Figure 7.

TRAP1 Favors Tumor Growth through Succinate-Dependent Stabilization of HIF1α

(A) Western immunoblot showing HIF1a stabilization in SAOS-2 cells kept either in culture or in focus-forming conditions in the presence of the cell-permeable succinate analog dimethyl succinate (20 mM, 48 hr). Extracts from focus-forming assays were obtained at the 15^th experimental day (i.e., 1–2 days before cells that did not form foci massively underwent death). Blots were probed with an anti-GAPDH to check for protein load. Cells are dubbed as in previous figures with respect to TRAP1 expression.

(B) Soft agar experiments performed on SAOS-2 cells treated with dimethyl succinate (5 mM). Data indicate the total colony area at the 25^th experimental day.

(C–F) Focus-forming assays on SAOS-2 cells (C) or MEFs (D) grown with or without TaKG and on SAOS-2 cells in which HIF1α (E) or HIF1β (F) expression had been knocked down by RNAi. Data are reported as in Figure 1A. In (E), CoCl₂ treatment is used to maximize HIF1α expression. In (F), knocking down of HIF1β is obtained with a mixture of three different shRNAs. Bar graphs report mean ± SD values (n ≥ 3); ^*p < 0.01 with a Student’s t test analysis. Cells are dubbed as in previous figures.