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Fig. 2 Induction of stem cell markers in Rb1−/− MEF sublines expressing RB and S18-2. (A) Analysis of S18-2 mRNA expression in mouse ESCs and in differentiated cells using the StemMapper database. Red: mouse ESCs; green: differentiated mouse cells. (B) Analysis of S18-2 mRNA expression in iPSCs and differentiated iPSCs using the StemMapper database. Red: iPSCs; green: differentiated iPSCs. (C) Western blotting of cell extracts using the indicated antibodies. Beta-Actin (b-Actin) was used as a loading control. (D) Immunostaining with anti-SSEA4 antibody. DNA is stained blue. (E) RH18RB cells were grown in bacterial petri dishes and cultured overnight on glass slides, followed by staining with antibodies against SSEA1 and SSEA4. DNA is stained in blue. (F) Heat map of data obtained from an expression array of 84 genes associated with mouse ESCs: red indicates genes that were expressed at higher levels and green indicates genes that were expressed at lower levels in RH18, RHRB, and RH18RB cells compared with RH cells. (G) The expression of the indicated set of genes was assessed by qPCR using TBP, beta-actin, or gapdh as endogenous controls and is presented as fold change compared to the internal controls. (H) Expression pattern of stemness-related genes or proteins in human mesenchymal stem cells after siRNA treatment. (Left) qPCR of the indicated genes presented as fold change compared to GAPDH which served as the internal control. *0.03 < P < 0.05; **0.01 < P < 0.03; ***P < 0.01. (Right) Western blot analysis using the antibodies for proteins encoded by the genes analyzed in the qPCR as indicated. b-Actin was used as a loading control.