2.1 Cell lines and cell cultures
Two human HER2+ breast cancer cell lines SK-BR-3 and BT-474 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Two human HR+, HER2-, PIK3CA mutated breast cancer cell lines T47D and MCF-7 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). SKBR-3-PR was a pyrotinib resistant cell line obtained by exposing SKBR-3 cells to increasing concentration of pyrotinib continuously. SK-BR-3 and SKBR-3-PR were cultured in Mcoy’5A (Gibco, US) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), BT-474 and T47D were cultured in RMPI-1640 (Gibco, US) with 10% FBS. MCF-7 was cultured in DMEM (Gibco, US) supplemented with 10% FBS.
All cell lines have recently been tested and confirmed with no mycoplasma contamination.
2.2 Chemicals
Pyrotinib dimaleate (SHR-1258 dimaleate) was obtained from Hengrui Medicine Co. Ltd (China). Alpelisib (BYL-719) and PI-3065 (PI3K p110δ inhibitor) were purchased from MCE (MedChemExpress, US). Agents were dissolved in dimethylsulfoxide (DMSO) and were kept at −20 °C for future use.
2.3 Cell viability assay and drug combination study
CCK-8 cell viability assay was performed to identify the 50% inhibitory concentrations (IC50) and combination index (CI). Cells were seeded in a 96-well plate at a density of 6000 - 12000 cells/well and cultured overnight, then cells were added with drugs at different concentrations. After 72h of treatment, Cell Counting Kit (CCK)-8 reagent (MCE, USA) was added at a concentration of 10%, and cells were incubated in a CO2 incubator for 3h, OD450 was measured. Cell inhibitory rate was calculated as (OD450negative control – OD450treatment)/ (OD450negative control – OD450black control), and the 50% inhibitory concentration was determined as IC50 of the drug. The combination index was calculated by using CompuSyn (ComboSyn Inc.) according to the theory of professor Ting-Chao Chou 24,25. CI values could indicate antagonistic (> 1.2), additive (1–1.2), and synergistic (< 1) effects of two or more drugs combination.
2.4 Cell proliferation assay and colony formation assay
Cells were seeded in a 96-well plate and cultured overnight. Then cells were added with DMSO, pyrotinib, alpelisib, or combinations. Every 24h, cells were added with CCK-8 and incubated for 3h, then OD450 was measured. The experiment was performed three times, and the cell proliferation curves were drawn.
Cells were cultured in a 6-well plate at a density of 3000 cells/well, then cells were added with different treatments the other day. The culture medium containing corresponding drugs was renewed every three days, and cells were cultured for three weeks. Finally, cells were fixed with 4% paraformaldehyde and stained with 0.1% Crystal Violet. The colony (>50 cells/ colony) was counted and compared to the controlled group. The experiment was performed in triplicate, and the data were representative of three separate experiments.
2.5 Transwell assay
Cells (10^5) suspended in 300 µL serum-free medium containing different drug treatments were transferred into the upper chamber of 24-well transwell chambers (#3422, Corning, NY, USA), and 600μL medium containing 20% FBS was added to the lower chamber. After incubation for 24 h, cells crossed the membrane were fixed with 4% PFA and stained with 0.1% Crystal violet. For each sample, 5 fields of view were obtained, and cell numbers were counted. The experiment was performed in triplicate, and the data were representative of three separate experiments.
2.6 Cell cycle assay
Cells were starved for 24h and treated with the corresponding treatments for 72h, and then cells were harvested and fixed with 75% ethanal at -20℃ overnight. Fixed cells were washed with PBS, resuspended in 100μL PBS, stained with 20μL 7-AAD in darkness for 15min at room temperature, and finally measured using a flow cytometer (BD Accuri C6 Plus, USA) according to the manufacturer’s instruction. The data were analyzed using Flowjo software (v.10.5; TreeStar, CA, USA). The final data were obtained from three independent experiments.
2.7 cell apoptosis assay
Cells were obtained after 72h of treatment and washed twice with PBS. Then cells were re-suspended in 100μL 1× binding buffer (BD Biosciences, NJ, USA), added with 5μL of Annexin V-FITC and 7-AAD and incubated in darkness for 15 minutes at room temperature. Samples were added with another 400μL 1× binding buffer before being measured with a flow cytometer (BD Accuri C6 Plus, USA). The data were analyzed using Flowjo software (v.10.5; TreeStar, CA, USA).
Tunel assays were performed according to the manufacturer’s instructions (Beyotime, Shanghai, China). Cells were plated in a 24-well plate and treated with different treatments for 72 hours. Then cells were fixed with 4% PFA for 30 minutes and permeabilized with 0.3% Triton-X-100 for 5 minutes. Cells were incubated with terminal deoxyribonucleotidyl transferase (TdT) at 37℃ in darkness for an hour and added with fluorescent mounting media (Beyotime, Shanghai, China). Pictures were taken by using DMi8 (Leica, German).
All assays were performed in triplicate, and the data were obtained from three separate experiments.
2.8 NA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted with Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. RNA was reverse-transcribed with ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Quantitative real-time PCR analysis was performed on Applied Biosystems Quantstudio5 (Thermo Fisher Scientific, USA) with PowerUp SYBR Green Master Mix (#A25742, Thermo Fisher Scientific, USA) following the manufacturer's protocol. The relative expression of mRNA to GAPDH was calculated. The data were obtained from three separate experiments. The sequences of primers are enlisted in Supplementary Table 1.
2.9 Protein extraction and western blot analysis
Total protein was extracted using Cell lysis buffer for Western and IP (Beyotime, Shanghai, China) supplemented with proteinase and phosphatase inhibitors cocktail tablet (Pierce, Thermo, USA). Protein quantification was performed using the BCA reagent (Pierce, Thermo, USA) according to the manufacturer’s instructions, and then samples were denatured with 5× loading buffer at 100℃ for 5min. The extracted protein samples were loaded into 8% - 15% SDS-PAGE gel for separation and transferred to 0.45μM polyvinylidene fluoride (PVDF) membrane (Millipore, Massachusetts, USA). The membrane was blocked with 5% skim milk or BSA for an hour. Primary antibodies were added, and the membrane was incubated at 4℃ overnight. Subsequently, the membrane was added with fluorescent secondary antibodies (LI-COR Biosciences, USA) and incubated in darkness for an hour at room temperature. The immunoreactive bands were obtained and analyzed using the LI-COR Odyssey CLx (LI-COR Biosciences, USA). The primary and secondary antibodies used are enlisted in Supplementary Table 2.
2.10 Statistical analysis
Student's t-tests and Analyses of variance models were used to compare mean values between the tested and controlled groups. Statistical analyses were performed with IBM SPSS version 22 (SPSS, NY, USA) and GraphPad Prism version 7 (GraphPad Software Inc., CA, USA). Results were presented as mean ± SD of three independent tests. The statistical significance of the difference between tested and controlled groups was assessed at significance thresholds of * (P < 0.05), ** (P < 0.01), and *** (P < 0.001).