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. 2017 Feb 14;18(7):1699–1712. doi: 10.1016/j.celrep.2017.01.051

Figure 5.

Figure 5

PP2A/B55α Promotes Colorectal Cancer Growth in a PHD2-Dependent Manner

(A–I) Focus formation assay with DLD1 cells stably silenced for control, B55α, PHD2, or both (shCTR, shB55α, shPHD2, and shPHD2∣shB55α, respectively) (A–C), with DLD1 shCTR and shB55α stably transduced with an empty vector or a hydroxylation-insensitive HIF1α (EV, shB55α, HIF1α, and shB55α∣HIF1α) (D–F), or with DLD1 cells stably silenced for control, B55α, Atg5 or both (shCTR, shB55α, and shAtg5, shAtg5∣shB55α, respectively) (G–I). Representative images from one of three independent experiments are shown (A, D, and G). The graphs show the size (B, E, and H) and total number (C, F, and I) of colonies per well.

(J and K) In vivo growth curves of xenograft tumors derived by subcutaneous injection in nude mice of HT29 (J) or DLD1 (K) cells stably silenced for control, B55α, PHD2, or both (shCTR, shB55α, shPHD2, and shPHD2∣shB55α, respectively).

(L and M) Tumor weights of HT29 (L) or DLD1 (M) cells 24 or 23 days after tumor cell injection, respectively.

(N) Morphometric quantification of DLD1 tumor sections stained for ApopTag showing apoptosis in the PIMO-positive area of the tumor.

(O) Morphometric quantification of DLD1 tumor sections stained for LC3 and PIMO, showing autophagy in hypoxic areas of the tumor. The data combine two or three independent experiments using seven or eight mice per condition in each experiment.

(P) WB analysis for HIF1α and PHD2 from control HT29 (guide RNA [gRNA]CTRL) and HT29 cells knocked out for PHD2 by means of a gRNAPHD2 and then reconstituted with a lentiviral vector encoding PHD2WT, PHD2S125A, and PHD2S125D or an EV.

(Q) In vivo growth curve of xenograft tumors derived by subcutaneous injection into nude mice of PHD2 knockout HT29 cells (gRNAPHD2) stably expressing PHD2WT, PHD2S125A, and PHD2S125D. The data combine two independent experiments using six mice per condition in each experiment. The WB was repeated twice.

∗p < 0.05 versus all other conditions; #p < 0.05 versus PHD2S125A. The graphs show mean ± SEM. See also Figure S4.