Fig. 4. HIF-1α activity is not affected by RACK1 levels. a) HCT116 cells were reverse transfected with RACK1 siRNA, which led to a 92-94% decrease in RACK1 mRNA expression. b) RACK1 protein levels decrease 68% following RACK1 siRNA treatment. c) RACK1 knockdown does not restore HIF-1 mRNA expression for either CA9 or LDHA (d) in HCT116 cells. e) RACK1 knockdown does not restore LDHA protein expression. f) HEK293T cells were reverse transfected with RACK1 siRNA, which led to a 72-79% decrease in RACK1 expression. g) RACK1 protein levels in HEK293T cells following RACK1 siRNA treatment. RACK1 knockdown does not restore HIF-1 gene expression for (h) VEGFA, (i) SLC2A1, (j) CA9, (k) LDHA, or (l) HK2 in HEK293T cells. m) Protein levels of HK2 or LDHA are not affected by RACK1 knockdown in normoxia. mRNA results are the mean ± SEM of independent experiments run in duplicate (n ≥ 3). mRNA data normalized to 18s RNA. Data analyzed using an ordinary one-way ANOVA with multiple comparisons to compare control siRNA to RACK1 siRNA in a given condition for each graph (Graphpad Prism). Statistically significant results are indicated as follows: ** = p <0.01 vs siRNA control, *** = p <0.001 vs siRNA control and **** = p <0.0001 vs siRNA control.