α il 6

The uninfected, LV-NC and LV-miR-137 groups of HTR-8/SVneo cells were plated at a density of 600-800 cells/well into a 6-well culture plate (Corning Incorporated) and cultured at 37°C in an atmosphere containing 5% CO₂, with exchange of the culture medium every other day. The uniformly plated uninfected HTR-8/SVneo cells were cultured with IL-6 antibody (α-IL-6; 1:1,000; BioLegend, Inc.) supplementary medium at 37°C, with HTR-8/SVneo cells not treated with α-IL-6 as a control group. The LV-NC and LV-miR-137 HTR-8/SVneo cells were cultured with normal and HG medium. After 10 days, the medium was removed from each well, and the plate was washed with PBS. The cells were then fixed with 4% paraformaldehyde (Biotech Well, Shanghai, China) for 10 min. Following the fixation process, crystal violet hydrate solution (Sangong Biotech, Co., Ltd.) was added for cell staining for 30 min. Finally, images of each well were captured by a camera (E-M1 Mark II; Olympus Corporation;) and colonies were counted, with a colony defined as >15 cells.

HL-1 medium supplemented as described previously (Rachitskaya et al., 2008 (link)) plus 4% heat-inactivated fetal calf serum (Hyclone) was used for all cell cultures. FACS-isolated T cell populations were plated between 2 and 5 × 10⁴ cells per well. All cultures were stimulated using plates precoated with αCD3 (3–4 µg/ml) and where indicated, 10 ng/ml recombinant murine IL-23 or 20 ng/ml IL-1β was added (R&D Systems). For neutralization experiments, we used αIL-6 (LEAF purified clone MP5-20F3) and αIL-23p19 (LEAF purified clone MMp19B2) from BioLegend at a concentration of 10 µg/ml. In addition, we used αIL-6Rα, αIL-21R, and IL-23R from R&D Systems at a concentration of 10 µg/ml. Cultures typically were harvested after 72 h.

The LV-NC and LV-miR-137 groups of HTR-8/SVneo cells were plated in a 96-well culture plate (Corning Incorporated) at the same density, and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. The uninfected HTR-8/SVneo cells were uniformly planted in several 96-well plates (Corning Incorporated), and different concentrations of IL-6 (15.0, 30.0, 37.5, 45.0 and 52.5 pg/ml; BioLegend, Inc., San Diego, CA, USA), PRKAA1 inhibitor (Dorsomorphin; MedChemExpress, Princeton, NJ, USA), PRKAA1 agonist (AICAR; MedChemExpress) and IL-6 antibody (α-IL-6; BioLegend, Inc.) were solely or jointly added to the medium for 24 h of treatment. The viability of the HTR-8/SVneo cells was determined using a CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). At the time of detection, the medium was removed and the cells were washed with PBS, following which 10 µl CCK-8 and 90 µl medium was added into each well of the plate. Following incubation at 37°C for 1-4 h in the dark, the plates were transferred to a microplate spectrometer (BioTek Instruments, Inc., Winooski, VT, USA) to measure optical density (OD) at a wavelength of 450 nm. The viability index was calculated as follows: Treatment group^{OD450 nm}/Control group^{OD450 nm} × 100%.