Article Title: Hyperactivated mTORC1 downregulation of FOXO3a/PDGFRα/AKT cascade restrains tuberous sclerosis complex-associated tumor development
Figure Lengend Snippet: mTORC1 downregulates PDGFRɑ/AKT pathway through inhibition of FOXO3a ( A ) Tsc2−/− or Tsc1−/− MEFs were infected with lentivirus harboring a vector encoding FOXO3aTM (LV-FOXO3aTM) or the empty vector (LV). ( B ) Tsc2+/+ or Tsc1+/+ MEFs were transfected with two independent siRNAs targeting FOXO3a or the control siRNAs (siNC) for 48 h. A and B. Cell lysates and RNA were subjected to immunoblotting (upper panels) and qRT-PCR (lower panels), respectively. ( C ) Schematic representation of the putative wild-type (WT) and mutated (mut 1 and mut 2 ) FOXO3a-binding sites in the promoter of mouse PDGFRɑ gene. ( D ) HEK293T cells were co-transfected with pPDGFRɑ-Luc reporter plasmid plus FOXO3aTM expression vector (pLVX-FOXO3aTM) or control vector (pLVX) and the internal control plasmid pRL-TK. ( E ) Tsc2−/− MEFs were co-transfected with pLVX-FOXO3aTM plus pPDGFRɑ-Luc, pPDGFRɑ mut1 -Luc, or pPDGFRɑ mut2 -Luc reporter plasmid and pRL-TK plasmid. D and E. Relative luciferase activity was examined 24 h after transfection. ( F ) Tsc2−/− MEFs were transduced with LV-FOXO3aTM or LV lentiviruses. ( G ) Tsc1+/+, Tsc1−/−, or rapamycin-treated (20 nM 24 h) Tsc1−/− MEFs. F and G. Cells were subjected to ChIP assay using an anti-FOXO3a antibody. Normal rabbit IgG antibody served as the negative control. PCR was performed to amplify regions surrounding the putative FOXO3a-binding regions (PBR1 and PBR2) and a nonspecific FOXO3a-binding region (NBR). Representative data from two independent experiments were shown. ** P
Article Snippet: For the cell viability assays, Tsc1- or Tsc2-null MEFs were seeded in 96-well plates at a density of 3, 000 cells/well and treated with DMSO, rapamycin, AG1295, or combination of rapamycin and AG1295 for 48 h. Followed by added 10 μl of Cell Counting Kit-8 reagent (Beyotime, China) to per well and incubated the plates for 1 h, the optical density (OD value) of each well was measured at 450 nm.
Techniques: Inhibition, Infection, Plasmid Preparation, Transfection, Quantitative RT-PCR, Binding Assay, Expressing, Luciferase, Activity Assay, Transduction, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction