Animals and Cell lines
Athymic female nude mice (4 weeks) were obtained from HUNAN SJA LABORATORY ANIMAL CO.,LTD and housed under specific pathogen-free conditions. Animal experiments followed the Guidelines for the Care and Use of Laboratory Animals of the TMMU, and all procedures were approved by the Animal Care and Use Committee of the TMMU. The human breast cancer cell lines (SK-BR-3, MDA-MB-361, MDA-MB-231, MDA-MB-453) were purchased from the American Type Culture Collection (ATCC, USA) and the Cell Bank of the Chinese (Shanghai, China). All cells were cultured in DMEM medium (Hyclone, USA), supplemented with 10% FBS (Biological Industries, USA) and 1% streptomycin/penicillin (Beyotime, Shanghai, China), and incubated in 5% CO2 at 37 °C.
Drug treatment of cells
Oxa (Oxa, S1224) used in the experiment was purchased from Selleck Chemicals Company (USA). For mice, Oxa or PBS was given through intraperitoneal injection at 5 mg/kg every three days when the tumor volume reaches 75 mm3 for two weeks. In vitro studies, MDA-MB-231 cells were treated with 20 µM and MDA-MB-453 cells were treated with 15 µM Oxa for 48 h and then incubated in drug-free medium. MHY1485 was purchased from Selleck Chemicals Company (USA), which was used to incubate 1 µM for 24 h before Oxa treatment.
Cell proliferation assay and colony formation assay
The cells were seeded into 96-well plates with a density of 4000 cells each well and 100 µl medium. After the cells had attached to the wall, the cells were washed twice using PBS, and added with Oxa at gradient concentrations (0 µM, 5 µM, 10 µM, 15 µM, 20 µM, and 40 µM, 60 µM). Afterwards, the cells were cultured in a cell incubator for 48 h. The supernatant was discarded after incubation, and 110 µl of Cell counting kit-8 (CCK-8) (CK04, DOJINDO, Japan) working solution (V culture medium: V CCK-8 stock solution = 10: 1) was added to each well to continue the culture for 3 h. The OD value of each well was read using a DG-3022A microplate reader (Nanjing Huadong Electron Tube Factory, China) at 450 nm to calculate relative cell viability (%) (relative cell viability = cell viability of drug-feed wells/the cell viability of drug-free wells), and further fitted to a dose–response curve in Graphpad Prism 8 software. To determine their clonogenic ability, MDA-MB-231 and MDA-MB-453 cells were trypsinized and seeded in 6-well plates (1000 cells per well). After the cells had attached to the wall, the cells were washed twice using PBS, and added with Oxa. The medium was changed every three days, and cells were cultured for up to 14 days until colonies were clearly visible. At the endpoint, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with crystal violet (Beyotime, China) for 30 minutes, and then colonies with >50 cells were counted.
The shRNAs targeting human PC4 were purchased and constructed by GenePharma (Shanghai, China). According to the manufacturer’s protocol, MDA-MB-231 and MDA-MB-453 cells were transfected with shRNAs or plasmid according to the manufactures’ instructions. The transfected cells were labeled as shPC4-1 and shPC4-2. The detailed sequence of shRNA listed in supplementary materials Table 1
Western Blotting analysis
The cells were harvested, washed, and lysed with RIPA buffer (Beyotime, China) containing protease inhibitor cocktail (Roche) for 30 minutes on ice. Total protein was extracted, and quantitated by a BCA kit (Beyotime, China) according to the manufacturer’s instruction. The protein samples were separated by electrophoresis in 10%~12% gel, and then transferred onto PVDF membranes (Millipore). Blotted membranes were blocked and incubation with primary antibodies overnight at 4 °C. The membranes were washed 5 minutes for 3 times with TBST, and subsequently incubated 1 h with HRP-linked secondary antibody (Cell Signaling Technology, USA) at room temperature. The band intensities were visualized and detected by an enhanced chemiluminescence detection system (Bio-Rad Laboratories). Besides, the detailed information of primary antibody is shown in supplementary Table 3.
Total RNA was extracted from breast cancer cells using Trizol (Cwbiotech, China). 1 μg RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis kit (#K1622, Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. As described in our previous work, RT-qPCR was performed using a SYBR Green qPCR master mix (Takara) according to the manufacturer’s protocol. After the reactions were completed, the comparative threshold cycle (Ct) method was used to calculate the relative gene expression. expression was used as the internal control. The utilized primer sequence for real-time qPCR listed in supplementary Table 2.
Apoptosis was evaluated by an Annexin V–APC apoptosis detection kit (BD Biosciences). Briefly, cells were collected, washed twice with PBS and resuspended in 500 µl of binding buffer. Subsequently, the cells were stained with 5 µl of Annexin V–APC and 5 µl of PI, then incubated for 15 minuets in the dark. The percentage of apoptotic cells was detected by flow cytometry. Experiments were performed at least three times.
Replicate cultures of 1×106 cells per well were plated in a 24-well plate. After Oxa treatment, the cells were incubated with 1 µl of Hoechst 33342 1000× (C0081S-6, Beyotime) and 1 ml PBS solution per well at 37°C for 10 minutes, followed by observation under a fluorescence microscope. Strong fluorescence can be observed in the nuclei of apoptotic cells, while weak fluorescence was observed in non-apoptotic cells.
For tissue section, the paraffin-embedded sections were dewaxed, rehydrated and add 20 µg/ mL DNase - free Proteinase K (ST533, Beyotime) at 37°C for 20 minutes. For vitro experiments, the cells were cultured in 48-well plates, incubated 24 h, then goes through a series of treatments, then fixed in 4% paraformaldehyde and permeabilized in 0.3% Triton X-100. Then, a One Step TUNEL Apoptosis Assay Kit (C1090, Beyotime) was performed according to the manufacturer’s instructions and observed under a fluorescence microscope (Leica, DMi8, Wetzlar, Germany).
Mice were inoculated subcutaneously with 5 x 106 respective MDA-MB-231 cells with stable PC4 knockdown or control cells in 100 µl PBS at one dorsal site. Meanwhile, these mice were divided into four groups randomly (Control, n=5; Control+Oxa, n=5; shPC4-1, n=5; shPC4-1+Oxa, n=5). Tumor growth was grossly monitored and measured with sliding calipers every 3 days. Volume of tumors were calculated according to the formula: volume (mm3) = (width2 x length)/2. Oxa or PBS was given through intraperitoneal injection at 5 mg/kg every three days when the tumor volume reaches 75 mm3 for two weeks, weight changes were recorded during the drug treatment. Then the mice were sacrificed, and the xenograft tumors were dissected, weighed and fixed in 4% paraformaldehyde for subsequent experiment.
Paraffin-embedded tissue sections were dewaxed, rehydrated and immersed in Sodium citrate repair solution (pH 6.0) for 15 minutes at 98°C for antigen retrieval. The slides were incubated with primary antibody overnight at 4°C. The slides were washed thrice with PBS (5 minutes each time), incubated with the appropriate secondary antibody for 1 h at 37°C and then detected by fluorescence microscope (Olympus BX51). The ratio of target protein-positive cells to DAPI-positive cells in Random field o per group was used for quantification by ImageJ software.
the results of this research are presented as the means ± standard. The data were analyzed in Excel, GraphPad 8.0 and SPSS 25.0 (IBM, Chicago, IL, USA). Comparisons between two groups were performed using the Student’s t-test. Comparisons among three or more groups were performed using a one-way analysis of variance (ANOVA). P<0.05 indicated a statistically significant difference.