Tritontm x 100

For in vitro analysis of the CrkL levels in the breast cancer cells by immunofluorescence, the cells were cultured in eight-chamber slides (BD Biosciences, CA, USA) at 80% confluency and allowed to attach overnight. The next day, the cells were fixed with 4% paraformaldehyde (PFA, Thermo Scientific, IL, USA), and the slides were randomly divided into two groups. In the first group the cells were permeabilized with 0.2% Triton^TM X-100 (Sigma-Aldrich Co., MO, USA) for 5 min and washed extensively. In the second group, to assess the fraction of CrkL bound to the cell membrane, the permeabilization step was not performed. Both permeabilized and non-permeabilized cells were further incubated with CrkL antibody (Santa Cruz Biotechnology, Inc., TX, USA, catalogue #SC-9005) at 1:500 dilution for 1 h at room temperature, washed, and then incubated with Dylight 594-conjugated goat anti-rabbit secondary antibody (Thermo Scientific, IL, USA, catalogue #35560) for 45 min. After the cells were washed, they were fixed again with 4% PFA, mounted with Prolong gold containing 4′,6-diamidino-2-phenylindole, DAPI (Life Technologies^TM, NY, USA), and the immunofluorescence was detected using a Nikon fluorescence microscope (Nikon Instruments Inc., NY, USA). All the experiments were repeated three times.

To perform immunofluorescence experiments, cells were fixed using Immunofix (Bio-Optica) for 15 min, washed, and maintained in PBS+ at 4 °C. Before antibody staining, cells were permeabilized with PBS+ containing 0.5% Triton^TM X-100 (Sigma) for 10 min, then incubated with a blocking solution (PBS+ containing 1% BSA (Sigma) and 2% Donkey Serum (Merck)) for 1 h at RT, and washed with PBS+ containing 0.2% Tween 20 (Merck). At this point, samples were incubated overnight at 4 °C with the following primary antibodies diluted in blocking solution: anti-forkhead box A2 (FOXA2; rabbit polyclonal IgG; 1:400; Thermo Fisher Scientific (720061)), anti-SRY-box transcription factor 17 (SOX17; mouse monoclonal IgG2b (OTI2G8); 1:400; Thermo Fisher Scientific (MA5-24891)), or anti-HNF4 (rabbit monoclonal IgG (F.674.9); 1:400; Thermo Fisher Scientific (MA5-14891)). After extensive washing, Alexa fluorophore-conjugated secondary antibodies (donkey anti-rabbit IgG 594; 1:600; Thermo Fisher Scientific (A-21207); donkey anti-mouse IgG 488; 1:600; Thermo Fisher Scientific (A-21202)) were added and incubated for 1 h at RT. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) for 10 min at RT. Images was acquired at ×20 and ×63 magnification with a Zeiss Fluorescence Axiovert 200 M microscope (Carl Zeiss, Oberkochen, Germany).

Triton^TM X-100, Tween 20 and paraformaldehyde (PFA) were purchased from Sigma-Aldrich® (NSW, Australia). Isoflurane (IsoFlo^TM) was purchased from Abbott Australasia Pty Ltd., (NSW, Australia). Medical oxygen was purchased from Coregas Pty Ltd., (NSW, Australia). Triple antibiotic powder (Tricin®) was purchased from Jurox Pty Ltd., (NSW, Australia). Benzylpenicillin (BenPen^TM, benzylpenicillin sodium for injection) was purchased from CSL Ltd., (VIC, Australia). Pentobarbitone (Lethabarb®, pentobarbitone sodium) was purchased from Virbac (Australia) Pty Ltd., (NSW, Australia). Eye ointment (Refresh Night Time®) was purchased from Allergan Australia Pty Ltd., (NSW, Australia). 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), Prolong® Gold antifade reagent, phosphate-buffered saline (PBS), medium 199 (1X), horse serum, Dulbecco's phosphate-buffered saline (DPBS, 1X) and 0.25% trypsin-EDTA (1X) were purchased from Thermo Fisher Scientific Australia Pty Ltd., (VIC, Australia). Normal goat serum (NGS) was purchased from Cell Signaling Technology® (MA, USA). Tissue-Tek® O.C.T. Compound was purchased from ProSciTech Pty Ltd., (QLD, Australia). Sodium Chloride injection BP (British Pharmacopeia) (0.9%) was purchased from Pfizer Australia Pty Ltd., (NSW, Australia). J-2156 was obtained from Boehringer Ingelheim Pharma GmbH & Co. KG, (BW, Germany).

