Paraformaldehyde phosphate buffered solution

Colony formation assays were conducted of 2 × 10³, 1 × 10⁴, 2 × 10⁴, and 1 × 10⁵ cells per well of HCC78, H3122, JFCR-028-3, and JFCR-168, respectively, into 12-well plates. After 48 hours of seeding, cells were treated with the indicated inhibitors. The medium was changed every 2–3 days, and cells were cultured with inhibitors for 9 days to 2 weeks. Colonies were fixed in 4% paraformaldehyde phosphate-buffered solution (Wako) for 15 minutes at room temperature and stained with 0.5% crystal violet (Sigma) for 30 minutes at room temperature. After staining, pictures of the wells were taken. The crystal violet dye was solubilized in 30% ethanol and 1% acetic acid, then measured by absorbance at 570 nm using a Multiskan GO Microplate Spectrophotometer.

MDA-MB-231 and MDA-MB-453 cells were treated with EPI (10 μM) for 30, 60, and 180 min and fixed with 4% paraformaldehyde phosphate-buffered solution (Fujifilm Wako), followed by permeabilization with 0.1% Triton X-100 (Fujifilm Wako) in PBS. After blocking with 10% FBS in PBS, the cells were incubated overnight with HSP70 primary antibody (Table S1) at 4 °C. The cells were incubated with Alexa Fluor 594-AffiniPure anti-mouse IgG (1:100, cat. no. 115-585-003, Jackson Immuno Research, West Grove, PA, USA) and 0.5 μg/mL DAPI (Fujifilm Wako) for the counterstaining of nuclei. The fluorescence was visualized using an Olympus FSX100 microscope (Olympus, Tokyo, Japan). The fluorescence intensity of HSP70 was analyzed using Image J 1.52a software.

Biosheets were fixed in a 4% paraformaldehyde phosphate-buffered solution (pH 7.4) (Wako pure chemical; Osaka, Japan), embedded in paraffin, and sectioned at 3 to 5 µm thickness. The sections were subjected to routine hematoxylin and eosin (H&E) staining for nuclear detection and Masson’s trichrome staining for collagen fiber detection.
For immunohistochemical analysis, the deparaffinized sections were heated in Immunosaver solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 90 °C for 1 h for antigen retrieval. Subsequently, the sections were washed in distilled water twice for 10 min and blocked in 1% bovine serum albumin (Wako pure chemicals) in PBS at 24–26 °C for 1 h and incubated with anti-SSEA4 mouse monoclonal antibody (1:100, ab16287; Abcam, Cambridge, UK) and anti-CD105 rabbit polyclonal antibody (1:200, bs-0579R; Bioss Inc., Boston, MA, USA) overnight at 4 °C. After being washed twice with distilled water for 10 min, the sections were incubated with Alexa Fluor 488 rabbit anti-mouse IgG antibody (1:1000, ab169345; Abcam) or Alexa Fluor 594 goat anti-rabbit IgG antibody (1:1000, ab150080; Abcam) at 24–26℃ for 2 h. DAPI (ProLong Gold Antifade Mountant with DAPI; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used as a nuclear counterstain. The sections were analyzed using fluorescence microscopy (ECLIPSE-Ti; Nikon Corporation, Tokyo, Japan).