The image presents a series of Western blot assays examining the degradation of H I F-1 alpha and H I F-2 alpha proteins in H e p 3 B cells treated with S S 1.21 and S S 3.2 under various conditions. Panels A and B show the effects of 10 micromolar S S 1.21 or S S 3.2 on H I F-1 alpha, H I F-2 alpha, and H I F-1 beta levels at 20 percent and 1 percent oxygen for 24 hours. Panels C through G display the results of treatments with 1 millimolar D M O G and either S S 1.21 or S S 3.2 for 6 or 24 hours. Panels H and I illustrate the impact of 5 micromolar S S 1.21 or S S 3.2 in the presence of vehicle, 10 micromolar M G 132, or 10 nanomolar bafilomycin at 20 percent or 1 percent oxygen for 6 hours. Panels J and K show the effects of 5 micromolar S S 1.21 or S S 3.2 with vehicle or 500 nanomolar T A K 243 for 6 hours. Panels L and M depict immunoprecipitation assays using anti-H I F-1 alpha antibody after treatment with 5 micromolar S S 1.21 or S S 3.2 with vehicle or M G 132. Panels N and O present the expression of full-length H I F-1 alpha and deletion mutants in H e p 3 B cells treated with 5 micromolar S S 1.21 or S S 3.2. Panel P lists I C 50 values for inhibition of target gene expression in various human and mouse cancer cells treated with the indicated H I F inhibitors. All immunoblot assays were performed 2 to 4 times, and representative results are shown. All data is approximate.
SS1.21 and SS3.2 trigger degradation of HIF-1α and HIF-2α to inhibit HIF target gene expression. (A and B) Hep3B cells were treated with 10-μM SS1.21 (A) or SS3.2 (B) at 20% or 1% O2 for 24 h, and immunoblot assays were performed. (C) Cells transfected with vector encoding HIF-1α-DM (P402A/P564A) were treated with vehicle, 10-μM SS1.21 (top), or SS3.2 (bottom) for 6 h. (D–G) Cells were treated with vehicle or 1 mM DMOG and vehicle or either 10-μM SS1.21 (D and E) or SS3.2 (F and G) for 6 h (D and F) or 24 h (E and G). (H and I) Cells were treated with 5-μM SS1.21 (H) or SS3.2 (I) in the presence of vehicle (V), 10-μM MG132, or 10-nM bafilomycin (Baf) at 20% or 1% O2 for 6 h. (J and K) Cells were treated with 5-μM SS1.21 (J) or SS3.2 (K) with V or 500-nM TAK243 for 6 h. (L and M) Cells were treated with 5-μM SS1.21 (L) or SS3.2 (M) with V or MG132, and immunoprecipitation was performed using an anti-HIF-1β antibody. Inputs and immunoprecipitates were subjected to immunoblot assays. (N and O) FL HIF-1α and deletion mutants (ΔbHLH and ΔPAS; N) were transiently expressed in Hep3B cells, which were treated with 5-μM SS1.21 (O, left) or SS3.2 (O, right). (P) IC50 values are shown for inhibition of target gene expression in cancer cells treated with indicated HIF inhibitor. All immunoblot assays were performed two to four times, and representative results are shown. See Fig. S2 A for data used to calculate IC50 values. Source data are available for this figure: SourceData F3.