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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: Correction to: Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells

Fig. 3

MCF-7 ALDH1A1 regulates angiogenic factor output via retinoic acid signalling. a Angiogenic factors release evaluated by ELISA plate array in supernatants of MCF-7 treated with CM037 (1 μM) for 48 h. The experiment was performed 2 times in duplicate. b MCF-7 cells were exposed to CM037 at different concentrations (1 and 10 μM) for 18 h and western blot was carried out. β-Actin was used to normalize loading. c Cells were treated with CM037 (1 μM, 18 h) and VEGF levels were measured by ELISA assay in MCF-7 conditioned media. After 18 h supernatants were harvested and cells fixed, stained and counted. The number of counted cells was not significantly different. Data are reported as pg/ml. **p < 0.01 vs untreated cells. d RT-PCR analysis of VEGF in MCF-7 Scr, MCF-7 ALDH1A1KD and MCF-7 ALDH1A1+ cultured in medium with 1% FBS for 48 h. Data are reported as ΔCt (Ct gene of interest-Ct Housekeeping gene). ***p < 0.001 vs MCF-7 Scr. ###p < 0.001 vs MCF-7 ALDH1A1KD. e Western blot analysis of VEGF and HIF-1α in MCF-7 exposed or not to CoCl2 (100 μM, 72 h, 1% FBS). β-Actin was used as loading control. Gel shown is representative of three experiments with similar results. f Quantification of blots reported in e. *p < 0.05 vs MCF-7 Scr. **p < 0.01 vs MCF-7 Scr. ### p < 0.001 vs MCF-7 ALDH1A1KD. g Soluble VEGF was detected by ELISA in media conditioned by MCF-7 cells. Cells were seeded in 24-well plates at density 3 × 104 cells/well. After 48 h the supernatants were harvested and cells fixed, stained and counted. The number of counted cells was not significantly different. Data are reported as pg/ml. **p < 0.01 vs MCF-7 Scr. ##p < 0.01 vs MCF-7 ALDH1A1KD. h HIF-1α and VEGF expression evaluated by western blot in MCF-7 ALDH1A1KD cells exposed for 48 h (1 μM) to exogenous retinoic acid. i HIF-1α and VEGF expression in MCF-7 ALDH1A1+ treated with RAR antagonist (AGN193109) and RXR antagonist (UVI 3003) for 48 h (each at 1 μM). β-Actin was used as loading control. Gel shown is representative of three experiments with similar results. j VEGF and CD133 expression in MCF-7 transiently silenced for HIF-1α. β-Actin was used as loading control. Gel shown is representative of three experiments with similar results

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