Enhanced Rb/E2F and TSC/mTOR Pathways Induce Synergistic Inhibition in PDGF-Induced Proliferation in Vascular Smooth Muscle Cells

Figures

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  Fig 1. Rb/E2F pathway regulates PDGF-induced proliferation in VSMCs. (A) Measurement of E2F luciferase activity with or without PDGF stimulation. Human VSMCs were transfected with E2F luciferase constructs, and incubated with or without PDGF for 48 hrs. The E2F luciferase activity was measured by dual luciferase reporter system with BD monolight 3010 luminometer; (B) mRNA expression of Proliferating cell nuclear antigen (PCNA), Cyclin E1 (CCNE1), Cyclin A2 (CCNA2) and Cyclin-Dependent Kinase 1 (CDK1) relative to the level of GAPDH in human VSMCs with or without PDGF stimulation for 48 hrs; (C) Western blot of human VSMCs showing decreased Rb proteins after transduction with shRNA against Rb in comparison to vector control cells. Actin serves as a loading control; (D) Luciferase measurement showing increased E2F activity after transduction with shRNA against Rb in comparison to vector control cells; (E) Cell proliferation measured by MTT assay showing increased cell growth in shRb cells in comparison to vector control cells. (F) Cell proliferation measured by cell counting showing increased cell growth in shRb cells in comparison to vector control cells. *<0.05, **<0.01.

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  Fig 2. TSC/mTOR pathway regulates PDGF-induced proliferation in VSMCs. (A) and (B) Cell proliferation measured by MTT assay showing decreased cell growth in cells treated by G418 (400ug/ml) or rapamycin (10 nM) in comparison to control cells; (C) Western blot of human VSMCs showing increased phospho-S6K proteins after stimulated with PDGF in comparison to vector control cells. S6K and Actin serves as loading controls; (D) Western blot of human VSMCs showing decreased TSC2 proteins and increased phospho-S6K proteins after transduction with shRNA against TSC2 in comparison to vector control cells. (E) Cell proliferation measured by MTT assay showing increased cell growth in shTSC2 cells in comparison to control cells; (F) Cell proliferation measured by cell counting showing increased cell growth in shTSC2 cells in comparison to control cells; (G) Cell proliferation measured by MTT assay showing decreased PDGF-induced cell growth after treatment of rapamycin. *<0.05, **<0.01.

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  Fig 3. Enhanced Rb/E2F and TSC/mTOR pathways induce synergistic growth inhibition in PDGF-induced proliferation in VSMCs. (A) Cell proliferation measured by cell counting showing cell growth is back to normal after transduction with shRNA against Rb and TSC2 in comparison to vector control cells; (B) Sequence analysis of indel mutations in targeted regions of Rb (Up panel) and TSC2 (Bottom panel) locus in A7r5 cell clones. sgRNA sequences is shown in purple and the PAM sequence is in yellow. Inserted sequence is in orange. Deleted sequence is shown in dashed line; (C) and (D) Western blot of CRISPR single clones of rat A7r5 cells showing the lost expression of Rb (C) and TSC2 (D) proteins in comparison to vector control cells; (E) Cell proliferation measured by MTT assay showing increased cell growth in cells with single Rb or TSC2 knockout in comparison to control cells; (F) Western blot of CRISPR Rb and TSC2 double knockout clones showing the lost expression of both pRb and TSC2 proteins in comparison to control cells; (G) Cell proliferation measured by cell counting showing synergistic growth inhibition in cells with Rb and TSC2 double knockout in comparison to control cells; (H)…

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  Fig 4. Enhanced Rb/E2F and TSC/mTOR pathways induce oxidative stress in VSMCs. (A) Cell stained with Dihydroergotamine (DHE) showing increased DHE fluorescence in Rb and TSC2 double knockout cells; (B) Quantification of the percentage of DHE positive cells in (A); (C) Cell stained with H2DCFDA showing increased DCF fluorescence in cells with Rb and TSC2 double knockout analyzed by flow cytometry; (D) Cell proliferation measured by MTT assay showing increased cell growth in cells with Rb and TSC2 double knockout after treated with N-acetyl cysteine (NAC, 5mM) in comparison to control cells; (E-H) mRNA expression of Sod2 (E), Catalase (F), Bcl2l1 (G) and insulin-like growth factor 1 receptor (Igf1r) (H) relative to the level of GAPDH in cells with Rb and TSC2 double knockout. **<0.01.