Journal: Cells

Article Title: Old Passengers as New Drivers: Chromosomal Passenger Proteins Engage in Translesion Synthesis

doi: 10.3390/cells13211804

Figure Lengend Snippet: Survivin accumulates in centromeric regions after irradiation. ( A ) Schematic representation of centromeric heterochromatin. In contrast to transcriptionally silenced heterochromatin, mainly characterized by Heterochromatin protein-1 (HP1) bound to the methylated lysine 9 residue of histone H3 (H3K9me), constitutive or centromeric heterochromatin additionally relies on CENP-A as a stable centromere component and CENP-B that also belongs to the centromere and is also recognized by CREST. ( B ) A431 and HeLa cells were co- transfected with plasmids coding for EGPF-HP1α (green) and Survivin-tdTomato (red), permeabilized, fixed, and subjected to confocal microscopy. DNA was stained with Hoechst (blue). Scale bar: 7.5 μm. ( C , D ) A431 cells stably expressing Survivin-GFP ( C ), as well as parental A431 cells ( D ), were irradiated with 6 Gy, fixed 30 min and 24 h after IR, permeabilized, and immunostained with CREST serum, detecting centromeric chromatin (red) for confocal microscopy. Cells in ( D ) were additionally incubated with a Survivin-specific antibody (green). DNA was stained with Hoechst (blue). Non-irradiated cells (w/o IR) were used as controls. Scale bar: 7.5 μm. The insets show higher magnifications of the areas outlined in the main panels. Representative images are shown.

Article Snippet: The antibodies used were as follows: human CREST serum (Antibodies Incorporated, Davis, CA, USA, 15-234; 1:400), rabbit anti-53BP1 (Novus Biologicals, Centennial, CO, USA, NB100-304; 1:2000), rabbit anti-Aurora B (Sigma Aldrich/Merck KGaA, Darmstadt, Germany, A5102; 1:2000), mouse anti-pATM (Santa Cruz Biotechnology, Heidelberg, Germany, sc-47739; 1:500), mouse anti-Borealin (MBL/Biozol, Eching, Germany, M147-3; 1:200), rabbit anti-CENP-C (Biozol, Eching, Germany, A06766-1; 1:100), rabbit anti-CENP-F (Novus Biologicals, Centennial, CO, USA, NB500-101; 1:750), mouse anti-cyclin A2 (Santa Cruz Biotechnology, Heidelberg, Germany, sc-271682; 1:100), rabbit anti-pDNA-PKcs (Abcam, Cambridge, UK, ab18192; 1:1000), mouse anti-DSN1 (Novus Biologicals, Centennial, CO, USA, 2A7; 1:800), mouse anti-HA (BioLegend, San Diego, CA, USA, 901501; 1:1000), mouse anti-Hec1 (GeneTex, Irvine, CA, USA, GTX70268; 1:1000), rabbit anti-INCENP (NEB/Cell Signaling, New England BioLabs, Frankfurt am Main, Germany, 2807; 1:400), mouse anti-myc (NEB/Cell Signaling, New England BioLabs, Frankfurt am Main, Germany, 2276; 1:1500), mouse anti-NSL1 (Origene, OTI4D3; 1:800), mouse anti-PCNA (NEB/Cell Signaling, New England BioLabs, Frankfurt am Main, Germany, 2586; 1:3200), rabbit anti-Survivin (Novus Biologicals, Centennial, CO, USA, NB200-201; 1:300), and mouse anti-γH2AX (BioLegend, San Diego, CA, USA, 613402; 1:10,000).

Techniques: Irradiation, Methylation, Residue, Transfection, Confocal Microscopy, Staining, Stable Transfection, Expressing, Incubation