FIGURE 8.

Influence of hnRNP-B1 on ASCL1/hASH1 expression. a, Kelly cells were grown under control (21% O₂, C) or hypoxic (1% O₂, Hy) conditions for 24 h. Western blot analysis was performed to assess relative hnRNP-A2 and hnRNP-B1 protein levels. Tubulin served as a loading control. Shown is a representative Western blot and statistical quantification for hnRNP-A2 and -B1 protein levels following 24 h of hypoxia. b, ASCL1 mRNA half-life measurements following control (si-control) or hnRNP-A2/B1 (si-hnRNP-A2B1) knockdown conditions. c, schematic of the cis-element deletions within the ASCL1 mRNA 5′-UTR. The putative hnRNP-A2/B1 binding site as predicted by the catRAPID fragments module is indicated. d, Western blot analysis for hnRNP-A2/B1 after RNA pulldown using the ASCL1 mRNA 5′-UTR and the mutated variants as indicated in c. hnRNP-A2/B1 binding was severely impaired following deletion of the predicted binding site. A Coomassie Blue-stainable band served as a loading control. e, reporter gene assays following forced hnRNP-B1 expression. (For a schematic of constructs used see also Fig. 3a.) Values were normalized to Renilla activity. f, representative Western blot analysis following forced hnRNP-B1 expression under control and hypoxic conditions. g, statistical analysis for f. Asterisks indicate significant changes between mock and hnRNP-B1 transfection. Number signs indicate a significant alteration between control and hypoxia (n = 4; ***/###, p < 0.001; ##, p < 0.01).