Figure 7.

p21^Cip1 and p27^Kip1 downregulation affects proliferation and viability of GSC-ECLs. (A) mRNA expression levels of p21^Cip1 and p27 ^Kip1 in NT-, p21^Cip1-, p27^Kip1-, or dual-siRNA–transfected G08 cells were analyzed 48 hours posttransfection by real-time RT-PCR. RPL7 expression was used as normalizer. Graph shows mRNA fold change relative to NT-siRNA transfectants, arbitrarily set as 1. (B) Representative immunofluorescent images of NT-, p21^Cip1- or p27^Kip1-siRNA transfectants exposed to CPT (1 μM, 24 hours) 24 hours posttransfection. Cells were stained with antibodies against p21^Cip1 and p27^Kip1. Nuclei were counterstained with DAPI. (C) G07, G08, and G09 cells were transfected with NT-, p21^Cip1-, p27^Kip1-, or dual-siRNA and exposed to CPT 24 hours posttransfection. Bar charts show the mean of PI-stained cells 48 hours after CPT addition. Each bar represents the mean ± S.E.M. of three independent experiments. Student's t test was used to compare p21^Cip1-, p27^Kip1-, or dual- to NT-siRNA transfectants (*P < .05). (D) Twenty-four hours posttransfection with the corresponding siRNA, G08 and G09 cells were left untreated or subjected to CPT for 24 hours. A representative flow cytometry plot is shown for each experimental condition. Dot plots show incorporation of BrdU into DNA against DNA content. The percentage of cells in S phase was determined by quantifying BrdU⁺ events.