Cell scraper

The discs treated with each solution for 5 min were placed in one of the wells of a 24-well plate, and 1 mL of PBS was added to each well. The biofilm was scraped from each disc using a cell scraper (Iwaki Co., Tokyo, Japan), and the dissociated cells were suspended by pipetting up and down [24 (link), 25 (link)]. The fungal suspensions were serially diluted with PBS, and 100-μL aliquot of each suspension was inoculated on Sabouraud glucose agar plates. After aerobic incubation for 48 h at 37°C, the CFUs were counted. Regarding the discs incubated for 24 h after the treatment, CFU assay was also conducted in the same way. Twenty discs from each group were used for this assay.

To generate KI and KO lines, we first dissociated hiPSCs (1390D4) into single cells using 0.5× TrypLE Select and a cell scraper (IWAKI), and then electroporated the vector complex into 2 × 10⁶ cells using the Neon® Transfection System (Thermo Fisher Scientific) (100 μL Kit, 1200 V, 20 ms, 2 pulses). Cells were seeded on two iMatrix-511-coated 6 cm dishes (1 × 10⁶ cells/dish) in AK02N medium supplemented with 10 μM Y-27632 for 1 day. On the next day, we changed the medium to AK02N medium without Y-27632. On day 3, we changed the medium to AK02N medium supplemented with 50 μg/mL hygromycin to select positive clones for 3 days. On days 10–14, we picked the survived colonies and expanded and analyzed them.
To eliminate the selection elements, we electroporated piggyBac Transposase mRNA (Transposagen) into 1 × 10⁵ hiPSCs using the Neon® Transfection System (10 μL Kit, 1200 V, 20 ms, 2 pulses) and seeded the cells on a iMatrix-511-coated 6 cm dish in AK02N medium supplemented with 10 μM Y-27632 for 1 day. On the next day, we changed the medium to AK02N medium without Y-27632. On day 3, we changed the medium to AK02N medium supplemented with 200 nM 1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl)-5-iodouracil (FIAU) for 4 days. On days 10–14, we picked the survived colonies and expanded and analyzed them.

For EB formation assays, 1.8 × 10⁵ cultured PSCs were detached from the plates using a cell scraper (Iwaki), dissociated by pipetting, transferred to low-attachment 6-well plates (Corning), and cultured with Es6 medium with 10 µM RI for 1 day and with Es6 medium alone for 13 days for EB formation. The medium was changed every 3 days. The sizes and numbers of EBs derived from PSCs on days 2 and 14 were measured using a CX5 high-content screening platform (Thermo Fisher). Gene expression levels were determined using RT-qPCR with a TaqMan Scorecard Panel (A15870), and gene expression profiles were determined using a RT-qPCR device (QuantStudio 12 K Flex).

Both Ca²⁺-ATPase and Na⁺/K⁺-ATPase activities were measured as previously described (26 (link)). Briefly, HLE cells were washed with ice-cold pH 7.4 Ca²⁺, Mg²⁺-free buffer (290 mOsm) and harvested using a cell scraper (Iwaki Co., Ltd., Tokyo, Japan). The composition of the pH 7.4 Ca²⁺, Mg²⁺-free buffer was as follows: 0.5 mM EDTA, 5 mM NaHCO₃, 5 mM KCl, 8 mM Tris, 15 mM HEPES, and 145 mM NaCl in water. The collected cells were homogenized in 600 ml of hypotonic buffer (pH 7.4) consisting of 10 mM mannitol, 5.75 mM HEPES, and 6.25 mM Tris base. Unbroken cells were pelleted at a low speed (2,040 × g, 10 min, 4°C), and the supernatant obtained was assayed for ATPase activity (assessed as Pi liberated from ATP). Ca²⁺-ATPase activity was calculated as the difference in phosphate release measured in the presence or absence of 0.1 mM Ca²⁺. Na⁺/K⁺-ATPase activity was calculated as the difference in phosphate release measured in the presence or absence of 1 mM ouabain. The activity of both plasma membrane Ca²⁺-ATPase (PMCA) and sarco (endo) plasmic reticulum Ca²⁺-ATPase (SERCA) was measured as Ca²⁺-ATPase activity in this study.