Collagen 1

The human laryngeal epithelioma cell line HEp-2 (ATCC CCL-23; American Type Culture Collection, USA), the human ileocecal carcinoma cell line HCT-8 (ATCC CCL-244), and the human colonic carcinoma cell line T84 (ATCC CCL-248) were used for investigating STEC adhesion profiles. The three cell lines were grown with DMEM supplemented with 10% fetal bovine serum (FBS; Lonza) and antibiotics (2% penicillin G-streptomycin–amphotericin B) at 37°C, in an atmosphere of 5% CO₂. The T84 cells were differentiated by growing five days in 24-well plates coated with collagen I (Roche). About 10⁶ viable cells were used for each bacterial infection in 24-well plates.

Human primary ADSCs were obtained from the eyelid adipose tissue of patients. The study was approved by the independent ethics committee of the Shanghai Ninth People’s Hospital and the informed consent form has been signed. Briefly, the adipose tissue was cut into small pieces and digested with collagenase A for 8 h. The digested tissue was centrifuged for 15 min (1,200 rpm, 37°C). Then, the precipitate was resuspended in a complete culture medium (DMEM with 10% FBS and 1% penicillin/streptomycin, Gibco) and seeded into a 10-cm culture dish. The culture medium was changed every 3 days. The culture plates with different Young’s modulus of 64 kPa and 0.2 kPa (Advanced Biomatrix Co. Ltd., USA) were covered with collagen I (0.1%, Roche) for 30 min to collect the conditioned medium of ADSCs on different substrates. Then, ADSCs were seeded into plates at a density of 1.67 × 10⁴/cm² (8 × 10⁴/mL). The medium was changed into DMEM without FBS after cell adhesion. The conditioned medium was collected 24 h later, filtered with a 0.22-μm filter, and stored at −80°C.

Upon adherence of calvarial osteoblasts or OCY454 cells on Collagen I (Roche)-coated surfaces of 8-well chambers, cells were fixed in 1.5% formaldehyde and immune-stained with rabbit anti-Paxillin antibody (1:150, Abcam) or mouse anti-Talin antibody (1:100, Sigma), followed by an incubation with a 488-Alexa Fluor conjugated secondary antibody (1:500, Thermo Fisher Scientific). Hoechst 33258 (Thermo Fisher Scientific) was used at a 1:10,000 dilution for nuclear staining. 633-Alexa Fluor conjugated phalloidin (Thermo Fisher Scientific) was used at a 1:20 dilution to stain actin. Cover slips were mounted with Fluoromount G (Southern Biotech).