Calcein am

Analytical-grade glyphosate (N-(phosphonomethyl) glycine, CAS No: 1071-83-6) was purchased from VWR International Kft (Debrecen, Hungary). Samples of three GBHs, namely
were kindly provided by pesticide applicators. Composition data for each formulation were retrieved from the material safety data sheets (MSDS). Chemicals used for the assays and human liver-derived metabolic activation system (S9 fraction) were obtained from Sigma-Aldrich Chemie GmbH (Heidelberg, Germany). Cell culture medium and its supplements were obtained from Biowest (Nuaillè, France). The acetomethoxy derivative of calcein (Calcein AM) and propidium iodide (PIO) fluorescent dyes were purchased from Biotium (Hayward, CA, USA). Heparin-containing vacutainers were purchased from BD Vacutainer Systems (Plymouth, UK).

Early passages (between 3 and 6) of hS/PC cells were encapsulated at 3 × 10⁶ cell/mL in HyStem® hydrogel (GS311; BioTime/Ascendance Biotechnology). According to manufacturer's instructions, hydrogels were formed by mixing reconstituted thiol-modified hyaluronic acid (5.9 mM) and polyethylene glycol diacrylate (1.5 mM) at a 4:1 volume ratio, and plated on microscope glass slides fitted with pre-sterilized arrays of 50 μL wells made from laser-cut polydimethylsiloxane (PDMS; Sylgard™ 184; Dow Corning) sheets (Figure S1). Hydrogels were removed from the mold then each transferred into individual wells of a 48-well plate and cultured as described above. A typical hydrogel formed a low-profile cylinder measuring 6 mm in diameter and 2 mm in height. Hydrogels also were formed directly on confocal glass bottom dishes (P50G1.54F; MatTek) for live-cell imaging. 3D cultures were maintained until microstructures reached up to ~50 μm in diameter (between 6 and 16 days), a time when most live-tracking experiments were initiated. Viability assays using Calcein AM (80011-3; Biotium) and Ethidium Homodimer-III (40050; Biotium) in accordance with manufacturer guidelines were performed in cell-laden hydrogel cultures. IMARIS 9.2 software (Bitplane) was used to separate and quantify objects (Movie S1).

Adhesion Assays: Detached HET‐1A cells were seeded in 96‐well plates. Simultaneously 100 μl of medium containing DCA or ursodeoxycholic acid (UDCA; Sigma‐Aldrich, St. Louis, MO, USA) was added to each well. After allowing 2 hrs for adhesion, the medium was aspirated, the cells washed, and 100 μl of medium containing 2.5 μM calcein AM (Biotium, Hayward, CA, USA) was added to each well for 1 hr at 37°C. Fluorescence was determined using a Victor luminometer (Perkin Elmer, Waltham, MA, USA). The Millicoat™ ECM screening kit (Millipore, Billerica, MA, USA) was used to determine adhesion to specific ECM proteins.
Detachment and Re‐Adherence Assays: cells were seeded in 12‐well plates and allowed to adhere overnight. After 2 hrs treatment with DCA, the growth medium was aspirated and the wells washed twice with medium to ensure capture of all detached cells. Detached cells were re‐suspended in fresh medium and placed in a new well. Wells containing the residual adherent cells were washed twice, and fresh medium was added to each well. After 24 hrs, images were acquired and cell viability determined using MTT (Sigma‐Aldrich, St. Louis, MO, USA). The original untreated well was used as the reference for comparison.