Fig. 5.

Phenotypic analysis of lentiviral-mediated increases in SOX9 levels. A: schematic of lentiviral construct generated. The control lentiviral vector contains all the elements as the SOX9 lentiviral vector except the Sox9 cDNA. EGFP is translated from an internal ribosomal entry site (IRES) as a reporter gene to assess infection. The puromycin resistance (PURO) gene is included to allow positive selection for viral integration into the genome. cDNA expression is driven by strong constitutive promoters [either cytomegalovirus (CMV) or phosphoglycerate kinase (PGK)]. LTR, long terminal repeats. B: validation of the SOX9 lentivirus. 48 h postinfection, intestinal epithelial cell (IEC)-18 cells were assessed for SOX9 by immunostaining (red), and EGFP autofluorescence (green). C: IEC-18 cells were infected with equivalent titers of either control lentivirus or SOX9 lentivirus and selected with puromycin to deplete the cultures of untransduced cells. Equivalent numbers of cells were plated and allowed to grow for an additional 5 days. Images (left and middle left) depict monolayers of IEC-18 cells that emerged in the control infected cells that express low endogenous levels of SOX9 (red). Other images (right and middle right) depict cells infected with the SOX9 lentivirus. No proliferation (as detected by clonal populations) was observed in SOX9 lentivirus-infected cells. Immunostaining for SOX9 (red) validates high expression of SOX9 in these cells. D: immunoblotting analysis of whole cell extracts made from control-virus (left lane) or SOX9-virus infected (right lane) IEC-18 cells. Blots were probed for c-MYC and proliferating cell nucleus antigen (PCNA) using β-actin expression as an internal control. E: morphological phenotypes of SOX9 lentivirus-infected IEC-18 cells. Note neuroendocrine-like morphologies (axonal/dendritic processes and dense vesicle formations) were evident 6–8 days postinfection.