Extended Data Fig. 3: Displacement of H3K9me2 from the nuclear periphery is reversible.
From: The nuclear periphery confers repression on H3K9me2-marked genes and transposons to shape cell fate
Immunofluorescence of H3K9me2 localization compared to the INM protein (LAP2β) in lamin + LBR QKO mESCS expressing Halo-NLS (a), Halo-LBR (b), Halo-LBR nucleoplasmic domain (NP) (c), and Halo-LBR transmembrane domain (TMD) (d). Central z-slices (XY) shown. Scale bar, 5 μm. (e) Radial intensity analysis of H3K9me2 position in QKO + Halo-NLS (n=27), QKO + Halo-LBR (n=16), QKO + Halo-TMD (n=17), and QKO + Halo-NP (n=26) 2 days after electroporation of plasmids. ** p < 0.05, Halo versus. Halo-LBR, shells 12–19; **** p < 0.0001, Halo versus. Halo-LBR, shells 22–25, unpaired two-sided t-test. ns, p > 0.05, Halo versus. Halo TMD and Halo versus. Halo NP in all shells. Points indicate mean and error bars indicate 95% confidence intervals. (f) Western blot showing LBR knockdown in lamin TKO mESCs expressing doxycycline-inducible LBR miR-E. Immunofluorescence of H3K9me2 (cyan) and LBR (magenta) in lamin TKO mESCs expressing LUC miR-E (g) or LBR miR-E (h). Scale bar, 5 μm. (i) Radial intensity analysis of H3K9me2 position in TKO + LUC miR-E untreated (NT) (n=107), TKO + LUC miR-E + dox 2d (n=140), TKO + LBR miR-E NT (n=102), TKO + LBR miR-E + dox 2d (n=83), unpaired t-test. **** p < 0.0001, 2d LBR versus 2d LUC miR-E, shells 1–13, 16–21, 23–25. **** p < 0.0001, LBR NT versus 2d LBR miR-E, shells 11–20, 22–24, unpaired two-sided t-test. Points indicate mean and error bars indicate 95% confidence intervals. (j) Radial intensity analysis of H3K9me2 position in TKO + LUC miR-E NT (n=107), TKO + LUC miR-E + dox 4d (n=97), TKO + LBR miR-E NT (n=102), TKO + LBR miR-E + dox 4d (n=95), unpaired two-sided t-test. **** p < 0.0001, 4d LBR versus 4d LUC miR-E, shells 14–20, 22–25. **** p < 0.0001, LBR NT versus 4d LBR miR-E, shells 4–20, 22–24, unpaired two-sided t-test. Points indicate mean and error bars indicate 95% confidence intervals.
