Research Paper Volume 13, Issue 7 pp 10490—10516

Nuclear envelope tethering inhibits the formation of ALT-associated PML bodies in ALT cells

The enhancement of nuclear envelope anchorage inhibits APB formation. (A) Schematic diagrams of cells overexpressing SUN1, RAP1-RCT-domain-deleted-SUN1 (RAP1ΔC-SUN1), or RAP1-SUN1 fusion chimera protein are shown. NE, the nuclear envelope. (B) U2OS and VA13 cells were infected with lentivirus expressing the empty vector control (EV), SUN1, RAP1ΔC-SUN1, or RAP1-SUN1 fusion and then selected in medium containing G418 for 5 days. Cell lysates were analyzed by immunoblotting with anti-RAP1, anti-SUN1, and anti-GAPDH antibodies. The arrowhead indicates the RAP1-SUN1 fusion protein. The arrow indicates endogenous SUN1. The asterisk indicates endogenous RAP1. The ladders under the major protein band show possible products of protein degradation. GAPDH was used as the loading control. (C) Representative images show the colocalization of TRF2 and PML in U2OS cells (upper panel) and VA13 cells (bottom panel), as shown in Figure 1. Scale bar, 20 μm. (D) Quantification of APBs (%) in the U2OS and VA13 cells shown in (C). Approximately 200-300 cells were analyzed for each independent experiment. Error bars denote SD; n=3 (independent experiments); *P

Figure 2. The enhancement of nuclear envelope anchorage inhibits APB formation. (A) Schematic diagrams of cells overexpressing SUN1, RAP1-RCT-domain-deleted-SUN1 (RAP1ΔC-SUN1), or RAP1-SUN1 fusion chimera protein are shown. NE, the nuclear envelope. (B) U2OS and VA13 cells were infected with lentivirus expressing the empty vector control (EV), SUN1, RAP1ΔC-SUN1, or RAP1-SUN1 fusion and then selected in medium containing G418 for 5 days. Cell lysates were analyzed by immunoblotting with anti-RAP1, anti-SUN1, and anti-GAPDH antibodies. The arrowhead indicates the RAP1-SUN1 fusion protein. The arrow indicates endogenous SUN1. The asterisk indicates endogenous RAP1. The ladders under the major protein band show possible products of protein degradation. GAPDH was used as the loading control. (C) Representative images show the colocalization of TRF2 and PML in U2OS cells (upper panel) and VA13 cells (bottom panel), as shown in Figure 1. Scale bar, 20 μm. (D) Quantification of APBs (%) in the U2OS and VA13 cells shown in (C). Approximately 200-300 cells were analyzed for each independent experiment. Error bars denote SD; n=3 (independent experiments); *P<0.05 (two-tailed Student’s t-test). N.S., no significance.