Evos fl cell imaging station

Cells transiently expressing GFP, GFP-RelA302-316, mCherry-H2B and mCherry-SETD6 were analyzed using EVOS FL cell imaging station (ThermoFisher) without fixation. For imaging of the synthetic peptide, either a biotin or a FITC tagged peptide was introduced into the cells. For the biotin tagged peptide cells were fixed with 4% PFA, permeabilized with 0.5% Triton X-100 and blocked with 10% fetal bovine serum for 30 min. Cells were then stained with a strep conjugated Alexa647 fluorophore (Jackson). Following staining, cells were mounted on slides with the DAPI fluoromount-G mounting solution (SoutherBiotech). For the FITC tagged peptide, cells were prepared same however without permebilization and staining. Images were captured using the Olympus FV1000 Inverted Confocal IX81 Microscope.

Apoptosis detection assay was performed by staining the DOX treated HCT-116 cells with Annexin V-FITC and propidium iodide (PI) using a MEBCYTO Apoptosis kit (MBL, Nagoya). Briefly, cells were collected with trypsin, washed once with PBSx1 and then stained with 2 µl of Annexin-V FITC and 1 µl PI to each sample in 500 µl of binding buffer (100 mM HEPES, 140 mM NaCl, and 25 mM CaCl₂, pH 7.4. After 15 min incubation in dark, samples were analyzed by flow cytometry (Guava® easyCyte flow cytometer). Apoptosis was defined as the total percentage of cells positive for both, PI and Annexin V. Similar protocol was used for the microscopic experiments. Briefly, cells were remained at their wells, washed once with PBSx1 followed by binding buffer addition with the mentioned volume of Annexin V and PI. Cells were incubated in dark, at room temperature for 20 min and then visualized under the microscope (EVOS FL cell imaging station Thermo Fisher).

The expression of cytokeratin-18 (CK-18) was detected by immunofluorescence to confirm the epithelial GEC phenotype. 4 × 10⁴ cells/wells were cultured on pre-coated collagen type I on sterile glass microscopic slides and incubated at 37 °C, 5% CO₂ for 24 h. Then, cells were fixed with 4% paraformaldehyde in PBS for 15 min at RT, washed three times in PBS 1X and permeabilized in 0.02% Triton X-100 for 20 min. GECs were blocked with 5% BSA and 2% goat serum in PBS for 1 h at RT. Thereafter, GECs were incubated at RT for 1 h using the CK-18 primary antibody (sc- 32329-Santa Cruz Biotechnology Dallas, TX, USA), diluted 1:100 in PBS with 5% BSA, washed three times and then incubated at RT for another hour with the Alexa Fluor 555 goat anti-mouse secondary antibody (A-21422, Thermo Fisher Scientific, USA), diluted 1:500 in PBS with 5% BSA. After washing the slides three times in PBS 1X, the samples were treated using the UltraCruz Aqueous Mounting medium with DAPI (sc-24941, Santa Cruz Biotechnology Dallas, TX, USA). Human epithelial cell lines, known as HeLa cells (ATCC® CCL-2), and human embryonic kidney 293 T cell lines (ATCC® CRL-3216) were used as positive and negative controls, respectively. The slides were analyzed using the EVOS FL Cell Imaging Station (Thermo Fisher Scientific, USA).