Fig. 4.

Punicalagin promotes BCL2 phosphorylation and consequently the dissociation of the BCL2-BECN1 complex through the ROS-JNK pathway. (A) HeLa cells treated with 140 μM punicalagin or dimethyl sulfoxide (DMSO; control) and 10 mM N-acetyl-L-cysteine (NAC) for 4 h were stained with 10 μM of 2′,7′-dichlorofluorescein diacetate and subjected to flow cytometric analysis. (B) The mTOR, phosphorylated mTOR (p-mTOR), JNK, and phosphorylated JNK (p-JNK) levels in HeLa cells treated with 140 μM punicalagin for 2, 4, or 6 h were detected using western blotting. (C) The phosphorylated and total BECN1, BCL2, and total PIK3C3 levels in HeLa cells treated as in B were determined using western blotting. (D) The p-BCL2 levels in HeLa cells treated with 140 μM punicalagin or DMSO (control) or the combination of 20 μM SP600125 treatment for 1 h and punicalagin treatment for 6 h were detected using western blotting. (E and F) The lysates of HeLa cells treated with 140 μM punicalagin or DMSO (control) for 6 h were subjected to immunoprecipitation with anti-BECN1 (E) or anti-BCL2 (F) antibodies. Additionally, the levels of BECN1 and BCL2 in the immunoprecipitate were analyzed using western blotting. All experiments were repeated three times.