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Fig. 7. Symplekin depletion inhibits cyclin D1 expression and intestinal cell proliferation. Control and Symplekin RiT cells were grown for 4 days with (grey bars) or without (white bars) addition of tetracycline. (A) Cyclin D1 mRNA expression was quantified using qRT-PCR from total RNA, relative to the expression of GAPDH mRNA. Values are the average from three experiments performed on two symplekin-depleted clones. Since addition of tetracycline in control clones resulted in a slight increase of cyclin D1 mRNA expression, results are expressed relative to the expression in their respective (untreated or tetracycline-treated) control (*P<0.05 compared with untreated cells, Student's t-test). (B) Proliferation was determined by measuring absorbance at 595 nm after cells were stained with Crystal Violet and solubilised in 1% SDS. Shown is a representative experiment performed in quadruplicates (*P<0.05 compared with untreated clones, Student's t-test). (C) Apoptosis was measured using the TUNEL assay (Roche Diagnostics) in Sym RiT cells before (-TC) and after (+TC) treatment with tetracycline, as well as DNase-treated positive controls (pos). Bar, 20 µm.
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