Lamin A/C depletion leads to Bmi1 delocalization and PRC intranuclear diffusion. (A) Representative confocal microscopy images of C2C12 MBs transfected with indicated siRNAs and immunostained using Bmi1, Ring1b antibodies, and DAPI (left), with the relative PLA experiments (right). Bar, 10 µm. Each fluorescent dot represents the colocalization of Bmi1 and Ring1b within the cells (B). n > 191 from four independent experiments. Two-tailed t test was applied for statistical analysis. SEM is indicated. Green, Alexa Fluor 488; red, Alexa Fluor 594. (C, left) Representative EM images of anti-Bmi1 immunogold labeling in C2C12 MBs transfected with indicated siRNAs (three different magnifications are shown). In the control, anti-Bmi1 antibody is specifically localized to condensed regions of heterochromatin (HE), whereas euchromatic regions (EU) are unlabeled. The gold particles are present at the border of the nucleolus (*). In lamin A/C–depleted C2C12 MBs, a significant increase of the labeling is diffused in the nucleus over HE and EU regions. Intranucleolar label (arrowheads) is also present. A few gold particles are randomly scattered in the cytoplasm (arrows). (right) Quantification of Bmi1 dot distribution in C2C12 cells transfected with siRNAs against lamin A/C or control. (top) Percentage of dots localized in the junction between heterochromatin and euchromatin (Het/Eu) or aberrantly localized in euchromatin (Eu). (bottom) Percentage of dots localized on the edge of nucleoli (outside) or aberrantly localized in nucleoli (inside) are shown. n > 84 from two independent experiments. Two-way analysis of variance was applied for statistical analysis. Statistically relevant differences (α = 0.05): *, P < 0.05; ***, P < 0.001. N, nucleus; NU, nucleolus; cy, cytoplasm; IF, immunofluorescence.