Figure 1.

Morphological changes and gene expression analyses of BMI1 and GAPDH in RNA interference (RNAi)-treated and scrambled (scr)-treated control cells. (A) Representative morphological changes of SH-SY5Y neuroblastoma (NB) cells (a), HCN-2 cortical neurons (b), NCCIT embryonic carcinoma cells (c), and H1 ES cells (d) 3 days after RNAi treatment (magnification: × 200). All cells were grown following providers' guidelines. For all experiments, the cells were seeded at 5 × 10⁴ cells/ml in fresh medium. To generate dsRNA targeting BMI1 mRNA, a DNA fragment was PCR amplified from human leukemic cell cDNA using primer sequences specific for the BMI1 gene (see below) but with an addition of T7 primer sequences at the 5′ end of both forward and reverse primers. The amplified DNA fragment was used to generate dsRNA targeting BMI1 mRNA using the MEGAscript RNAi kit (Ambion) following the manufacturer's instructions. Purified dsRNA was transfected into cultured cells (8 μg dsRNA/10⁶ cells) using the X-tremeGENE siRNA Transfection Reagent (Roche Applied Science). For RNAi specificity control, an equal amount of mixed pool of scrambled (scr) siRNA molecules (commercially available from Ambion) was used for transfection in place of the BMI1 dsRNA. Detection of alkaline phosphatase activity was performed using a semi-quantitative Alkaline Phosphatase Histochemical Staining system (Sigma Diagnostics) according to the manufacturer's instructions. (B) Representative gene expression analysis of BMI1 and GAPDH in RNAi-treated and scr-treated control EC cells, normal ES cells, NB cells, and normal neurons. Extraction of total cellular RNA and cDNA synthesis were performed as described previously (Liu et al., 2004). cDNA was amplified with primers specific for either BMI1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PCR conditions were: 94°C for 4 min, 28–30 cycles of 94°C for 45 s, 56°C for 30 s, 72°C for 40 s, and a final extension step at 72°C for 5 min. The primer sequences for BMI1 are 5′-GTCCAAGTTCACAAGACCAGACC-3′ and 5′-ACAGTCATTGCTGCTGGGCATCG-3′. GAPDH primers were the same as used previously (Liu et al., 2004). (C) Representative Western blotting analysis of BMI1 and GAPDH in RNAi-treated and scr-treated control EC cells, NB cells, and normal neurons. Western blotting was performed using primary antibody against GAPDH (Santa Cruz Biotechnology) and monoclonal antibody against BMI1 (Abcam). Bound antibody was detected with the Immun-Star™ HRP chemiluminescent system following the manufacturer's instructions (Bio-Rad).