Research Paper Volume 13, Issue 7 pp 10490—10516

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

SUN1 knockdown induces APB formation and C-circle levels. (A) U2OS and VA13 cells were infected with control (shLuc) or shSUN1 lentivirus and selected with 1 μg/ml puromycin for 3 days. Cell lysates were subjected to immunoblot analysis with anti-SUN1 and anti-GAPDH antibodies. GAPDH was used as the loading control. (B) Representative images show the colocalization of TRF2 and PML in U2OS cells (upper panel) and VA13 cells (bottom panel). Virus-infected and puromycin-selected cells were subjected to immunofluorescence staining with anti-TRF2 and anti-PML antibodies. DNA was stained with DAPI. Cells containing at least three large TRF2 and PML colocalization foci (yellow) in the nucleus were counted as APB-positive cells. Scale bar, 20 μm. (C) Quantification of APBs (%) in the U2OS and VA13 cells shown in (B). Approximately 200-300 cells were analyzed for each independent experiment. Error bars denote SD; n=3 (independent experiments); *PD) Depletion of SUN1 stimulates the formation of C-circles in U2OS cells. (E) Quantification of the level of C-circles in the cells in (D). The signals were quantified with ImageJ software. The level of C-circles is represented in an arbitrary unit (a.u.). Error bars denote SD; n=3 (independent experiments); *P

Figure 1. SUN1 knockdown induces APB formation and C-circle levels. (A) U2OS and VA13 cells were infected with control (shLuc) or shSUN1 lentivirus and selected with 1 μg/ml puromycin for 3 days. Cell lysates were subjected to immunoblot analysis with anti-SUN1 and anti-GAPDH antibodies. GAPDH was used as the loading control. (B) Representative images show the colocalization of TRF2 and PML in U2OS cells (upper panel) and VA13 cells (bottom panel). Virus-infected and puromycin-selected cells were subjected to immunofluorescence staining with anti-TRF2 and anti-PML antibodies. DNA was stained with DAPI. Cells containing at least three large TRF2 and PML colocalization foci (yellow) in the nucleus were counted as APB-positive cells. Scale bar, 20 μm. (C) Quantification of APBs (%) in the U2OS and VA13 cells shown in (B). 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). (D) Depletion of SUN1 stimulates the formation of C-circles in U2OS cells. (E) Quantification of the level of C-circles in the cells in (D). The signals were quantified with ImageJ software. The level of C-circles is represented in an arbitrary unit (a.u.). Error bars denote SD; n=3 (independent experiments); *P<0.05 (two-tailed Student’s t-test).