Hoechst 33342

T3M-4 human pancreatic adenocarcinoma cells (RCB1021) were provided by the RIKEN BioResource Research Centre (Ibaraki, Japan) and were cultured in RPMI 1640 containing 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO₂. The uptake of BPA into T3M-4 cells was performed with several modifications to the previously reported method [26 (link)]. Briefly, T3M-4 cells were cultured in a 35 mm dish with a glass bottom (Matsunami Glass Ind., Osaka, Japan) 2 days before the experiment. After washing twice with Hank’s balanced salt solution (HBSS: 125 mM choline chloride, 4.8 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 1.3 mM CaCl₂, 5.6 mM D-glucose, and 25 mM HEPES), 1 mL of BPA (1 mM in HBSS) was added and incubated at 37 °C for 10 min. For the BPA-absent group, only HBSS was added and incubated. After washing three times with PBS(-), cells were fixed with 4% paraformaldehyde for 30 min at room temperature and incubated for 30 min, followed by incubation with 1 mL of BS-631 (100 μM in 0.5% DMSO/PBS(-)) for 30 min at room temperature. For nuclei staining, cells were incubated with Hoechst 33342 (5 μg/mL, Nacalai Tesque, Kyoto, Japan) for 10 min at room temperature. Fluorescence images were acquired using a BZ-X810 (Keyence, Osaka, Japan) instrument.

The human cervical cancer cell line HeLa and human gastric cancer cell line N87 or human breast cancer cells expressing firefly luciferase SK-BR-3/Luc were obtained from the RIKEN Bio-Resource Center (Tsukuba, Japan) or JCRB Cell Bank (Osaka, Japan), respectively, and maintained at 37 °C in 5% FBS-DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml) (Nacalai Tesque). The cells (1.0 × 10⁴ cells/well) were seeded on a slide chamber plate, cultured for 16 h and fixed by treatment with a 4% paraformaldehyde solution (Nacalai Tesque) for 10 min at 25 °C. The cells were blocked with 1% BSA/PBS for 1 h at 25 °C. After the addition of 50 nM purified His-tagged FLAPs or 5 nM trastuzumab in 1% BSA/PBS to the chamber, the cells were incubated for 16 h at 4 °C. Thousand-fold diluted Alexa Fluor 488-conjugated mouse anti-His tag secondary antibody (Medical & Biological Laboratories, Aichi, Japan) or Alexa Fluor 488-conjugated mouse anti-human IgG Fc secondary antibody in 1% BSA/PBS were used to fluorescently label the His-tagged FLAPs or trastuzumab, respectively. After washing three times with PBS, the stained cells were mounted with Fluoromount (Diagnostic ByoSystems, CA, USA) containing 1/1000 Hoechst 33342 (Nacalai Tesque). All photos were taken using a BZ-X700 microscope (Keyence, Osaka, Japan).

AT5BIVA, an SV40-transformed AT fibroblast cell line, and 11-4 cells, which are AT5BIVA cells with the addition of chromosome 11 to restore ATM, were gifts from Satoshi Tashiro (Hiroshima University, Japan) (Sun et al., 2010 (link)). The expression levels of ATM in these cells were confirmed by western blotting. HEK293T cells were obtained from Kei Fujinaga (Sapporo Medical University, Japan) and its ability to produce lentivirus was confirmed by infection assays. AT5BIVA, 11-4 and HEK293T cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% L-glutamine-penicillin-streptomycin solution (GPS; Sigma-Aldrich) at 37°C under 5% CO₂. Mycoplasma-free conditions were routinely confirmed by Hoechst 33342 (Nacalai Tesque) staining.

Cells transfected with vector, pCI-neo/FABP5 or pCI-neo/NES-FABP5 were fixed with 4% PFA for 20 min (Nacalai Tesque). Cells were incubated with the following primary antibodies overnight at 4°C: anti-FABP5 (1:100) (Cell Signaling Technology), anti-α-tubulin (1:1000) (Santa Cruz Biotechnology), and anti-ERRα (1:100) (Cell Signaling Technology). After washing with TBS twice, cells were incubated with the following secondary antibodies overnight at 4°C: Alexa Fluor^® 488-conjugated donkey anti-rabbit IgG H&G (1:2000) (Abcam) and Alexa Fluor^® 647-conjugated goat anti-mouse IgG H&G (1:2000) (Abcam). Cells were stained with Hoechst 33342 (Nacalai Tesque). Laser scanning confocal microscopy was performed on an Olympus FV1000.

Differentiated AT2 cells on a cell culture insert were removed with a membrane using biopsy punches (BP-50F) (Kai Corporation, Tokyo, Japan), fixed with 4% paraformaldehyde in PBS, and pre-incubated in PBS with 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) and 0.1% Triton X-100 for 30 min at 20°C to 25°C. Primary antibodies in 3% BSA/PBS were applied overnight at 4°C, followed by 1 h at 20°C to 25°C with the secondary antibodies. The antibodies used in this study are listed in the antibody list. The nuclei were stained with Hoechst 33342 (Nacalai Tesque). The cells were observed using a fluorescence microscope (model BZX710; Keyence). Analysis was performed using three independent samples. A TCS SP8 (Leica Microsystems) was used for confocal imaging.