Ms 100

DNA was extracted from the Sterivex filters (i.e., 0.22- to 5-μm size fraction) using an AllPrep DNA/RNA minikit (catalog no. 80204; Qiagen) with a modified protocol: the filter paper removed from a Sterivex cartridge was put into a lysing matrix E tube (catalog no. 6914050; MP Biomedicals) with a mixture of 400 μL RLT plus buffer (containing 1% β-mercaptoethanol in accordance with the kit’s protocol) and 400 μL phenol-chloroform/isoamyl alcohol (25:24:1, vol/vol/vol); bead-beating was performed at 3,500 rpm for 30 s (MS-100; TOMY Digital Biology), followed by cooling on ice for 1 min, and then again at 3,500 rpm for 30 s; the supernatant after centrifugation (16,000 × g for 5 min at room temperature) was mixed with 500 μL chloroform-isoamyl alcohol (24:1, vol/vol) to remove the residual phenol and then centrifuged again; the supernatant was then used as the loading material for the AllPrep DNA spin column and processed in accordance with the manufacturer’s instructions. The quantity and quality of the DNA were measured using a Qubit double-stranded DNA (dsDNA) HS assay kit (catalog no. Q32851; Thermo Fisher Scientific) and a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific). Consequently, at least 2 μg purified DNA was obtained from each sample.

The epithelial-stromal separation method was re-designed in accordance with the methods described in the previous studies (9, 11, 27). The dissected vagina was digested in 1 mL of 1% trypsin (1:250 powder, 9002-07-7, Gibco, Canada) in Hanks’ balanced salt solution (H6648, SIGMA, US), in a 37°C water bath, for 30 min. After digestion, the vagina was placed into cold Hanks’ balanced salt solution to stop the trypsin action. In cold Hanks’ balanced salt solution, the tube-shaped vagina was cut vertically into a sheet shape with dissection scissors. The vaginal epithelium located at the inner side of the tube-shaped organ was scratched down from the stroma by a flat stick, such as the flat side of a spatula. The separated epithelium and stroma were transferred into tubes with Hanks’ balanced salt solution and collected by centrifugation (3,000G, 1 min, 4°C). Remove the Hanks’ balanced salt solution, add 750 μL of Sepasol (Sepasol-RNA I Super G, NACALAI TESQUE, Japan) for each tube, and mix it vigorously to disperse the cells in Sepasol. The epithelium and stroma were homogenized in Sepasol by Micro Smash™ (MS-100, TOMY, Japan) with 4 pieces of 3.0φ beads (ZB-30, TOMY, Japan), 3 times, each time 10 sec, 3,000 rpm. These procedures were conducted to make sure that Sepasol penetrated the cells to avoid RNA degradation. Then the samples can be stored at -30°C.

Whole-cell lysates were prepared by homogenizing brain and other tissues for 60 s in lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EGTA, and 1% Triton X-100) supplemented with 5 μg/mL leupeptin (nacalai tesque; 43449-62) on ice using a Micro Smash (TOMY; MS-100) (4500 rpm, 4 °C). Whole-cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk in TBS-T (50 mM Tris–HCl pH 8.0, 150 mM NaCl, and 0.05% Tween-20), the membranes were probed with the indicated antibodies, and immunolabeling was detected using an enhanced chemiluminescence (ECL) system. Antibodies are listed in Supplementary Table S1. Band intensity was measured using ImageQuant TL (GE Healthcare).

Western blotting was performed as described previously [88 (link)]. 1×10⁸ cells from log-phase cultures in YE media were collected. Cells were suspended in 150 μl of 10% trichloroacetic acid (TCA) and disrupted by a bead neater (TOMY, MS-100) in the presence of acid-washed glass beads. After adding 250 μl of 5% TCA, cell extracts were kept on ice for 30 min. The pellet recovered after centrifugation at 5,000 rpm for 10 min at 4°C (TOMY, Kitman) was suspended in 200 μl of SDS-elution buffer (0.5 M Tris base, 28.125 mM Tris-HCl (pH6.8), 11.25% glycerol, 0.9% sodium dodecyl sulfate, 5% β-mercaptoethanol, 0.0045% bromophenol blue) and incubated at 95°C for 2 min. The supernatant was recovered after centrifugation at 12,000 rpm for 10 min at room temperature. The cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (acrylamide to bisacrylamide ratio, 37.5:1) and transferred onto PolyScreen PVDF Transfer Membrane (Perkin Elmer, NEF1002001PK). Anti-PCNA antibodies (1:2,000) and Peroxidase AffiniPure HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 111-035-003; 1:10,000) were used as the primary and secondary antibodies, respectively. The blots were developed using Supersignal West Femto substrate (ThermoScientific, 34095). Images were acquired using ImageQuant LAS 500 (GE Healthcare).