Figure 3. HIF-1α upregulates exosomal miR-210 and mediates Hypoxic^EXO-induced sphere formation.

(A) Western blot of HIF-1α in hypoxic A673 and SK-ES- cells transfected with Non-Targeting control (NTC) and HIF-1α-siRNA. α-Tubulin was used as the internal loading control. (B) Analysis of miR-210 expression levels in A673 and SK-ES-1 cells transfected with NTC and HIF-1α-siRNA, and (C) their respective exosomes using qRT-PCR (Mean ± SEM, n = 3, ^* P ≤ 0.02, ^** P ≤ 0.05, ^*** P ≤ 0.0002). RNU6B and miR-16 was used as a control to normalize miRNA expression in cells and exosomes, respectively. (D) Assessment of sphere formation in A673 and SK-ES-1 EWS cells cultured in normoxic conditions with 20 μg/ml normoxic and hypoxic exosomes derived from their respective cells transfected with NTC and HIF-1α-siRNA. Quantification of spheres was performed (Mean ± SEM, n = 3, ^* P ≤ 0.0004, ^** P ≤ 0.0001).