Figure 2. Inhibition of basonuclin expression via siRNA reduces pre-rRNA level in the HaCaT cells.

A, HaCaT cells were transfected with siRNA and controls. b, siBnc1 targeting siRNA, c, control siRNA, f, mock transfection (Lipofectamin only). Basonuclin mRNA and protein levels were monitored by PCR and Western blot at four post-transfection time points, 12, 24, 36 and 48 hours. No difference among the experimental groups was detected at 0 h (not shown). A beta-actin RT-PCR served as a control for the quantity and quality (integrity) of RNA. A protein band detected by Brilliant Blue R (B.B.R) was chosen as a loading control because of its consistent quantity in cells receiving different treatments. The protein levels shown in the Western blot were quantified in relation to the loading control (histogram). B, Immunocytochemical staining of transfected cells. HaCaT cells were cultured in chamber slides and transfected with siBnc1-s and siCb-s. Cells were fixed at 48 h post-transfection and stained with anti-basonuclin (anti-Bnc1) and anti-UBF antibodies simultaneously. Microscopic photographs in each column represent the same field, visualized by different secondary antibodies, basonuclin, red (cy5), UBF (FITC) and DNA (DAPI). C, Effect of basonuclin siRNA on the level of 47S pre-rRNA. 47S pre-rRNA was measured by RT-PCR or Northern analysis, with beta-actin and mature rRNA as references, respectively. HaCaT cells were seeded at 1.5×10⁵ cell/35 mm dish and transfected after cells were attached. RNAs were prepared at post-transfection 12, 24, 36 and 48 hours. The treatment groups b, c, and f are as described in (A). The results of Northern analysis was quantified in relation to methylene-green-stained mature rRNA (histogram), n = 3. Note that a reduction of 47S pre-rRNA level was seen only at 48 h post-transfection. M.G., methylene green. D, The effect of basonuclin knock-down could be seen only at high cell density. 47S pre-rRNA level was monitored by Northern analysis. HaCaT cells were seeded at densities: low, 2×10⁴, medium, 5×10⁴ (performed once hence not shown) and high, 1.5×10⁵ and RNA harvested at 48 h post-transfection. The relative level of 47S pre-rRNA to mature rRNA was quantified (histogram) (n = 2). Mock transfection was not performed in every experiment, hence its omission from the quantitative analysis.