Deltavision image restoration microscope

The protocol used for ExM was as described previously (Chozinski, Halpern, et al., 2016 ). RPE-1 cells were enriched for mitotic cells by treatment with 0.1 μg/ml colcemid (Roche) for 2 h. Cells were trypsinized, washed in PBS, and cytospun onto coverslips. The cells were then fixed with 2% PFA (Sigma-Aldrich) and stained with monoclonal CENP-A antibody (Abcam ab13939) and Alexa-488–labeled secondary (Jackson ImmunoResearch 715-547-003). Stained coverslips were then cross-linked with 0.25% glutaraldehyde (Electron Microscopy Sciences 16120) for 10 min. Gelation was then performed with gelation solution (1× PBS, 2 M NaCl, 2.5% [wt/wt] acrylamide, 0.15% [wt/wt] N,N′-methylenebisacrylamide, 8.625% [wt/wt] sodium acrylate, 0.2% [wt/wt] ammonium persulfate, and tetramethylethylenediamine) for 30 min. The gelled coverslips were then subjected to protease digestion with 8 U/ml proteinase K (Roche RPROTK-RO) for 60 min at 37°C. Gels were then expanded in 50 ml of deionized water for three 30 min rounds. Gels were cut and imaged on glass-bottomed dishes (MatTek P35G-1.5-14-C) using the 60× silicone oil objective (Olympus UPLSAPO-XS) on a DeltaVision Image Restoration Microscope (GE Healthcare). Sample image z-stacks were deconvolved using SoftWorx (GE Healthcare).

HeLa cells grown on coverslips were fixed by 4% paraformaldehyde at 37°C for 10 min. Before staining by DAPI (Sigma), cells were permeabilized with 0.5% Triton and X‐100 for 5 min, then mounted on glass slides using gelvatol. Images were captured using a Delta Vision Image Restoration Microscope with a ×63 objective (DeltaVisionElite, GE).

Cells were first transfected with empty vector (EV) or Myc-tagged SENP1 for 24 hours. Thereafter, cytospins were prepared followed by fixation with 4% paraformaldehyde at the following time points: 0 minute, 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, and 48 hours. After fixation, cells were subjected to permeabilization with 0.2% Triton-X in PBS for 30 minutes at room temperature. Subsequently, cells were blocked with 0.3% Triton-X/2% BSA in PBS solution for 1 hour at room temperature. Thereafter, Myc-tag primary antibody was added at 1:2000 dilution to detect exogenous SENP1, and slides were incubated at 4°C overnight. The following day, slides were washed three times in PBS, and Alexafluor 647 fluorochrome-conjugated secondary antibody was added at 1:200 dilution for 1.5 hours at room temperature. Thereafter, slides were washed three times in PBS and counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Prolong Gold Antifade Mountant (Invitrogen) was added; slides were coverslipped and sealed with nail polish to prevent dehydration. Images were captured using Deltavision Image Restoration microscope (Olympus IX71; GE Healthcare; total magnification is × 400).