Figure 2
CX 3 CR1 is required for keratinocytes differentiation. (A) Expression of CX3CR1 in parental PA-1 cell line and that stably transfected with either CX3CR1 or scrambled shRNA constructs was examined with Western blot. Actin served as a loading control. CX3CR1 expression levels were quantified using digital densitometry and normalized to the levels of actin expression. (B) Expression of cytokeratin 14 in PA-1 cells stably transfected with either CX3CR1 or scrambled shRNA constructs treated with recombinant BMP-4 or vehicle, as indicated, was examined with immunocytochemistry. CK14 in PA-1 was probed with anti-CK14 and Alexa555-conjugated anti-rabbit antibodies, and DNA was stained with 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI); CK14 – red, DNA – blue. Images were taken using 5× magnification on the objective. The histogram demonstrates the average intensity of CK14 signal across the field as determined by the Zeiss AxioVision software. Data is an average of three independent experiments. *p < 0.05. (C) Expression of cytokeratins 14 and 18 in PA-1 cells stably transfected with either CX3CR1 or scrambled shRNA constructs treated with recombinant BMP-4 or vehicle, as indicated, was examined with Western blot. β-Tubulin served as a loading control. The histogram shows CK14 and CK18 expression levels. Expression of CK18 in CX3CR1shRNA-transfected PA-1 cells was arbitrarily set as 1 and expression of both CK14 and CK18 in other conditions was calculated accordingly. The data represent a typical Western blot image, and quantitative analysis was performed using three independently performed experiments. *p < 0.05. (D) Expression of CX3CL1 in SKOV-3 and PA-1 cell lines was examined with Western blot. Actin served as a loading control. SKOV-3 cell line was used as a positive control.