Figure 3. MST1-AUF1 pathway inhibits mitochondrial function and mTOR activity to promote senescence. (A) Measurement of the oxygen consumption rate using proliferating HDFs transfected with control or AUF1 siRNAs after treatment with oligomycin, FCCP, and Antimycin A and Rotenone over a given time course. (B) Western blot analysis of phosphorylated 4EBP on Thr37/46 and Thr70, indicating mTOR activity, a critical regulator of glucose metabolism and senescence. The results in (A) and (B) represent three independent experiments. (C) Western blot analysis of phosphorylated MST1 on Thr183 and total MST1 in proliferating and senescent HDFs. (D) Western blot analysis of AUF1, p-AUF1, MST1, and p16 in proliferating HDFs transfected with an empty vector or the cDNA plasmid of HA-tagged MST1. (E) Western blot analysis showing the levels of p-AUF1 and AUF1 in cell lysates from proliferating and senescent HDFs. The results in (B–E) represent three independent experiments. (F) RT-qPCR levels of PGAM1 and PDP2 mRNAs from total RNAs purified from proliferating fibroblasts transfected with an empty vector or the cDNA plasmid of HA-tagged MST1 (left). Media levels of IL-6 and TNF-α isolated from proliferating fibroblasts transfected with an empty vector or the cDNA plasmid of HA-tagged MST1 (right). (G) RT-qPCR levels of Pgam1 and Pdp2 mRNAs from total RNAs purified from Mst1+/+ and Mst1−/− mouse lung fibroblasts. (H, I) RT-qPCR levels of co-purifying PGAM1 (H) and PDP2 mRNAs (I) from immunoprecipitated AUF1 in HDFs transfected with an empty vector or HA-tagged MST1, compared to IgG control precipitates. The graphs are averages ± standard deviations (S.D.) of four independent experiments (*p < 0.001). (J) Number of β-GAL-positive fibroblasts in the presence or absence of a chemical inhibitor against PGAM1, PGMI-004A (20 μM), in proliferating fibroblasts transfected with an empty vector or the cDNA plasmid of HA-tagged MST1. The graphs in (J) are an average ± S.D. of three independent experiments (*p < 0.001). (K) Schematic representation of the MST1-AUF1-PDP2/PGAM1 pathway in the regulation of glycolysis and senescence.