Type 1

Distillers’ grains were generously supplied by the Suzhou Bridge Wine & Spirits Co. (Suzhou, China). Peptides YPLPR, AFEPLR, and NDPF (purity ≥ 98%) were composed by All Peptide Biotechnology Co., Ltd. (Hangzhou, China). Dulbecco’s Modified Eagle Medium (DMEM) medium, embracing 10.0% fetal bovine serum (FBS) and 1.0 mol/L phosphate buffer (PBS) was bought from Gibco (Life Technologies, Inc., Carlsbad, CA, USA). Alkaline protease (2.4 U/g), penicillin-streptomycin (100.0 U/mL penicillin-100.0 mg/mL streptomycin), 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) powder, 2′,7′-dichlorofluorescence yellow diacetate (DCFH-DA), and trypsin solution were purchased from Solarbio (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). The HepG2 cell line was acquired from the Kunming Institute of Animal Science, Chinese Academy of Sciences (Kunming, China). Rosiglitazone, insulin, ABTS, DPPH, α-glucosidase derived from Saccharomyces cerevisiae (type I, protein content of at least 30.0 units per milligram), and p-nitrobenzene α-D-glucopyranoside (pNPG, with a minimum purity of 99.0%) were purchased from Sigma-Aldrich (located in Shanghai, China). The glucose kit was purchased from Nanjing Jiancheng Co. (Nanjinng, China). Other reagents applied were all of analytical rating.

Glass slides were cleaned by ultrasonication in an acetone bath for 10 min, washed with isopropanol, and dried with nitrogen gas. Then, the surface was made hydrophilic by oxygen plasma treatment. 300 μl of 2% (w/v) agarose solution (Type I, Sigma, USA) at 50 °C was spin-coated on top of clean glass slides followed by 2 μm-thick SU-8 photoresist (SU8 2, Microchem, USA) spin coating. The SU-8 layer was patterned by standard photolithography techniques; in brief, the SU-8 layer was exposed to a pattern of ultraviolet light through a photomask, developed by SU-8 developer (Microchem, USA), washed with isopropanol twice, and dried using nitrogen gas. Surface-patterned glass slides were kept under vacuum until use. The SU-8 thickness was determined to be 2 μm using a profilometer. The SU-8 thickness was varied by using various volumes of SU-8 during spin-coating and was optimized such that the patterned surfaces were consistent and planar.

Human ECM proteins, including fibronectin, type I, and type IV collagen (Sigma), were coated at 100 μl/well on Maxisorp 96-well microtiter plates at the concentration of 10 μg/ml by overnight incubation at +4°C. A volume of 100 μl/well of 2% (w/v) bovine serum albumin (BSA) (Sigma) was added as a negative control. Before adding the bacteria, the wells were blocked with 2% BSA at room temperature for 2 h, followed by washing three times with PBS. Bacterial cells, metabolically labeled with ³H-thymidine (Perkin Elmer), were normalized to an OD₆₀₀ of 0.5 (0.5 × 10⁹ cells/ml) in PBS, added at 100 μl/well, and incubated for 2 h at RT. After washing three times with PBS, 100 μl/well of 0.1 mol/L NaOH–1% SDS was added and the plates were incubated at +37°C overnight. The lysed cell suspension was collected into vials containing 1 ml of Optiphase Hisafe III scintillation liquid (Perkin Elmer) and the radioactivity was measured as previously described (von Ossowski et al., 2010 (link)). The proportion of adhered cells was determined by comparing the measured radioactivity of the lysed cell suspension with that of the cell suspension added to the wells.