Figure 5.

14-3-3η repressed LDHA expression in the presence of rotenone. (A) HEK293T cells were transfected with vector or HA-14-3-3η plasmids, and treated with rotenone (100 nM) for 48 h. Concurrent 14-3-3η overexpression and rotenone treatment induced overexpression of c-Myc and p-AMPKα, and reduced expression of LDHA. LDHA expression remained inhibited in the presence of DFO (100 μM)-induced HIF-1α upregulation. (B) HEK293T cells were transfected with vector or HA-14-3-3η plasmids, and treated with rotenone (100 nM) with or without CoCl₂ (500 μM) for 48 h. 14-3-3η decreased CoCl₂-induced HIF-1α upregulation. (C) HEK293T cell lysates were immunoprecipitated by anti-LDHA antibodies, and an interaction between HA-14-3-3η and LDHA was detected. (D) HEK293T cells were treated as indicated for 48 h. CoIP was performed with anti-FLAG antibodies. (E) HEK293T cells were treated as indicated for 48 h, and MG-132 (10 μM) was added for another 6 h. Cell lysates were immunoprecipitated by anti-LDHA antibodies, and ubiquitinated LDHA was detected. (F) HEK293T cells were treated as indicated for 48 h, and the concentration of lactate in the medium was determined. n = 3. *p < 0.05. IB, immunoblotting; IP, immunoprecipitation; WT, wild type.