Figure 2.

PHD2 Depletion Prevents Degradation of B55α in Response to Glucose Starvation

(A) WB analysis of MCF7, MDA-MB231, and SKBR3 cells transiently transfected with small interfering RNA (siRNA) specifically targeting PHD2 (siPHD2) or control (siCTR) for 48 hr.

(B and C) qPCR of RNA extracted from MDA-MB231 cells (B) and MCF7 cells (C) transfected with siRNA specifically targeting PHD2 (siPHD2) or control (siCTR).

(D and E) MDA-MB231 cells (D) and MCF7 cells (E) were cultured in complete (Mock) or glucose-deprived (GS) medium at the indicated time points. Then WCEs were collected and analyzed by WB.

(F and G) MDA-MB231 cells (F) and MCF7 cells (G) were transfected with siRNA targeting PHD2 (siPHD2) or control (siCTR). Cells were cultured in complete or glucose-deprived medium (GS) for 16 hr. WCEs were then collected and analyzed by WB.

(H–K) MDA-MB231 cells (H and J) and MCF7 cells (I and K) were cultured in complete (Mock) or glucose-deprived (GS) medium for the indicated time points. LC-MS was then performed for metabolite quantification on biological triplicates as described in Experimental Procedures. The α-KG-to-fumarate ratio in MDA-MB231 or MCF7 cells is shown in (J) and (K), respectively.

(L) MCF7 were transfected with plasmids expressing ODDD (oxygen degradation domain) alone (EV) or together with PHD2 (PHD2). After 8 hr, medium was replaced with complete or glucose-deprived medium (GS) for 16 hr. Then cells were lysed and luciferase activity was measured and normalized for protein concentration. ^#p < 0.05 versus other conditions; ^∗p < 0.05 versus Mock. All graphs show mean ± SEM.

See also Figure S2.