Fig. 6.

ALDH1A1 influences tumor angiogenesis and VEGF production in vivo. a Evaluation of VEGF, HIF-1α and ALDH1A1 RNA in tumor samples. Frozen tumors were homogenized and RNA was extracted to perform RT-PCR analysis of VEGF, HIF-1α and ALDH1A1 mRNA. Data are reported as ΔCt (Ct gene of interest-Ct Housekeeping gene). Each bar is the mean of 6 different tumors. The experiment was repeated two times. *p < 0.05 vs Scr group. **p < 0.01 vs Scr group. #p < 0.05 vs ALDH1A1KD group. ###p < 0.001 vs ALDH1A1KD group. b Evaluation of VEGF and ALDH1A1 proteins in tumor samples. Tissues were harvested, homogenized and sonicated. Subsequently, proteins were extracted and western blot was performed. β-Actin was used as loading control. The experiment was repeated two times. c Evaluation of mRNA for CAIX (HIF-1α target gene) and stemness markers (SOX2, NANOG, OCT-4 and TWIST) in tumor samples. Each bar is the mean of 6 different tumors. The experiment was repeated two times. #p < 0.05 vs ALDH1A1KD group. ##p < 0.01 vs ALDH1A1KD group. ###p < 0.001 vs ALDH1A1KD group. d Evaluation of HIF-1α and stemness markers (CD133, KLF4 and SOX2) proteins in tumor samples. The experiment was repeated two times. e Quantification of blots reported in d. *p < 0.05 vs Scr group. #p < 0.05 vs ALDH1A1KD group. ##p < 0.01 vs ALDH1A1KD group. f Quantification of microvessel density by human CD31 staining (magnification 20x) was done counting 5 random fields for section, each slide having five sections. **p < 0.01 vs Scr group. ##p v 0.01 vs ALDH1A1+ group. g Representative images of immunostaining for CD31 (red) and DAPI (blue) in tumor sections from Scr (left), ALDH1A1KD (center) or ALDH1A1⁺ (right) mice. Pictures report different vessel densities in tumors. Magnification 20x. Scale bar, 50 μm. h Representative images of double-immunostaining for CD31 (red) and NG2 (green) in tumor sections from Scr (left), ALDH1A1KD (center) or ALDH1A1⁺ (right) mice. DAPI staining is blue. Magnification 40x. Scale bars, 50 μm