Glass coverslips

HEp-2 epithelial cells were grown to 70–90% optical confluence in 24-well plates on glass coverslips (8 mm, Thomas Scientific, Germany) in DMEM FCS (with 0.45% glucose and 10% FCS; Lonza, Germany). Before adding bacteria, HEp-2 cells were washed three times with DMEM. Bacteria were grown overnight in LB broth, and 10 µl (~MOI of 100) were added to the HEp-2 cells with 1 ml DMEM containing 1% mannose and incubated for 3 h statically at 37 °C with 5% CO₂. HEp-2 cells were washed five times with PBS, followed by fixation with 3% PFA for 20 min at room temperature. Samples were subsequently air dried and mounted for phase contrast microscopy at 1000-fold magnification on a Nikon Eclipse inverted microscope. For the quantitative HEp-2 adherence assay, 1 ml of PBS and 10 µl of 10% saponin was added after washing with PBS, followed by pipetting up and down approximately ten times, stepwise dilution in PBS and plating on LB agar to determine colony forming units (CFU). As reference for the amount of bacteria multiplied within the 3 h, the washing step with PBS was omitted and the counted CFUs was set to 100%.

Naïve HUH7 cells or HUH7 cells expressing ORF1 WT, WT-HA-tag, C483A-HA-tag, C563-HA-tag, D248A-HA-tag, or H249A-HA-tag were seeded onto separate glass coverslips (#1.5; 10 mm; Thomas Scientific, Swedesboro, NJ, USA) in a 24-well plate at 100,000 cells per well. 2 days post seeding, the cells were fixed with 4% PFA for 15 min and subsequently permeabilized in 0.25% Triton x-100 for 15 min. The rabbit anti-HA tag, C29F4 (Cell Signaling Technology, Danvers, MA, USA) primary antibody was used at a ratio of 1:1000 (V/V), and the AlexaFluor647 (goat anti-rabbit IgG [H+L], ThermoFisher Scientific, Waltham, MA, USA) secondary antibody was used at a final concentration of 1 µg/mL. All antibodies were diluted with PBS and incubated for 40 min at room temperature (RT). Hoechst 33342 (ThermoFisher Scientific, Waltham, MA, USA) was incubated at a final concentration of 1 µg/mL for 10 min at RT. The coverslips were then mounted onto glass microscopic slides (VWR International, Radnor, PA, USA) with 5 µL of ProLong gold antifade reagent (ThermoFischer Scientific, Waltham, MA, USA). The stained samples were imaged using the Nikon A1R-Si microscope (Nikon, Melville, NY, USA) in the Princeton University Confocal Microscopy Facility. The images were taken at 40× magnification. Images were then analyzed using Fiji (ImageJ2) image analysis software.

Gelatin from porcine skin (type-A, 300 bloom), methacrylic anhydride, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), Opti-MEM I reduced serum medium (Opti-MEM), fetal bovine serum (FBS), goat serum, trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA), antibiotic-antimycotic solution stabilized (Anti-Anti, 100X), 4′,6-diamidino-2-phenylindole (DAPI), formalin (10% w/v), Live/Dead^® Viability/Cytotoxicity Kit, Alexa Fluor^® 594-phalloidin, dialysis membrane (M_w cutoff: 12–14 kDa) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The CellTiter 96^® AQueous One Solution Cell Proliferation Assay solution was obtained from Promega (Madison, WI, USA). Mouse cytochrome P450 3A (CYP3A4/CYP3A5 monoclonal) antibody and Alexa Fluor^® 594-conjugated goat anti-rabbit secondary antibody were purchased from Abcam (Cambridge, MA, USA). Ruthenium visible light photocrosslinking kit was purchased from Advanced BioMatrix (San Diego, CA, USA). Glass coverslips of 8 mm in diameter were obtained from Thomas Scientific (Swedesboro, NJ, USA). All other chemicals used in this study were obtained from Sigma-Aldrich unless otherwise mentioned.