Figure 2. HDHB depletion impairs homologous recombination repair in vivo.

(A) Schematic of in vivo recombination assay. I-SceI-induced double-strand break can be repaired by homologous recombination and result in short tract gene conversion (STGC) or long tract gene conversion (LTGC)/sister chromatid exchange (SCE). Arrows indicate the target positions of PCR primers used in Fig. 2G. (B) Western blotting of cell extracts with or without HDHB silencing. Cells were co-transfected with pEGFP and sorted by FACS. Quantification of reaction products was conducted by densitometric scanning, using IPLabgel 1.5. HDHB knockdown efficiencies were 75% for HDHB-shRNA-1 and 80% for HDHB-shRNA-2. (C) Cell cycle analysis of SW480 cells transfected with control shRNA or HDHB shRNA. (D) Transfection efficiency of control shRNA and HDHB shRNA into SW480 cells evaluated by GFP co-transfection and expression. (E) G418-resistant colonies formed after co-transfection of I-SceI with control shRNA or HDHB shRNA. (F) Frequency of neo-resistant colonies per 10⁴ cells. pFLAG was used as a control vector for I-SceI. The mean values ± s.d. from three independent experiments are plotted. *P<0.01, Student t-test. (G) PCR products amplified from the genomic DNA of surviving colonies were cut with NcoI and analyzed by agarose-gel electrophoresis. Lane 6 of the lower panel indicates a long tract gene conversion or sister chromatid exchange (LTGC/SCE). (H) Three silent third-codon HDHB mutations which confer resistance to shRNA-1. Red letters are the mutant nucleotides. (I) Western blotting of whole cell extracts of SW480/SN.3 cells. SW480/SN.3 cells bearing doxycycline-inducible silent mutant HDHB were transfected with HDHB shRNA-1. To induce the expression of silent mutant HDHB, different concentrations of doxycycline (Dox) were added to cell culture for 48 hours. Ctl, control cells without shRNA-1 transfection and doxycycline treatment. (J) Frequency of neo-resistant colonies per 10⁴ cells. SW480/SN.3 cells bearing doxycycline-inducible silent mutant HDHB were transfected with HDHB shRNA-1 or control shRNA, together with I-SceI expression plasmids, and then treated with or without 50 ng/ml doxycycline. The mean values ± s.d. from three independent experiments are plotted. *P<0.01, Student t-test. Doxycyline treatment rescued the reduced homologous recombination in HDHB shRNA transfected cells.