Image lab software ver 5

To determine the efficacy of TIG or TIG-R EVs on recurrent TIG-R, TIG-S cells (ATCC 17978) were initially cultured under different concentrations of TIG. To determine MIC, aliquots from individual samples were spotted on an LB-agar plate, followed by incubation at 37 °C (day 1; passage 0). The next day, the colonies grown at a sub-lethal concentration (0.125 µg·mL⁻¹) were further cultured with or without TIG-R EVs (5 µg) under different concentrations of TIG for 16 h in LB broth. This step was performed for 10 days (passage 9) and the recurrence efficacy of TIG or TIG/TIG-R EVs was evaluated. All LB-agar plates were imaged and processed by ChemiDoc^TM MP Imaging System (Bio-Rad, Hercules, CA, USA) and Image Lab Software (ver.5.2.1, Bio-Rad, Hercules, CA, USA).

On POD 1, skin samples (n = 6) from CVZ were collected and stored at −80 °C before Western blot analysis. Total protein was extracted using RIPA lysis buffer, supplemented with protease inhibitor (Beyotime Biotechnology, China). After homogenization and centrifugation of the samples, BCA Protein Assay Kit (Beyotime Biotechnology, China) was used to determine the protein concentrations of the supernatant. Proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gels and transferred onto the PVDF membranes. After blocking with 5% non-fat milk or bovine serum albumin for 2 hours at room temperature, the membranes were incubated overnight with primary antibodies HIF-1α (1:200, Abcam), VEGF (1:1000, Abcam), Beclin 1 (1:1000, Cell Signaling Technology), p62(1:1000, Abcam), LC3 (1:1000, Sigma), Bcl-2 (1:1000, Cell Signaling Technology), Bax (1:1000, Cell Signaling Technology), cleaved Caspase-3 (1:1000, Cell Signaling Technology) and GAPDH (1:2000, Bioworld Technology) at 4 °C. Then the membranes were incubated with goat-anti-rabbit or goat-anti-mouse secondary antibodies for 2 hours, followed by detection with ECL plus reagent kit (Thermo Fisher Scientific, Rockford, IL). Finally, the band intensity was quantified using the Image Lab software (ver.5.2, Bio-Rad).

Equal amounts of protein (30 µg) from treated and untreated Caco2 cells were electrophoresed through an SDS-polyacrylamide gel (10% resolving gel) in Tris-glycine buffer at 90 V for 2 h. The proteins were transferred from the SDS-polyacrylamide gel to Immun-Blot PVDF membrane (Bio-Rad, Hercules, CA, USA) using a wet transfer system. Membranes were developed using WesternBreeze Chromogenic Anti-Rabbit Kit (WB7105, Invitrogen, Carlsbad, CA, USA) and rabbit polyclonal primary anti-Nrf-2 antibody (sc-722, Santa Cruz Biotechnology, Heidelberg, Germany) according to the manufacturer’s instructions. The protein bands were detected by staining with the BCIP/NBT substrate and visualized with the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). Quantification of bands on Western images was done using the Image Lab software (ver. 5.2, Bio-Rad, Hercules, CA, USA). To normalize Nrf-2 levels, Nrf-2 band intensity was divided by β-actin (A1978, Sigma-Aldrich, St. Louis, MO, USA) band intensity (which serves as reference protein), and represented as percentages of control.