Figure 6. Overexpression of GPC1 increases proliferation of T24 cells.

(A) Overexpression of GPC1 as measured by immunofluorescence microscopy and slot blot assays. T24 cells were transfected with no vector (Mock) or a GPC1 overexpression vector containing a GFPSpark (GFP-GPC1) as indicated in the images. After fixation with acetone and counterstaining with DAPI (to visualized the cells nuclei, blue), the overexpression of GPC1 was detected by fluorescence microscopy (GFP-GPC1). Representative cells are shown for Mock and GFP-GPC1. Exposure times were the same in all cases. Bar, 10 μm. Insets in (A), cell extracts containing the same amounts of protein were blotted onto PVDF membranes and probed using anti-GPC1 primary antibody followed by horseradish peroxidase-conjugated anti-rabbit IgG. Loading consistencies were controlled and adjusted after probing with β-tubulin using anti-β-tubulin primary antibody followed by horseradish peroxidase-conjugated anti-mouse IgG. GPC1 signals in the blots were quantified by densitometry. The amount of immuno-reactive GPC1 was significantly increased in GFP-GPC1 transfected cells compared to the Mock (Student’s t-test, two-tailed unequal variances, N = 5, ^** P ≤ 0.01). Values shown are means ± SE. (B) Effect of overexpression of GPC1 on proliferation of T24. T24 cells were transfected with GFP-GPC1 or Mock (no vector). Untreated cells containing only medium were included as control. The cell density was determined after 4 days of proliferation. The relative cell number was calculated as % of untreated cells. The results are presented in graphs where n = 5. The data points are shown as the means ± SE. Proliferation of T24 cells significantly increased in the GFP-GPC1 transfected cells compared to the Mock (Student’s t-test, two-tailed unequal variances, N = 5). Error probabilities of P ≤ 0.05 were considered statistically significant. Indication of P-value summaries: ns P > 0.05 and ^*** P ≤ 0.001.