Fig. 8. Kazrin knockdown promotes proliferation and impairs terminal differentiation. (A) MTT proliferation assay with keratinocytes stably expressing kazrin siRNAs 426 or 1077 or the scrambled control. Data are the means ± s.d. (B-D) Flow cytometry analysis of keratinocytes stably expressing the scrambled control or kazrin siRNA426. Keratinocytes were plated in the absence of a feeder layer and incubated in high Ca²⁺ medium 4 days prior to harvesting. (B) Forward and side scatter; (C,D) labelling with antibodies to involucrin (C) or transglutaminase 1 (D). (E-F) Validation of siRNA specificity with a mutated kazrinA construct (KA-mut) that is resistant to siRNA426. (E) Keratinocytes were infected with siRNA426 or the scrambled control, and then either GFP, wild-type kazrinA (KA-wt) or KA-mut. Protein extracts were subjected to immunoblotting for kazrin or for GAPDH as a loading control. (F) The siRNA426-resistant kazrinA construct (KA-mut) rescues the decrease in differentiation in siRNA426-expressing keratinocytes plated in the absence of a feeder layer and incubated in high Ca²⁺ medium 4 days prior to analysis. The percentage of keratinocytes with low forward and side scatter is indicated.