Research Paper Volume 17, Issue 7 pp 1746—1761

Figure 2. Senescent fibroblasts overexpress PGAM1 and PDP2 mRNAs to promote cellular senescence and glycolysis. (A) Fold changes in mRNAs encoding proteins involved in glycolysis and the TCA cycle. The comparison was performed in senescent HDFs and proliferating HDFs or proliferating HDFs transfected with siRNAs targeting AUF1, HuR, TTP, or TIAR and proliferating HDFs transfected with control siRNAs. (B, C) Western blot analysis of PGAM1, PDP2, p16, and Tubulin using cell lysates purified from proliferating HDFs transfected with cDNA plasmids of Flag-PGAM1 or PDP2, compared to an empty vector. The results in (B) and (C) represent three independent experiments. (D and E) Senescence-associated β-galactosidase assay using proliferating HDFs transfected with Flag-PGAM1 or PDP2 plasmids, compared to an empty vector. The graphs in (D) and (E) are averages ± standard deviations (S.D.) of three independent experiments (^*p < 0.001). (F) Colorimetric measurement of a subset of glucose metabolites using cell lysates (for 2-PG, PEP, and pyruvate) or culture media (for lactate) from HDFs transfected with control or AUF1 siRNAs. The graphs are averages ± S.D. of four independent experiments (^*p < 0.001).