Figure 1.
Cell cycle transition of conditionally reprogrammed HFKs.
A, HFKs at passage 10 were grown in the presence of either Y-27632 (Y), J2 cells (J2) or J2 cells and Y (J2+Y). Conditional reprograming of HFKs increased G2-M transition. FACS indicated that the combination of J2 cells and Y (J2+Y) increased the percentage of cells in G2-M to 32.3%. B, Co-culture of HFKs with J2 cells maintained the level of cell cycle regulators. J2 cells up-regulated cyclins A and E, MCM4 and pCDK1, as well as the stem cell marker p63 vs. Y alone, and reduced expression of the squamous epithelial marker Involucrin.
Figure 2.
Gene expression signature of conditionally reprogrammed HFKs.
A, Heatmap of the top 30 up- and down-regulated genes based on the changes in the ratio of Y/F, J2/F and J2Y/F. B, Gene expression signatures related to the effects of Y, J2 or J2+Y. A list of changes in gene expression is in Table C in S1 File.
Figure 3.
Functional siRNA library screening of J2 cells for secreted factors.
J2 cells were grown in the presence of Y, and transfected with a library of 332 siRNAs. The growth response of HFKs to the each conditioned medium (CM) resulting from each RNA ‘knockdown’ was evaluated two days after transfection by colony assay. Listed are genes whose ‘knockdown’ resulted in ≥50% reduction in growth.
Figure 4.
Post-translational changes associated with conditional reprogramming.
A, Heatmap of the post-translational changes occurring in HFKs after 2 days in culture with J2 cells (J2), Y or J2 cells and Y (J2+Y). Shown are proteins that were modified ≥50% by each condition as determined by RPPA analysis. B, Summary of RPPA analysis. Co-culture of HFKs with J2 cells and Y preferentially increased pAkt, and reduced phosphorylation of 13 proteins, including Smad2. C, Western analysis of conditionally reprogrammed HFKs. J2 cells prevented the reduction of pSmad1/5, pSmad2, TGF-β receptor II (TβRII), nuclear β-catenin, c-Myc, EGFR, ERBB2, VEGFR2, pSrc, pERK, pGSK3β, eIF4G and p4EFBP by Y.