Figure 5

siRNA silencing of HP1β reduced UBF1/2 accumulation after UVA irradiation. (A) Recruitment of endogenous UBF1/2 (red) to locally induced, γH2AX-positive (blue) DNA lesions (yellow frames) in 3T3 cells stably expressing GFP-HP1β (green) in control sample relevant to siRNA experiments. (B) Effect of HP1β siRNA on UBF1/2 (red) accumulation after UVA irradiation by a 355-nm UVA laser. Induced DNA lesions were positive for γH2AX (blue). (C) Percentage of irradiated cells with accumulated UBF1/2. Thirty cell nuclei were evaluated for each event. Asterisks show statistically significant differences from control values (P ≤ 0.05) using Student’s t tests. (D) Efficiency of HP1β siRNA was determined by (a) Western blots showing the level of HP1β and UBF1/2. Samples were loaded according to identical total protein levels. Results of (b) qRT-PCR experiments show up-regulation of HP1β in UVA-irradiated cells. Results were obtained from two biological replicates. The transcripts were normalized to two housekeeping reference genes (GAPDH and β-actin). Irradiation was performed by UVA lamp (E) Analysis of CPD accumulation at DNA lesions in (a) control cells and (b) cells exposed to UBF siRNA. Image acquisition was performed 7 min, 15 min, 30 min, 1 h, 2 h, and 3 h after local microirradiation by a 355-nm UVA laser. For panel E, 40 nuclei were examined. C, control; Exp 1, first independent experiment; Exp 2, second independent experiment; IF, immunofluorescence; LCI, live-cell image.