Figure 4. 53BP1 depletion increases chromosomal instability in WI38 human fibroblasts. WI38 normal human fibroblasts were infected with one of two 53BP1 shRNA constructs (sh53BP1 A or sh53BP1 B) or a scrambled shRNA construct for 24 hours and selected in puromycin for 72 hours. (A) Representative Western blot and densitometric quantitation showing 53BP1 protein levels as a percentage of scrambled control (set as 1 on y-axis). β-actin levels are included as a loading control. (B) Quantitation of micronuclei-positive cells following infection with scrambled or 53BP1 shRNA constructs. Both control, non-damaged cells and cells damaged with 1.5ug/mL neocarzinostatin (NCS) for 2 hours are shown. Data are pooled from three replicates, and at least 400 cells were counted for each condition. Positive cells contain at least one micronucleus. Error bars indicate standard deviation. **P<0.01; ***P<0.001. (C) Representative images depicting micronuclei and 53BP1 foci in control and 53BP1-depleted human fibroblasts. Both non-damaged and damaged (NCS) cells were prepared as in (B) and stained with DAPI and anti-53BP1 (green). Images showing nuclear structure are depicted for both wild-type fibroblasts and fibroblasts infected with one of two 53BP1 shRNA constructs, and examples of micronuclei are marked with white arrows.