48 h (1% FBS) in presence of Bevacizumab (100 ng/ml); HUVEC were fixed, stained and counted (5 fields random for well). Data are reported as number of HUVEC counted/well (n = 3). **p < 0.01 vs HUVEC co-cultured with MCF-7 Scr without Bevacizumab. ###p < 0.001 vs HUVEC co-cultured with MCF-7 ALDH1A1⁺ without Bevacizumab. §§p < 0.01 vs HUVEC co-cultured with MCF-7 Scr without Bevacizumab. ^^^p < 0.001 vs HUVEC co-cultured with MCF-7 ALDH1A1KD. c Tumor cells were co-cultured with MCF-7 for 18 h (1% FBS) in presence of Bevacizumab (100 ng/ml). Data are reported as % area of migration ratio (% of area at 18 h/area at 0 h). **p < 0.01 vs HUVEC co-cultured with MCF-7 Scr without Bevacizumab. #p < 0.05 vs MCF-7 ALDH1A1⁺ without Bevacizumab. §p < 0.05 vs HUVEC co-cultured with MCF-7 Scr without Bevacizumab. ^^p < 0.01 vs HUVEC co-cultured with MCF-7 ALDH1A1KD. d Quantification of branching points of HUVEC seeded in Matrigel layer and co-cultured MCF-7 for 18 h (1% FBS). The results represent the media of 5 pictures. **p < 0.01 vs HUVEC co-cultured with MCF-7 Scr without Bevacizumab. ##p < 0.01 vs MCF-7 ALDH1A1⁺ without Bevacizumab. §§p < 0.01 vs HUVEC co-cultured with MCF-7 Scr without Bevacizumab. ^^^p < 0.001 vs HUVEC co-cultured with MCF-7 ALDH1A1KD. e Representative pictures of HUVEC network (4x magnification). f Tumor cells were seeded at the bottom of 12-well plates with HUVEC in transwells. The cells have been maintained in co-culture until HUVEC monolayer formation in presence or not of Bevacizumab (100 ng/ml) (n = 3). *p < 0.05 vs HUVEC co-cultured with MCF-7 Scr without Bevacizumab. ##p < 0.01 vs MCF-7 ALDH1A1⁺ without Bevacizumab. §p < 0.05 vs HUVEC co-cultured with MCF-7 Scr without Bevacizumab. ^^p < 0.01 vs HUVEC co-cultured with MCF-7 ALDH1A1KD. g HUVEC were co-cultured with MCF-7 until confluent in presence, or not of Bevacizumab (100 ng/ml). Immunofluorescent images for VE-Cadherin were obtained by confocal microscope (TCS SP5 Leica). Scale bars, 50 μm