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. 2020 Mar 13;295(16):5335–5349. doi: 10.1074/jbc.RA119.012260

Figure 5.

Figure 5.

PRR11 controls nuclear lamina assembly via F-actin cytoskeleton organization. a, H1299 cells treated with siNC or siPRR11 were stained for Lamin B1. Representative images are shown. White arrowheads indicate the cells with irregular shape. Bars, 10 μm. b, quantification of cells of irregular shape demonstrated in a. Results are shown as the mean ± S.E. (n = 3). Greater than 50 cells were counted per condition in every repeat. c, immunofluorescence staining for Lamin B1 of cells transfected with Flag-tagged pVN173 or WT PRR11. White arrowheads indicate the WT PRR11-expressing cells with disordered nuclear lamina as shown by Lamin B1. Representative images are shown. Scale bars, 10 μm. d, quantification of cells of disordered Lamin B1 as normalized to total Flag-positive cells demonstrated in c. Results are shown as the mean ± S.E. (n = 3). Greater than 50 cells were counted per condition in every repeat. e and f, CK-666 inhibited nuclear lamina reassembly induced by WT PRR11 overexpression. e, H1299 cells were overexpressed with WT PRR11 and treated with or without CK-666 (84 μm), and then cells were fixed 24 h after and stained for Flag and Lamin B1. Representative images are shown. Bar, 10 μm. f, quantification of cells of disordered Lamin B1 as normalized to total Flag-positive cells demonstrated in e. Results are shown as the mean ± S.E. (n = 3). Greater than 50 cells were counted per condition in every repeat. g and h, effects of PRR11 N- and C-terminal truncated mutations on nuclear lamina assembly. g, H1299 cells were overexpressed with Δ1–100 or Δ290–360, and then cells were fixed 24 h after and stained for Flag and Lamin B1. Representative images are shown. Bar, 20 μm. h, quantitative analysis of aberrant Lamin B1 distribution cells demonstrated in g. Ratio indicated that the number of cells with aberrant Lamin B1 distribution is relative to the number of Flag-positive cells. Results are shown as the mean ± S.E. (n = 3). Greater than 20 cells were counted per condition in every repeat. Cell nuclei were stained with DAPI (blue). ***, p < 0.001, or N.S., not significant (p > 0.05).