Bacterial cell leakage was evaluated using the SYTOX Green assay. E. coli cultures were grown to mid-exponential growth phase (OD₆₀₀ ~ 0.2) in LB medium and then centrifuged (5000× g for 2 min at 25 °C), washed and resuspended in phosphate buffer. Cell suspensions for OD₆₀₀ ~ 0.05 were incubated with 5 µM SYTOX Green (Invitrogen, Carlsbad, CA, USA) during 15 min in the dark before the influx assay. At 2–4 min after initiating data collection peptide concentrations from 0.625 to 5 μM were added to the cell suspension, and the increase in SYTOX Green fluorescence was measured (485 and 520 nm excitation and emission wavelengths respectively) for 40 min in a Cary Eclipse spectrofluorimeter, Agilent Technologies, Madrid, Spain. Maximum fluorescence was that resulting from cell lysis with Triton^TM X-100 (Sigma-Aldrich).

Cell proliferation assays were performed in 96-well plates (Sarstedt, Belgium). Cells were plated at 1500 cells/wells in 100 µl of culture medium. The next day, cells were supplemented with 100 µl of fresh medium containing bufalin or with vehicle for control. After 72 h, cells were washed and then fixed with 1% glutaraldehyde (Sigma) during 15 min. After washing, cells were stained with 4% crystal violet (Sigma) during 30 min, washed with tap water and leave on a bench at open air to dry. Cells were finally permeabilized with a solution of TritonTM X-100 (Sigma) during 90 min and the absorbance was determined at 570 nm with a spectrophotometer (VERSA max-SoftMax Pro, Molecular Devices, USA). Results were normalized to control (untreated cells).

Immunofluorescence analysis was performed according to standard protocols^{59 (link)}. Cells (1.5 × 10⁴) were seeded in triplicate on coverslips (WHB, Shanghai, China) in 24-well plates. After 48 h, the cells were fixed with 70% ethanol and permeabilized with 1.5 M HCl (Lanxi Zhongxing Chemical Reagent Co., Ltd., Lanxi, China). The cells were then washed twice with PBS and blocked with 5% goat serum (Absin Bioscience, Inc., Shanghai, China) and 0.3% Triton^TM X-100 (Sigma-Aldrich) in 1× PBS. Next, the cells were stained with primary antibodies against 5mC (#28692, 1: 1600, Cell Signaling Technology) or at 4 °C overnight^{34 (link)}. The next day, the cells were incubated with anti-rabbit IgG-Alexa Fluor^TM 488 antibody (#8878; 1:500, Cell Signaling Technology) in the dark on a gyratory shaker (Kylin-Bell Lab Instruments, Haimen, Jiangsu, China). Coverslips were then mounted with 5 μL of mounting medium containing DAPI (Beyotime). At least 100 cells from each biological replicate were captured at 600× magnification using a confocal laser-scanning microscope (Nikon Corporation). The fluorescence signal intensity was measured using ImageJ v. 2.4.1.7 (National Institutes of Health, MD, USA).

TiHo-0906 cells (P7 and P77) were seeded in 6-well plates (2 × 10⁵ cells/well) and incubated for 18 h at 37 °C in a humidified atmosphere of 5% CO₂. Afterwards, the cells were exposed to different dilutions of doxorubicin (1 nM, 10 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1000 nM, and 2000 nM [3 mL/well]) and then re-incubated for 72 h. After this period, media containing non-adherent and dead cells were collected. Adherent cells were dissociated with 1 mL TrypLE^TM and centrifuged (1000 rpm for 6 min) together with the previously collected media. The supernatant was discarded, and cell pellets were resuspended in 500 μL of 1X Binding Buffer. Cell suspensions were incubated (10 min at room temperature in the dark) with 5 μL Annexin V-FITC and 1 μL SYTOX Green Dye (Annexin V-FITC Detection Kit plus, PromoCell). Flow cytometry was performed using a MACSQuant® Analyzer 10 (Miltenyl Biotec). Dead cells treated with 1 mL 0.2% Triton^TM X-100 (Sigma-Aldrich) and non-treated viable cells were used to set the gates. Annexin V-FITC and SYTOX Green Dye were measured in the FL-1 channel, and results were analysed with FlowJo Version 7.6.5 (FlowJo, Ashland, OR, USA). For data analysis, cell debris on the left-side of the plot were set as negative gate. Afterwards, intact cells were gated for viability on three different populations: viable, apoptotic, and dead.