C myc

Western blotting: TIMELESS (Abcam, ab109512, 1/50000), β-actin (Abcam, ab49900, HRP-linked,1/50000), c-Myc (Abcam, ab32072, 1/1000) and PD-L1 (Cell Signaling Technology, #13684, 1/1000), Rabbit secondary antibody (Cell Signaling Technology, #7074, 1/2000).
Immunohistochemistry: TIMELESS (Abcam, ab72458, 1/200), CD8a (Servicebio, GB13429, 1/200), PD-L1 (Servicebio, GB11339A, 1/500), Ki-67 (Servicebio, GB111141, 1/500), Mouse secondary antibody (Servicebio, GB23301, 1/200), Rabbit secondary antibody (Servicebio, GB23303, 1/200).
Immunofluorescence: TIMELESS (Abcam, ab109512, 1/100), c-Myc (Abcam, ab32072, 1/100), CD8a (Servicebio, GB13429, 1/5000), Granzyme B (Abcam, ab255598, 1/2000), active caspase 3 (Servicebio, GB11532, 1/500), Hoechst 33342 (Beyotime, C1027, 1/100), CY3-TSA (Apex Bio, K1051, 1/200), Donkey Anti-Rabbit IgG H&L Alexa Fluor^® 647 (Abcam, ab150075, 1/200).
Flow cytometry: PD-L1 (Cell Signaling Technology, #13684, 1/200), CD8a-PerCP/Cyanine5.5 (Clone 53-6.7, Biolegend 1007341/100), CD45-AF700 (Biolegend 103128, 1/80), Fixable Viability Dye eFluor™ 450 (Invitrogen, 1/500).

The human TNBC cell lines MDA-MB-231 and MDA-MB-468 were from ATCC and stored in our laboratory. The docetaxel or vinorelbine-resistant MDA-MB-231 cell lines (MDA-MB-231/DR or MDA-MB-231/VR) were treated and established with docetaxel or vinorelbine for about 6 months in our laboratory. All these cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (HyClone, USA) in 5% CO₂ at 37 °C. Docetaxel and vinorelbine were purchased from Aosaikang and Haosen in China, respectively. Aspirin was purchased from Sigma in USA. The following antibodies were used for IHC and western blotting: YAP (Abcam, UK), β-catenin (Santa Cruze, USA), β-TrCP, Bcl-l2, Bax (CST, USA), C-Myc, CyclinD1 (Epitomics, USA), Ki67 (MXB biotechnologies, China), GAPDH (Biostar, China).

3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI) and other chemicals were purchased from Sigma (St. Louis, Missouri, USA). The specific anti-SHP1 short interfering RNA (siRNA) and nonspecific control siRNA sequences were purchased from GenePharma (Shanghai, China). Commercial antibodies to the following antigens were as follows: SHP-1, EGFR, p-GSK3β (Ser9), Cyclin D1, and c-Myc (Epitomics, Burlingame, USA); p-EGFR, β-actin, GAPDH, histone H3, and EGFR (Santa Cruz Biotechnology, California, USA); h-Ras, p-Erk1/2, Erk1/2, β-catenin, Snail, E-cadherin, and N-cadherin (Cell Signaling Technology, Massachusetts, USA); k-Ras, GSK3β (Proteintech Group, Chicago, USA); and SHP-1 (Abcam, Cambridge, UK).

Cell pellets from transfection procedures were lysed in Mammalian Protein Extract Reagent (M-PER; Pierce/Thermo Scientific, Rockford, IL, USA). Following pre-cleaning by centrifugation, protein concentrations of cell lysates were determined by using Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). Lysates equivalent to 25 μg of protein were separated on NuPAGE Bis-Tris (4-12%) gels (Invitrogen, Carlsbad, CA, USA) and transferred onto PVDF membranes. Membranes were blocked in Blocking Buffer (LI-COR, Lincoln, NE, USA) and incubated with specific antibodies against ERG splice variants (ERG MAb 9FY; Biocare Medical Inc., Concord, CA, USA), C-MYC (Epitomics, Burlingame, CA, USA) and GAPDH (Santa Cruz biotechnology, Santa Cruz, CA, USA). Membranes were washed in Tris-Buffered Saline + Tween 20 (TBST) before incubation with appropriate secondary antibodies (goat anti- Mouse IRDye 8000CW or goat anti-Rabbit IRDye 680CW, LI-COR). Signals of proteins detected were visualized and quantitatively measured using the Odyssey infra-red imaging scanner and software (LI-COR).