Research Paper Volume 13, Issue 7 pp 10490—10516

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

Disruption of the interaction between RAP1 and SUN1 does not interfere with APB formation in ALT cells. (A) U2OS cells were infected with the knockdown control (shLuc) or shRAP1 lentivirus and simultaneously complemented with control (EV), wild-type RAP1 (WT), RAP1 coil deletion (ΔCoil), nonphosphorylatable (8FA), or phospho-mimetic (8DE) RAP1 mutant lentiviruses. Virus-infected cells were selected for 5 days and subjected to further methionine restriction for 3 days. Cell lysates were analyzed by immunoblotting with anti-RAP1 and anti-GAPDH antibodies. The arrowhead indicates the location of endogenous RAP1, and the multiple lower-molecular-weight bands are degraded RAP1. Asterisk (*), RAP1 coil deletion mutant. GAPDH was used as the loading control. (B) Quantification of APBs (%) in the U2OS cells shown in (A). Approximately 200-300 cells were analyzed for each independent experiment. Error bars denote SD; n=3 (independent experiments); *P

Figure 5. Disruption of the interaction between RAP1 and SUN1 does not interfere with APB formation in ALT cells. (A) U2OS cells were infected with the knockdown control (shLuc) or shRAP1 lentivirus and simultaneously complemented with control (EV), wild-type RAP1 (WT), RAP1 coil deletion (ΔCoil), nonphosphorylatable (8FA), or phospho-mimetic (8DE) RAP1 mutant lentiviruses. Virus-infected cells were selected for 5 days and subjected to further methionine restriction for 3 days. Cell lysates were analyzed by immunoblotting with anti-RAP1 and anti-GAPDH antibodies. The arrowhead indicates the location of endogenous RAP1, and the multiple lower-molecular-weight bands are degraded RAP1. Asterisk (*), RAP1 coil deletion mutant. GAPDH was used as the loading control. (B) Quantification of APBs (%) in the U2OS cells shown in (A). 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.