Figure 4. Rapamycin regulates GUARDIN expression via FOSL2.

• A, B
  qPCR assays for GUARDIN expression in A549 cells treated with increasing doses of rapamycin for 24 h (A) or 100 nM of rapamycin for the indicated times (B).
• C
  Western blotting assays for p53, LRP130, FOXO4, and p21 expression in A459 and H1299 cells treated with 100 nM of rapamycin or vehicle (DMSO) for 48 h.
• D
  A549 cells with shCtrl or shGUARDIN were treated with 100 nM rapamycin or DMSO vehicle in the indicated combinations for 48 h. Western blotting was used to measure p21 levels (left) while conducting SA‐β‐gal staining in parallel (middle) with the percentage of senescent cells calculated from the SA‐β‐gal staining (right).
• E
  Mammalian two‐hybrid assays between pACT‐LRP130 and pBIND‐PGC1α in A549 cells treated with DMSO or 100 nM rapamycin. Samples were subjected to the luciferase activity assays.
• F
  SIX4 and FOSL2 mRNA (left) and protein levels (right) measured by qPCR and Western blotting, respectively, in A549 cells treated with DMSO or 100 nM rapamycin. TBP served as a control.
• G
  qPCR (upper) and Western blotting (lower) assays for GUARDIN, SIX4, and FOSL2 expression in A549 cells comparing shCtrl with shSIX4 (left), shFOSL2 (middle), or FOSL2 (right) after transduction for 48 h.
• H
  Luciferase assays in A549 cells transduced with shCtrl, shSIX4, or shFOSL2 using pGL3 (negative control) or pGL3‐FOXO4 promoter reporter plasmids.
• I
  ChIP assays comparing control IgG versus FOSL2 antibodies demonstrate specific recovery of the GUARDIN promoter by RT–PCR assay. UPe and UPk served as a positive and negative controls, respectively 54.

Data information: (A, B, D‐I) values are mean ± SEM (n = 3 biological replicates). (A, B, E–I) two‐tailed paired Student's t‐test; (D) two‐way ANOVA with Bonferroni's multiple comparison post‐test (*P < 0.05, **P < 0.01, ***P < 0.001).Source data are available online for this figure.