B2531

Cell proliferation and G₂ phase length were analyzed by specific experimental design described previously (Fig. 6a). Embryos were first treated with 10 mM 5-bromo-2′-deoxyuridine (BrdU, Sigma-Aldrich) pulses as described62 at 16 hpf and then fixed with fresh 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) every hour until 19 hpf. Double IHC was performed with mouse BrdU antibody (B2531, Sigma-Aldrich, 1:250) and rabbit P-H3 antibody (GTX128116, Genetex, 1:1000) to mark cells in S phase and in M phase with corresponding secondary antibodies (1:500). Images were collected utilizing LSM 780 confocal laser-scanning microscope (Carl Zeiss) with 20X lens scanning the dorsal midbrain. Every individual embryo was scanned reaching 25 μm deep from top (10 stacks, 2.5 μm per stack) and shown with represented z stack. Furthermore, G₂ phase length was determined by the percentage of labeled mitosis (PLM) paradigm28 (link) which reveal the progression of the BrdU and P-H3 co-labeled nuclei after BrdU incorporation. Due to its proportionality to G₂ length, it is simple to predict the duration of G₂ phase by 50% of co-labeled nuclei in average.

Proliferation of skin keratinocytes in vivo was assessed using BrdU (nakalai tesque). Mice were wounded on their dorsal skin and intraperitoneally administrated with 250 mg/kg body weight of BrdU at 3 days after wounding. After 2 hr of BrdU administration, sections of the wounded skin were prepared as described above in “In situ hybridization”. After sections were washed three times with PBS, endogenous peroxidase in the sections was inactivated with 3% H₂O₂/methanol solution at r.t. for 15 min, and subsequently antigens in the section was retrieved by heating the section in 0.01 M sodium citrate, pH 6.0, with microwave oven. The sections were washed three times with 0.1% Triton X-100/PBS, blocked with H-PHT at r.t. for 1 hr, then incubated with anti-BrdU antibody (B2531, Sigma-Aldrich) in H-PHT at 4 °C overnight. After washing three times with PBST, sections were incubated with biotinylated anti-mouse/anti-rabbit IgG antibody (BA-1400, VECTOR) in H-PHT at r.t. for 1 hr. The sections were washed three times with PBST, and incubated with ABC Reagent kit (PK-6100, VECTOR) at r.t. for 30 min. After being washed twice with PBS, sections were reacted with DAB (DAKO). Sections were also counterstained with haematoxylin for nuclear staining. Images were obtained by BZ-X710 microscope (Keyence). The number of BrdU-positive cells at wound site were measured by ImageJ (NIH).

Cell proliferation assay was performed on SAFs cultured for 24 hours under CTR (MEM + 10% FBS), ST (MEM + 0.5% FBS) and 50 TSA (MEM + 10% FBS + 50 nM TSA) conditions and evaluated by an indirect immunocytochemistry assay for 5-bromo-2'-deoxyuridine (BrdU) detection, a thymidine analogous incorporated during S-phase. Briefly, SAFs of CTR, ST and 50 TSA groups were cultured with 100 μM BrdU for 6 hours before the end of culture, fixed in cold methanol for 20 minutes, and permeabilized at room temperature (RT) with 0.1% Triton-X-100 (in PBS) for 15 minutes. Next, cells were treated with 4N HCl at RT for 30 minutes and incubated with a primary antibody (mouse anti-BrdU, monoclonal antibody, B2531, Sigma) at a rate of 1:100 in blocking solution (0.1% bovine serum albumin (BSA) in PBS) at 4°C, overnight. Cells were then incubated with a secondary antibody (rabbit anti-mouse IgG-FITC polyclonal antibody, F9137, Sigma) at a rate of 1:500 in BS at RT for 2 hours and counterstained with 0.5 μg/mL propidium iodide (PI) at RT for 5 minutes. Between every passage, cells were washed twice with PBS at RT for 5 minutes. The number of proliferative cells was calculated for each group by calculating the ratio between the number of BrdU-positive cells and the total number of nuclei. At least 200 nuclei were analyzed for each group for statistical significance.

For cell viability determination, 500 cells were plated in triplicate in 6-well plates. After 1–5 days, viable cell numbers were counted via trypan blue exclusion assays. For BrdU incorporation assays, cells plated on coverslips the day before were pulsed for 4 h with 10 μM BrdU (B5002, Sigma), fixed in 4% paraformaldehyde and incubated with mouse anti-human BrdU (B2531, Sigma) antibody at 4 °C overnight. After thorough washing, coverslips were incubated at room temperature for 1 h with Alexa Flour 594-conjugated goat anti-mouse IgG (1:500). Coverslips were then counterstained with DAPI (1:1000) and mounted with 10 μl Gold Antifade Reagent (936590, Prolong). Images were acquired under microscope (Nikon, Eclipse E800). A minimum of 1000 cells was counted for each condition.

BrdU assay was performed to determine proliferation. Cells were treated with concentrations of ATO ranging from 0 μM to 5 μM for 72 hours, and then cytospun onto slides. They were then fixed with 4% formaldehyde in PBS for 15 minutes, and permeabilized with 0.1% Triton/PBST for 15 minutes. Cellular proteins were then denatured with 2 N HCL, washed with PBST (PBS Tween-20), blocked with 5%NGS/PBST for 15 minutes and then incubated in primary antibody against BrdU (Sigma, B2531). Anti-BrdU antibody was used per the manufacturer’s instruction at 1:500 dilution. After washing 3 times with PBST, cells were incubated for 45 minutes in the dark with the appropriate cy-3 conjugated secondary antibody. Cells were then counterstained with 4’,6-diamidino-2-phenylindole (DAPI), mounted with Vectashield (Vector Laboratories), and visualized and pictures taken by fluorescence microscopy.

Primary fibroblasts were pulse-labelled with BrdU (10 µM final concentration) for 1 h at 47 h after initial treatment. For assessment of cell proliferation, cells were fixed with 4% paraformaldehyde (PFA) (15713-S) (Electron Microscopy Sciences, PA, USA) for 10 min. DNA was denatured using 2 N HCl containing 0.5% Triton X-100 (9410) (Merck Millipore, MA, USA) for 30 min at 37 °C followed by neutralization with 0.1 M borate buffer pH 8.5 for 10 min. Nonspecific staining was blocked with 5% normal goat serum (PCN5000) (Invitrogen, CA, USA) for 30 min before applying mouse monoclonal anti-BrdU antibody (1:1000, B2531) (Sigma-Aldrich, MO, USA) for 1 h. Thereafter, goat anti-mouse Alexa Fluor® 568 IgG (H + L) (1:500, A-11004) secondary antibody (Invitrogen, CA, USA) was incubated for 1 h, postfixed the cells with 4% PFA for 5 min, and stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (D1306) (Invitrogen, CA, USA) for nuclear visualization. Representative images were acquired using an Olympus Inverted Fluorescence Microscope Model IX83 (Olympus, Tokyo, Japan) equipped with ORCA-Flash 2.8 Digital CMOS Camera (C11440) (Hamamatsu Photonics, Hamamatsu, Japan). Ten images were randomized at magnification × 100 for quantitative analysis of % BrdU⁺ nuclei/total nuclei using image acquisition software (cellSens Dimension Desktop, Olympus, Japan).