Figure 3.

Silencing RNF44 in BR cells abrogates AMPK‐α1 degradation, switches metabolism to glucose addiction, and restores autophagosome formation upon arginine deprivation. BR cells were transfected with individual siRNAs against RNF44, nontargeting (NT) siRNA (50 nm), or transfection reagent alone (Veh, as a control group). Total cell lysates of these transfectants were subjected to immunoblotting (A) and immunoprecipitation of AMPK‐α1 (B). (C) A2058BR cells were transfected with RNF44 siRNAs and treated with ADI‐PEG20. Their autophagosomes and nuclei were separately stained with Cyto‐ID (green) and Hoechst 33342 (blue) and then visualized using fluorescent microscope (scale bar = 50 μm). Autophagy‐positive cells were quantitated and presented as a bar graph. (D) Cell viability of these transfectants was analyzed by MTT after treatment with ADI‐PEG20 (100 ng·mL⁻¹) or completed medium for 48–72 h. (E) Glucose uptake was analyzed by 2‐NBDG uptake with FACS. (F) CAT‐1 and CAT‐2 expressions were assessed by FACS, and data were shown in a bar graph (n = 3, *P < 0.05, **P < 0.01, and ***P < 0.005).