The four panels labeled A, B, C, and D depicting the effects of various inhibitors on H I F-1 and H I F-2 activity in H e p 3 B cells under different oxygen conditions. Panel A shows bar graphs of R P L 13 A m R N A expression levels in H e p 3 B cells treated with different concentrations of dual H I F inhibitors (S S 1.21, 1.21 S 9 N, S S 3.2, 3.2.16) and H I F-2-selective inhibitors (P T 2385, P T 2977) at 20 percent and 1 percent oxygen for 24 hours. The vertical axis represents R P L 13 A m R N A levels, and the horizontal axis represents the concentration of the compounds. Panel B includes two graphs: the upper panel shows relative fluorescence at different concentrations of acriflavine, and the lower panel shows the fraction bound with a derived K d estimate. Panel C presents a table comparing the binding affinities (K d) and inhibitory concentrations (I C 50) of dual H I F-1 over 2 inhibitors for H I F-1 alpha, H I F-1 alpha: H I F-1 beta, H I F-2 alpha: H I F-1 beta, C A 9 m R N A, and E P O m R N A. Panel D shows bar graphs of H I F-1 alpha and H I F-2 alpha m R N A expression levels in H e p 3 B cells treated with the same inhibitors and conditions as in Panel A. The vertical axis represents m R N A levels, and the horizontal axis represents the concentration of the compounds. The data are presented as mean plus or minus standard deviation (S D) with no significant difference (n s) versus vehicle-treated cells, analyzed using two-way A N O V A with Dunnett's multiple comparisons post-test. All data is approximate.
Molecular analyses . Related to Figs. 1, 2, and 3. (A) Hep3B cells were treated with 0–10 μM of the indicated dual HIF inhibitor (SS1.21, 1.21S9N, SS3.2, or 3.2.16) or HIF-2–selective inhibitor (PT2385 or PT2977) at 20% or 1% O2 for 24 h, and RPL13A mRNA expression was determined by RT-qPCR assays. Data are presented as the mean + SD (n = 3). ns, no significant difference versus vehicle-treated cells; two-way ANOVA with Dunnett’s multiple comparisons post-test. (B) Binding of acriflavine to fluorophore-tagged recombinant human HIF-1α was analyzed by MST. Data are presented as relative fluorescence at each concentration of added compound (nM; upper panel) and fraction bound with derived Kd estimate (mean ± SD, n = 4; lower panel). (C) Dual HIFi were characterized by: direct binding to purified HIF-1α using MST; inhibition of HIF-1α or HIF-2α dimerization with HIF-1β in cell lysates; and inhibition of HIF-1 target gene CA9 and HIF-2 target gene EPO expression in hypoxic Hep3B cells. (D) HIF-α mRNA expression was determined by RT-qPCR assays. Data are presented as the mean +/− SD (n = 3–6). ns, no significant difference versus vehicle-treated cells; two-way ANOVA with Dunnett’s multiple comparisons post-test.