Six well plate

The previously stored MAC-T cells (17 (link)) in liquid nitrogen were resuscitated and cultured with 10 mL Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium (Hyclone, UT, USA) containing 10% fetal bovine serum (System Biosciences, Mountain View, CA, USA) in a 5% CO₂ humidified incubator at 37°C and then passaged every 2 days.
MAC-T cells were identified by immunofluorescence analysis with epithelial marker cytokeratin 18 (18 (link)). The cells were inoculated in a six-well plate (NEST, Wuxi, Jiangsu, China). When the cell confluence was 80%, the medium was discarded. The cells were rinsed three times with PBS, and fixed at room temperature for 20 min with pre-cooled 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), then added 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), and incubated at room temperature for 5 min. After blocking for 1 h at room temperature in 2% BSA. the cells were incubated with primary antibody for cytokeratin 18 (1:500, Santa Cruz, Dallas, TX, USA) overnight at 4°C. The cells was incubated with Cy3-labeled goat anti-mouse IgG (1:200, Beyotime, Shanghai, China) for 1 h at room temperature, then incubated with DAPI (Beyotime, Shanghai, China) for 10 min at room temperature, followed by seal the coverslip and imaging under fluorescence inverted microscope (Olympus, Shinjuku-ku, Tokyo, Japan).

The tumor tissue was washed 3–5 times in sterile phosphate-buffered saline (PBS) (Gibco). Under sterile conditions, it was then finely shredded using a blade and transferred into a mixed solution containing Type II Collagenase (1 mg/ml) (Gibco) and Dispase (Invitrogen) (1 mg/ml) in RPMI-1640 (Gibco), and placed on a shaking table at 37 °C for enzymatic dissociation. The supernatant was sucked out when 1/3rd of the tissue was dissociated, filtered through a 100 mesh filter and centrifuged at 300×g for 3 min, and discarded. The precipitate was washed with PBS, centrifuged again, and resuspended in complete culture medium (RPMI-1640 [Gibco] + 10% fetal bovine serum [FBS; VivaCell] + 1% penicillin–streptomycin [BI]). The suspension was then inoculated in a six-well plate (NEST) and cultured at 37 °C under an environment of 5% CO₂. The culture medium was changed every 48 h. Fibroblasts were removed using a mechanical scraping method. Cell growth was regularly monitored under an optical microscope. When the cells reached 80% conflfluency, they were passaged using 0.25% trypsin digestion. From the third generation onward, cells were passaged at a 1:2 ratio and preserved in serum-free rapid cell cryopreservation solution (Mei5 Biotechnology).

HCT116 cells were transfected with scramble or CISD2 knockdown lentivirus using six-well plates (NEST Biotechnology) at an MOI of 20, as reported earlier (Shao et al., 2021 (link)). After 24 h of infection, cells were harvested via centrifugation, resuspended in preheated complete culture medium, and administered selective antibiotics. Following a 7-day screening, CISD2 expression was gauged through immunoblotting. Verified cells were then used for subsequent experimentation.

The cells were cultured in six-well plates (Nest, Biotechnology, Jiangsu, China) overnight. The cells monolayers were wounded by scratching with plastic 10-μl micropipette tips and washed 2 times with PBS. Fresh growth medium with no FBS was added to the plates. Subsequently, the cells were transfected as described above for 48 h. Images of the different stages of wound healing were photographed via microscopy at 0, 24 and 48 h. Relative cell motility was quantified using Image-Pro Plus.