Volocity version 6

All fluorescence images were captured on Perkin Elmer ERS 6FE spinning disk confocal microscope and images were processed and analyzed in Volocity version 6.1.1 software (Perkin Elmer, Shelton, CT, USA). Cilia were counted by detecting all objects with ACIII intensity greater than 7 SD above the mean image intensity that were between 1 and 10 µm³. All images were acquired using the same camera exposure, gain and laser power settings.

Cultures were fixed with a 4% PFA solution for 15 minutes, rinsed 3X with 1× PBS and permeabilized with a 0.1% triton solution. Cells were then blocked for an hour with donkey serum blocking buffer (2.5 mL Donkey Serum + 2.5 mL BSA + 40 mL sterile water + 5mLs PBS 10×) after a 15-min permeabilization period with 0.1% triton in PBS. Primary antibodies for MyoD (Cat# LS-C263888, LS Bio, WA, USA), Pax7 (Cat# PAS-117P, Invitrogen, MA, USA) and Ki67 (Cat# ab238020, Abcam, Cambridge, UK), MyHC (cat# A4.1025-s, DSHB, IA, USA) were added to the cultures and incubated overnight at 4 °C. The secondary antibodies (Thermofisher Scientific) were added the next day at a 1:250 dilution. Coverslips were rinsed 3X in 1× PBS and mounted on glass slides with Prolong gold mounting medium with DAPI (cat# P36931, ThermoFisher). Images were taken with an Axioscope spinning disk confocal microscope (Carl Zeiss) linked to XCite 120 Fluorescence Illumination system beam with laser scanning software, Volocity version 6.3.0 (Perkin Elmer, MA, USA).

Mutant and WT cultures on glass coverslips were fixed with 4% PFA solution for 15 min and rinsed 3× with 1× PBS solution. Cells were then blocked with donkey serum blocking buffer (2.5 mL Donkey Serum + 2.5 mL BSA + 40 mL sterile water + 5mLs PBS 10x) for 1 h after a 15-min permeabilization period with 0.1% triton in PBS. Primary antibodies for MyoD, Pax7, desmin (Thermofisher Scientific PA5-16705, MyHC DSHB, A4.1025-s), and Ryanodine receptor (RyR) (Millipore, AB9078), dihydropyridine receptor (DHPR) (Thermofisher Scientific,MA3920) were added to blocked cultures and incubated overnight at 4 °C. The secondary antibodies donkey anti-mouse and anti-rabbit (Thermofisher Scientific, A10037, A-21206) were added the next day at a 1:250 dilution. Coverslips were rinsed 3× in 1× PBS mounted on glass slides with Prolong gold mounting medium with DAPI (Life Technologies P36931) after a 2-h incubation period for imaging. Images were taken with Axioskop 2 mot plus upright spinning disk confocal microscope (Carl Zeiss) that had been connected to a XCite 120 Fluorescence Illumination system (EXFO) beam with multi-spectral laser scanning software, Volocity version 6.3.0 (Perkin Elmer).

Purified naïve CD4 T cells were incubated with 5 μM CFSE at 37 °C for 5 min, quenched with 50% FBS at 4 °C for 5 min and were washed in migration buffer. CFSE-labelled CD4 T cells (5 × 10⁴ cells per transwell) migrating across endothelial monolayers to CCL19 (50 ng/ mL) were visualized by EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific) with a ×20 objective. One image was captured every 5 min for 3 h. Cell tracks were analyzed with Volocity version 6.3 software (Perkin Elmer).

Cfse-labeled, FACS-sorted WT, PD-1^−/−, PD-L1^−/− iTreg cells (5 × 10⁴ cells per transwell) migrating across endothelial monolayers to CCL19 (50 ng/ mL) were visualized by EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific) with a ×20 objective. One image was captured every 5 min for 3 h. Cell tracks were analyzed with Volocity version 6.3 software (Perkin Elmer).