Figure 1 Effect of PXC on cell viability, proliferation and colony formation of breast cancer cells A, B: inhibition of PXC on breast cancer cells after 72 h (A) or 96 h (B) treatment, the final concentrations of PXC were 7.81, 15.63, 31.25, 62.50, 125.00, 250.00, 500.00, 1000.00 μg/mL, respectively; C, D: cells were treated with PXC for 72 h, then cells were incubated with EdU (green) and Hoechst 33342 (blue), and photographed, the scale bar represents 100 μm; C: representative images of EdU assay in MDA-MB-231 cells; C1-C3: Control group; C4-C6: PXC 0.50 mg/mL; C7-C9: PXC 1.0 mg/mL; C1, C4, C7: Hoechst 33342 staining, C2, C5, C8: EdU staining, C3, C6, C9: the merge images of Hoechst 33342 and EdU; D: representative images of EdU assay in MDA-MB-468 cells; D1-D3: control group; D4-C6: PXC 0.50 mg/mL; D7-D9: PXC 1.0 mg/mL; D1, D4, D7: Hoechst 33342 staining, D2, D5, D8: EdU staining, D3, D6, D9: the merge images of Hoechst 33342 and EdU; E: statistical diagram of the percentages of EdU-positive cells in C and D, each value is expressed as the mean ± standard error of mean (SEM) (n = 3); F, G: Effect of PXC on colony formation. Cells were treated with PXC for 14 d, the colonies were stained with crystal violet solution and photographed; F: effect of PXC on colony formation in MDA-MB-231 cells. F1-F4: image of the colonies; F5-F8: picture of the individual colony (× 200 magnification); F1, F5: control group; F2, F6: PXC 0.25 mg/mL; F3, F7: PXC 0.50 mg/mL; F4, F8: PXC 1.00 mg/mL; G: effect of PXC on colony formation in MDA-MB-468 cells. G1-G4: image of the colonies; G5-G8: picture of the individual colony (× 200 magnification); G1, G5: control group; G2, G6: PXC 0.25 mg/mL; G3, G7: PXC 0.50 mg/mL; G4, G8: PXC 1.00 mg/mL; PXC: Pingxiao capsule; CCK-8: cell counting kit-8. Data were represented as the mean ± SEM from three repeated experiments. aP < 0.05 vs the Control group.

Figure 2 Effect of PXC on migration, invasion and EMT of TNBC cells A: representative photographs of the cells migration through polycarbonate membrane stained by 0.2% crystal violet; A1-A4: MDA-MB-231 cells; A5-A8: MDA-MB-468 cells; A1, A5: control group; A2, A6: PXC 0.0625 mg/mL; A3, A7: PXC 0.25 mg/mL; A4, A8: PXC 1.00 mg/mL; B: representative photographs of the cell invasion through the Matrigel-coated polycarbonate membrane stained by 0.2% crystal violet; B1-B4: MDA-MB-231 cells; B5-B8: MDA-MB-468 cells; B1, B5: Control group; B2, B6: PXC 0.0625 mg/mL; B3, B7: PXC 0.25 mg/mL; B4, B8: PXC 1.00 mg/mL; C, F: representative image of Western blotting for the expression levels of EMT-related proteins in MDA-MB-231 and MDA-MB-468 cells after PXC treatment; D: quantitative assessment of C in MDA-MB-231 cells; E: quantitative assessment of C in MDA-MB-468 cells; G: quantitative assessment of F, GAPDH was used as internal control, each value is expressed as the mean ± standard error of the mean (n = 3). PXC: Pingxiao capsule; EMT: epithelial to mesenchymal transition; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MMP2: matrix metallopeptidase-2; MMP9: matrix metallopeptidase-9. aP < 0.01 vs the Control.

Figure 3 Inhibitory effect of PXC on orthotopic human breast tumor in nude mice and breast cancer metastasis in mice A-F: inhibitory effect of PXC on MDA-MB-231 orthotopic human breast tumor in nude mice (n = 10). PXC 750 mg/kg (oral gavage, daily) and paclitaxel 20 mg/kg (intraperitoneal injection, on alternate days) were given for 14 d, tumor tissues were removed, weighed, and fixed in 4% paraformaldehyde for Immunohistochemistry staining. A: photographs of tumor in each group at the end of treatment; B: changes in body weight during the whole experiment; C: change of tumor volume in each group was measured every day; D: changes in tumor weight; E: representative image of immunohistochemistry staining of tumor tissues. E1-E4: the Control group; E5-E8: the PXC 750 mg/kg; E9-E12: the Pac 20 mg/kg; E1, E5, E9: N-cadherin; E2, E6, E10: E-cadherin; E3, E7, E11: MMP2; E4, E8, E12: MMP9; the scale bar represents 50 μm; F: quantification of E; G, H: inhibitory effect of PXC on breast cancer metastasis in mice (n = 3). 4T1 cells were inoculated into mice by tail intravenous injection. After 7 d of inoculation, the animals administrated with PXC at doses of 375, 750, and 1500 mg/kg (oral gavage, daily) respectively and paclitaxel 20 mg/kg (intraperitoneal injection, on alternate days). On day 14 of treatment, the lungs were harvested and fixed in in 4% paraformaldehyde for HE staining. G: representative photographs of HE staining of lung in 4T1 pulmonary metastasis tumor model. G1: blank group; G2: control group; G3: PXC 375 mg/kg; G4: PXC 750 mg/kg; G5: PXC 1500 mg/kg; G6: Pac 20 mg/kg; H: quantification of metastatic nodules in transverse section of lung after PXC treatment; PXC: Pingxiao capsule; Pac: paclitaxel; HE: hematoxylin-eosin staining. Data were expressed as the mean ± standard error of the mean. aP < 0.05 vs the control.

Figure 4 Effects of combined PXC and X-ray irradiation treatment on the proliferation and apoptosis of breast cancer cells Cells were preincubated with PXC for 24 h, and then irradiated at 8 Gy. After irradiation, cells were cultured for 48 h and then subjected to CCK-8 and apoptosis assay. A, B: effects of treatment with PXC and X-ray irradiation on the viability of MDA-MB-231 (A) and MDA-MB-468 (B) cells; data were represented as the mean ± standard error of the mean from five independent experiments; C, D: apoptosis rate of MDA-MB-231 (C) and MDA-MB-468 (D) cells induced by the combined PXC and X-ray irradiation treatment, which assessed by flow cytometry; CCK-8: cell counting kit-8; PXC: Pingxiao capsule; Pac: paclitaxel. Data were represented as the mean ± standard error of the mean from three independent experiments. aP < 0.01, compared with the control.