Trition x 100

Grids carrying 50 nm sections were pretreated with 0.1% Trition X-100 (Sigma Aldrich) in phosphate-buffered saline (PBS) for 10 min and blocked with 1% BSA and 0.1% fish skin gelatin (Sigma Aldrich) in PBS for 1 hr. Sections were then incubated with a primary antibody mAb414 (Covance, Princeton, NJ; RRID:AB_10063490), which recognizes four nucleoporins (Nups 358, 214, 153, and 62), for 2 hr, a rabbit anti-mouse secondary antibody (Cat. No. Z0259; Dako, Hamburg, Germany; RRID:AB_2532147) for 1 hr, and 10 nm of gold-conjugated Protein A (CMC university Medical Center Utrecht) for 30 min. The antibodies and Protein A beads were diluted in PBS with 0.2% BSA and the sections were washed for five times with PBS containing 0.2% BSA between steps. After multiple washes with PBS, sections were fixed in 2.5% glutaraldehyde in PBS for 20 min in order to immobilize the antibodies and Protein A-gold beads on sections. After washing with water, sections were post-stained with 2% UA and lead citrate for contrast enhancement. All steps were carried out at room temperature. Images were taken on a TEM (CM 120 Biotwin; Phillips, Hillsboro, OR). For specificity analysis of immuno-EM labeling, the number of gold particles on assembly intermediates and ones nonspecifically attached within 50 nm under the inner nuclear membrane was counted.

For immunofluorescence, cells were plated into 24-well plates and allowed to grow until they reached an optimal density. After incubation with TGF-β and/or 1α,25(OH)₂D₃ for 72 h, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Trition X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1.5 h and blocked with 0.5% FBS in PBS for 1 h. The fixed cells were incubated with primary antibodies of E-cadherin and Vimentin overnight at 4 °C. Then, the cells were stained with 647-conjugated secondary antibodies in the dark room for 1.5 h and washed with PBS 3 times. Nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI) for 20 min. Fluorescent labeling was analyzed using a Confocal Laser Scanning Microscope (TCS SP2, Leica, Wetxlar, Germany).

Cells were collected and fixed with 4% PFA, followed by permeabilization with 0.1% Trition X-100 (Sigma-Aldrich, USA) and block with 5% BSA in PBS, and then incubated with primary antibody against γH2A.X (1:100, CST #9718) overnight at 4 °C. Cells were dropped in glass slides and then mounted using Antifade Mountant with DAPI (Invitrogen, USA) after incubated with secondary antibody (Invitrogen, USA) and phalloidin-FITC (Beyotime Biotechnology, China). Slides were scanned and photographed using a ×100 objective on Zeiss LSM780 confocal microscope (Zeiss, Jena, Germany).

Human iPSC colonies were fixed for 30 min in 4% paraformaldehyde (VWR) and washed 3X with PBS. Fixed colonies were permeabilized with 0.3% Trition X-100 (Sigma Aldrich) throughout blocking and antibody incubation steps. Samples were incubated in primary antibodies over night at 4°C, subsequently washed with PBS and incubated in secondary antibodies for an hour at room temperature. Primary antibodies used were: anti-OCT4 (SantaCruz 1:400), anti-SOX2 (AbCAM 1:400), anti-Zo1 (LifeTechnologies 1:400), NANOG (AbCAM 1:300), anti-β-catenin (BD Biosciences 1:200), anti-EpCAM (Millipore 1:200). All secondary antibodies were used at 1:1000 and purchased from Life Technologies.