Fig. 2: Silencing of SENP2 causes a rapid loss of proliferative capacity and differentiation in human primary epidermal cells.

A–I were performed 7 days post-infections; J, 5 days post-infections. A Top: expression of SENP2 quantitated by qRT-PCR 5 days post-infection, relative to CT (n = 3). Bottom: number of harvested cells 7 days post-infections, relative to CT (n = 3). B Immunofluorescence for SENP2 (green). Nuclei labelled with DAPI in blue. Arrow indicates a multinucleated cell. Scale bar 50 μm. Top: immunofluorescence for SENP2 (green) and γ-Tubulin (red) in human primary epidermal cells treated with DMSO only (C), Nocodazole (D) or Taxol (E) for 24 hours. Nuclei labelled with DAPI in blue. Bottom: γ-Tubulin. Arrows for SENP2 accumulation. Arrowheads for centrosomes. Scale bar: 10 μm. F Clonal expansion capacity of CT or shSP2 cells as monitored by clonogenicity assays. 7.500 total cells were plated per well and stained 8 days after plated (n = 3). G Representative flow cytometry dot plot displaying size and complexity light scatter parameters of CT or shSP2, as indicated. HS: the fraction of cells with high scatter values, typical of terminal differentiation. H Representative flow cytometry histogram for differentiation marker involucrin (invol) of CT or shSP2. I Percent of cells with high scatter values (HS), or positive cells for involucrin, as indicated. Quantitated by flow cytometry (n = 2). J Analyses by western blotting of the expression of Involucrin in primary keratinocytes.