S 4300 microscope

ARPE 19 cell monolayers were cultured in a TEER plate (0.4 μm pore size and 12 mm diameter; Corning Transwell, Sigma-Aldrich, St. Louis, MA, USA) and analyzed by (1) scanning electron microscopy using HITACHI S-4300 microscope operated at 5 keV (the detector features 1280 × 960 pixel), (2) the TEER values (Ωcm²) using an electrical resistance system (KANTO CHEMICAL CO. INC., Tokyo, Japan) and (3) FITC-dextran permeability measurements by measuring the fluorescence intensity of the amount of FITC that permeated through the membrane from the basal compartment to the apical compartment during a period of 60 min as described in a previous study [27 (link),28 (link),29 (link)].

HTM cell monolayer TEER was determined according to previously described methods^{50 (link)}. Briefly, HTM cells prepared in 150 mm 2D cultured dishes as above were washed with a PBS, and the cells were detached using 0.25% Trypsin/EDTA. After centrifugation for 5 min at 300×g, the cell pellet was re-suspended in grown medium A and HTM cells were seeded on 12 well plates for TEER (0.4 μm pore size and 12 mm diameter; Corning Transwell, Sigma-Aldrich) at a density of 2.0 × 10⁴ cells per well. In each well of the TEER plate, the apical side (inside of the membrane inserts) and basal side (outside of the membrane inserts) were maintained in 0.5 mL and 1.5 mL of growth medium A, respectively. When cells had reached approximately 80% confluence, 5 ng/mL TGFβ2 was added to the grown medium A of the apical side in the absence or presence of 10 µM Rip or 10 μM Y27632 (Day 1). This culture medium of the apical side in each experimental group was changed every other day. At Day 6, TEER (Ωcm²) was measured using an electrical resistance system (KANTO CHEMICAL CO. INC., Tokyo, Japan) according to the manufacturer’s instructions after washing twice with PBS. Alternatively, after washing with PBS as above, HTM cells on the membrane were processed by scanning electron microscopy (EM) using HITACHI S-4300 microscope operated at 5 keV (the detector features 1280 × 960 pixel).

Quantitative PCR using specific primers (Table S1) and statistical analyses using the Graph Pad Prism 8 (GraphPad Software, San Diego, CA, USA) were performed as described in a previous report [37 (link)]. Scanning electron microscopy (SEM) using a HITACHI S-4300 microscope was operated at 5 keV (the detector features 1280 × 960 pixel) as basically reported in our previous study [37 (link)]. For estimation of the statistical difference between study groups, the Student’s t-test for two group comparison or one-way ANOVA followed by a Tukey’s multiple comparison test were used.