35 mm collagen coated glass bottom dish

TMEV-infected cochleae were cultivated on a collagen-coated 35-mm glass-bottom dish (MatTek) for live imaging. For visualizing activated SCs and GERCs as macrophages, a Leica SP8 confocal laser-scanning microscope equipped with a HC PL APO 63×/1.40 CS2 oil objective was used. Images were captured every 5 min, from 8 to 25 h after TMEV infection, using a 488-nm argon laser with 10% intensity. While capturing images, the explant was maintained in a CO₂ (5%) stage-top incubator (INUBG2F-GSI2, Tokai Hit) at 37 °C. The movies were constructed using Leica LAS AF software (9–14 h and 14–21 h, respectively).

For live imaging of EB1-EGFP, cells were collected by standard trypsinization and 1.4 x 10⁶ cells were plated on a collagen-coated 35 mm glass-bottom dish (MatTek Corporation). After recovering overnight, the spent media was replaced with fresh media containing diluted DMSO/nocodazole. Each dish was treated and imaged separately at room temperature using an Eclipse TE2000 confocal microscope (Nikon). After imaging up to 15 min, treated plates were incubated in 5% CO₂ at 37°C for an additional 1.75 hours. Adhesion activation was then analyzed via brightfield microscopy with images collected using a IX-71 microscope (Olympus), the same as similar assays in this paper. Cells were then fixed in -20°C methanol for 10 min, washed twice with PBS, then blocked for 30 min in 5% milk/PBST before overnight incubation with mouse anti-α-tubulin DM1A monoclonal antibody (Pierce, #62204) [1:2,500] at 4°C. Cells were then washed twice with PBS before incubation with rabbit anti-mouse IgG (H+L) 546 [1:500] in the dark for 2 hours at room temperature. Cells were washed twice and left in a third wash of PBS for imaging on an Eclipse TE2000 confocal microscope (Nikon).

Previously isolated human skeletal muscle cells (HSkMCs) from severely obese, insulin‐resistant women (BMI ≥40 kg/m², HOMA‐IR ≥2.5, n = 6) and lean insulin‐sensitive women (BMI < 25 kg/m², HOMA‐IR < 2.5, n = 6) were used in this study (Kugler et al., 2020). In brief, human skeletal muscle cells (Passage 3) were thawed, pooled together, and grown in a humidified environment with 5% CO₂ at 37°C on a type‐I collagen‐coated flask (Greiner Bio‐one). At a confluence of ~80%–90%, myoblasts were subcultured onto type I collagen‐coated plates (Corning), 35 mm collagen‐coated glass‐bottom dish (MatTek), Seahorse XFp cell culture miniplate (Agilent Technologies), or 96 well clear bottom black polystyrene microplate (Corning) depending on experimental purposes. Upon reaching ~80%–90% confluency, myoblasts were switched to low‐serum (2% horse serum) media to induce differentiation into myotubes. On day 6 of differentiation, myotubes were treated with either Mdivi‐1 (20 μM) or vehicle (1% DMSO in PBS) for 12 h. For insulin signaling and glucose uptake measurements, myotubes were serum‐starved for 3 h, incubated with or without 100 nM of insulin for 10 min, and cell lysates were collected for further analysis as previously described (Bikman et al., 2010). All experimental procedures were repeated in three independent experiments.