Research Paper Volume 16, Issue 20 pp 12977—13011

Figure 6. Reduction of Lamin B1, LBR-associated interactions in WRN-depleted cells. (A) An example of LBR and WRN IF in situ in WI38hTERT cells expressing the indicated shRNAs. Scale, 10 µm. (B) Quantitations of the indicated IF in situ analyses in the same WI38hTERT cells as in (A). Left panel, mean nuclear levels of LBR (per cell) background-corrected by the means of the entire fluorescent signal outside the nuclei (per image). Right panel, ratios of mean nuclear to mean cytoplasmic LBR IF signals per cell. Note the log scale. Red triangles are distribution means. The graphs represent four (LBR, left panel), two (LBR, right panel) and over five (WRN) independent experiments each. (C) A Western blot of WI38hTERT expressing the indicated shRNAs and probed with antibodies against NCL (internal control), LBR, and HP1α. Quantitations below the image average two independent experiments. The values were normalized to NCL and shown relative to shNS control. (D, E) Quantitations of the indicated PLA or IF in situ analyses in the same WI38hTERT cells as above. The panels represent two (LBR/Lamin B1), three (Lamin B1/H3K9me3), and three (Lamin B1) independent experiments each. Nuclear MFI values were used instead of PLA foci numbers in cases where the latter numbers were too high to robustly count individual foci. (F) ChIP qPCR analyses of LBR levels on the indicated genomic loci performed in the same cells as elsewhere in the figure. The graphs summarize results of two independent experiments.