BIM upregulation reflects increased EGR1 activity. (A) Forty-eight hours after transfection of Jurkat cells with shRNAs targeting RAPTOR or RICTOR, knockdown was assessed by immunoblotting. (B-C) Plasmids containing the 0.8-kb BIM promoter or truncation constructs in front of firefly luciferase cDNA (C) were transfected into (B) 5 × 10⁶ log phase P388 cells. After treatment with diluent or 5 or 10 μM OSI-027, cells were assayed for firefly and Renilla luciferase activities. Panel is representative of 3 independent assays. (D-E) After Jurkat cells were treated with diluent or 10 μM OSI-027, RNAseq analysis was performed. Each mRNA count number was normalized to counts per million. Error bars in panel E, mean ± range of 2 independent assays. (F-G) After treatment with diluent, 10 μM OSI-027, 1 µM MLN0128, or 10 nM rapamycin, cells were harvested for (F) qRT-PCR and (G) immunoblotting. (H) Cells transfected with 40 µg empty vector or dnEGR1 construct along with 10 µg BIM promoter luciferase and 5 µg pTK-Renilla constructs were treated with diluent or 10 µM OSI-027 and assayed for luciferase activities. In panel H, *P = .011. (I-J) Cells were transfected with empty vector or dnEGR1 construct, treated with diluent or 10 µM OSI-027, and harvested for (I) qRT-PCR and (J) immunoblotting. Results of drug-treated samples were normalized to corresponding diluent-treated controls, which were set to 1. In panel I, *P = .02. (K) After treatment with diluent, 10 μM OSI-027, or 10 nM rapamycin, cells were harvested for ChIP assay. (L) DNA samples from ChIP assay subjected to qPCR. Results are expressed as relative % of input DNA precipitated. In panel L, *P ≤ .002 relative to dimethylsulfoxide or rapamycin. Diagram at right shows possible EGR1 binding sites in BIM promoter, with proximal site (−29 to −18) in larger font.