Down-regulation of Hiwi decreases DNA-methylation and limits tumorigenic growth.

<p>(A) Verification of inducible sh-Hiwi MFH clones by immunohistochemistry. sh-Hiwi MFH clones C and E were induced with doxycycline for 3 days before IHC analysis. (B) Day 21 in osteogenic media. Calcium matrix formation measured by Alizarin Red S stain is restored in sh-Hiwi MFH clones C and E (approximately 50% of cells staining), induced with doxycycline 7 days before addition of differentiation media and continued in doxycycline-spiked media during differentiation. (C) Doxycycline-induced sh-Hiwi MFH cells at 4 weeks in colony forming assay show decreased colony formation, as compared to uninduced sh-Hiwi MFH cells. p<0.001 for clone C and p<0.005 for clone E by Student's T Test (D) Global DNA methylation is decreased in induced sh-Hiwi MFH cells. Cells were induced with doxycycline for 3 days before DNA was collected and assayed. Error bars represent standard error. * = p<0.05 by Student's T Test (E) Untreated sh-Hiwi MFH cells or treated with 1 uM 5-azacytidine at 4 weeks in colony forming assay. 5-azacytidine treatment decreases colony formation capacity. Combined p<0.01 by Student's T Test. All experiments were performed in triplicate. Representative pictures are shown.