Fig 2: Schematic presentation of the human SUN1 isoforms annotated under O94901 at UniProt database as well as other relevant sequences deposited into GenBank. A novel isoform identified in this work is shown at the bottom of the schematic. The exon numbers are annotated in Ensembl database for SUN1-001 transcript (ENSG00000164828). The exons are shown as boxes with the corresponding number. The exons 10-19 are presented as a dash line as they are identical for all the isoforms containing the C-terminal half. A vertical bar in exon 6 represents the 10-aa peptide missing in the canonical isoform-1 that was identified during phosphoproteomics analysis by93 [Color figure can be viewed at wileyonlinelibrary.com]

Fig 3: Cloning of the LBR and SUN1 fragments generated from cDNA of HGPS T08 cells for sequencing analysis. Schematics of the in silico designed DNA fragments generated using primers designated by the arrows (A). The expected sizes of DNA fragments are shown in numbers of base-pairs. (B) Gel electrophoresis analysis of LBR fragments (lanes 2, 3, 4) and the SUN1 fragments (lanes 5, 6, 7) amplified from cDNA of T08 cells using the pairs of primers indicated above the lanes. Lanes 1 and 8 are the DNA molecular weight markers, BIOLINE Hyperladder II and I, respectively. The sizes of the DNA markers in base-pairs are shown on the left and the right side

Fig 4: SUN1 depletion increases APB formation in TOP3a-knockdown cells. (A) U2OS and VA13 cells were infected with control (shLuc), shSUN1, shTOP3a, or shSUN1 combined with shTOP3a lentiviruses and selected for 3 days. Cell lysates were analyzed by immunoblotting with anti-SUN1, anti-TOP3a, and anti-GAPDH antibodies. The arrowhead indicates the location of the TOP3a protein. 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), as shown in Figure 1. 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). N.S., no significance.

Fig 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 (?Coil), 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.