Figure 2

Suppression of H3-K9 or H3-K27 HKMT resulted in reduced cell proliferation in immortalized and transformed NHBE cells. A. Quantitative RT-PCR shows different level of knock-down efficiency for siRNAs designed specifically to each target HKMT gene in Ras transformed human bronchoepithelial cells at 48 hours after the transfection of siRNAs. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization of cDNA input. Relative expression compared to siRandom non-targeting control treated Ras-LT-hT-NHBE transformed cells. Columns, averages from duplicate wells.B.C. Colorimetric cell enumeration assay shows significant reduction of cell growth after 72 hours of the siRNA treatment of EZH2, G9A and SUV39H compared to the random non-targeting control siRNA in both immortalized (B) and Ras transformed bronchoepithelial cells (C) while interference of the other HKMTs did not affect cell growth rate in these cells. Points, averages from duplicate plates; bars, SD. D.E. Direct cell number counts after a longer incubation period shows significant reduction of cell growth with suppression of EZH2, G9A and SUV39H compared to the siRandom control in both immortalized (D) and transformed cells (E), consistent with colorimetric assay for a shorter incubation period. After a longer period of incubation, inhibitory effect of growth became more prominent in the cells interfered with EZH2 expression than cells suppressed with G9A or SUV39H expression. Meanwhile, siRNA treatment of SET9, SMYD3, SETDB1 and DOT1L did not alter the cell growth rate. (■, black filled square), siRandom, (□ empty square), siSET9, (△ empty triangle pointing up), siSMYD3, (◇ open diamond), siSETDB1, (▽ empty triangle pointing down), siSUV39H, (○ open circle), siG9A, (× cross), siEZH2, (◆ filled diamond), siDOT1L.