Heat shock protein 20 promotes sirtuin 1-dependent cell proliferation in induced pluripotent stem cells

Figure 1 Generation and molecular analysis of induced pluripotent stem cells expressing heat shock protein 20. A: Schematic diagram of the CMV-HSP20-P2A-copGFP overexpression gene construct, in which the heat shock protein 20 (HSP20) gene is under control of the cytomegalovirus promoter; B: Morphology of induced pluripotent stem cells overexpressing HSP20; C: Expression of green fluorescent protein (GFP) in passage 3; D: Expression of GFP in passage 18; E: Fluorescence enzyme-linked immunoassay image showing the expression of HSP20; F: Western blot showing the protein expression of HSP20; G: Flow cytometry analysis confirming the expression of HSP20; H: Real-time polymerase chain reaction analysis of the HSP20 gene. Data are expressed as the mean ± SD and were analyzed by the Student’s t-test, ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001.

Figure 2 Heat shock protein 20 induced proliferation in induced pluripotent stem cells. A: Cell counting of induced pluripotent stem cells (iPSCs) for 6 consecutive days. B: Methyl tetrazolium (MTT) assay showing the growth and metabolic activity of iPSCs; C: Proliferation was assessed by proliferative marker Ki67 in cultured iPSCs; D: Real-time polymerase chain reaction showing the expression of Ki67. Data are expressed as the mean ± SD and were analyzed by the Student’s t-test, ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. HSP: Heat shock protein.

Figure 3 Heat shock protein 20 induced the expression of pluripotent genes. Real-time polymerase chain reaction showed that overexpression of heat shock protein 20 (HSP20) in the transfected induced pluripotent stem cells (iPSCs) exhibited higher expression of sex determining region Y-box 2, homeobox transcription factor nanog, octamer-binding transcription factor 4, Kruppel-like factor 4, and stage-specific embryonic antigen 4 genes compared to the iPSC control. Data are expressed as the mean ± SD and were analyzed by the Student’s t-test, ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001.

Figure 4 Heat shock protein 20 stimulated expression of sirtuin-1 and proliferating cell nuclear antigen. A: Both western blot and flow cytometry analyses confirmed that overexpression of heat shock protein 20 (HSP20) in the transfected induced pluripotent stem cells (iPSCs) showed higher expression of sirtuin 1 (SIRT1); B: Real-time polymerase chain reaction showed that overexpression of HSP20 in the transfected iPSCs exhibited higher expression of the SIRT1 gene; C: Size analysis of the SIRT1 gene by agarose gel electrophoresis; D: Real-time PCR showing the expression of proliferating cell nuclear antigen (PCNA). Data are expressed as mean ± SD and were analyzed by the Student’s t-test, ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001.