Figure 5
The interference of a ROCK inhibitor with lentiviral gene transduction. (A-D) Flow cytometric analysis of keratinocyte culture on a feeder layer of 3 T3 fibroblasts in the presence (B) and absence (A) of Rho-associated kinase (ROCK) inhibitor Y-27632. Keratinocytes (5 × 105 cells) were seeded on mitomycin C-treated 3 T3 cells, and transduced with the enhanced green fluorescent protein (EGFP)-expressing lentiviral vector at multiplicity of infections (MOI) 1, with and without 10 μM of Y-27632. After seven days of cultivation, the expression levels of EGFP and ITGA6 were analyzed by flow cytometry. ITGA6-positive and -negative cells were recognized as keratinocytes and 3 T3 fibroblasts, respectively. Representative dot plot images of triplicate experiments in each experimental condition are shown. Numerical values indicate the percentage of cells in each subpopulation. (C-D) Negative controls of flow cytometric analysis of keratinocyte culture on a feeder layer of 3 T3 fibroblasts shown in Figure 3. Keratinocytes (5 × 105 cells) were seeded on mitomycin C-treated 3 T3 cells, grown for seven days, and analyzed by flow cytometry. Flow cytometric analysis of the untransduced suspended keratinocytes and 3 T3 fibroblasts incubated with only PE-conjugated secondary antibodies (C), or with PE-conjugated secondary antibodies after treatment with anti-ITGA6 antibodies (D). Numerical values indicate the percentage of cells in each subpopulation. (E) Quantitative PCR analysis of relative copy number of EGFP transgene in keratinocytes and 3 T3 fibroblasts in the presence (□) and absence (■) of 10 μM of Y-27632. (F) The number of EGFP-positive cells in the keratinocyte cultures after two days of lentiviral transduction in the presence and absence of 10 μM of Y-27632. P- value was calculated by Student’s t-test. n.s., not significant. The values (means ± SD) in E and F were determined based on results from triplicate experiments.