Bromodeoxyuridine (brdu)

Cell viability and proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS, G3580, Promega) and 5-bromo-2'-deoxyuridine (BrdU, #6813, Cell Signaling) assays, respectively. Cells were plated in 96-well plates at a density of 3,000 - 4,000 cells/well in complete medium for 24 h. Cells were then treated with respective agents the following day. All samples were assayed in quadruplicates.

To determine the growth ability of cells, indicated NSCLC cells were plated into 12-well plates for 1 × 10⁴ cells/well and the cell numbers were subsequently counted each day using an automatic cell analyzer Countstar (Shanghai Ruiyu Biotech Co., China, IC 1000). For BrdU incorporation, cells were seeded into 8-well plate for 24 h culture. Cells were treated with 10µM BrdU (Abcam) for 20 min and then fixed with 5% PFA. After permeabilization, cells were incubated with BrdU (Cell Signaling Technology) primary antibody overnight and secondary antibody (Thermo). The nucleus was stained with DAPI. In the colony formation assay, indicated cells were seeded into 6-well plate with 400 cells/well, and the medium was changed every 3 days for 2 weeks. After which, cells were fixed with 5% PFA and stained with crystal violet for images capture and counting.

Fixed and paraffin tissue sections were processed as described before [41 (link)]. The following primary antibodies were used: αSMA (Abcam, Cambridge, MA, USA), YAP (Santa Cruz Biotechnology, Dallas, TX, USA), V2R (Millipore Sigma, St. Louis, MO, USA), BrdU (Cell Signaling Technology, Danvers, MA, USA), and V2R (#V5514) from Sigma Millipore (St. Louis, MO, USA). For IHC, secondary antibodies were applied, followed by incubation with Streptavidin HRP conjugate (Invitrogen, New York, NY, USA) and slides were developed with DAB (Vector Laboratories, Burlingame, CA, USA) and counterstained with Harris Haematoxylin, dehydrated, and mounted with Permount (Fisher Scientific, Fair Lawn, NJ, USA). For IF, goat anti-Rabbit IgG fluor and Goat anti-mouse IgG Texas red (Invitrogen, New York, NY, USA), secondary antibodies were applied, incubated, washed with PBST, and stained with DAPI. Slides were mounted with Flour-G (Invitrogen, New York, NY, USA) and sealed with nail polish. All images were captured using a Nikon 80i upright microscope (Tokyo, Japan) in the KUMC Imaging Center.

hMSCs or IPCs were fixed in 4% paraformaldehyde, treated with 3% H₂O₂ and blocked with horse serum, then incubated with monoclonal antibodies against specific CD markers, insulin, PDX-1 or c-peptide (diluted 1:1000) and then incubated with the relevant IgG conjugated with fluorescence CY3 or FITC (1:200). The cell clusters from three independent inductions were digested and re-cultured for 24 h in a lysine coated 24-well plate before fixation. Cells were then stained with anti-human c-peptide and PDX-1 antibodies, followed by incubation with the relevant secondary antibodies labeled with CY3 or FITC.
For staining of tissue sections, slides were deparaffinized in xylene and rehydrated in graded alcohol solutions. They were then treated with retrieval solution (Dako, Carpinteria, CA) and incubated with the primary antibodies including BrdU, PCNA, insulin and PDX-1 (1:1000, Cell Signaling) and then with the secondary antibodies of CY3 or FITC. Fluorescence signals were then detected by laser scanning confocal microscopy (Olympus FV500, Japan).