Fig 1: A combination of Z734 and lapatinib inhibits the growth of breast cancer. (A) MCF–7 cells were incubated with Z734 (10 µM) and/or lapatinib (0.5 µM), and p–ERK2, ERK2, p–p53, p53 and HERC3 protein expressions were detected by WB. (B) Representative images of ERK2 and p53 immunofluorescence staining in MCF–7 treated with Z734 (10 µM) or lapatinib (0.5 µM). DAPI was used to stain the nuclei. Quantification of fluorescence was performed on the Average Optical Density using the ImageJ software. Scale bars = 10 µm. (C) ELISA was used to detect the expression levels of Ki67 in MCF–7 to reflect cell proliferation after Z734 and/or lapatinib treatment. (D) Caspase3 activities in overexpressing of ERK2 in MCF–7 treated with Z734 (10 µM) and/or lapatinib (0.5 µM) were detected. (E) MCF–7 were treated with Z734 (10 µM) and/or lapatinib (0.5 µM) for 12 h, followed by flow cytometry to detect cell apoptosis. (F) Migration of MCF–7 cells were evaluated using the transwell assay after being treated with Z734 (10 µM) and/or lapatinib (0.5 µM). Scale bars = 20 µm. (G) Body weights of female Balb/c mice with different treatment groups (n = 5 for Control group; n = 5 for Z734 group; n = 5 Lapatinib group; n = 5 for Z734 + Lapatinib group). (H) Picture (n = 5 for Control group; n = 5 for Z734 group; n = 5 Lapatinib group; n = 5 for Z734 + Lapatinib group, combined results of 2 independent experiments) of MCF–7 breast tumor across four groups. (I,J) The volumes (Figure 6I) and weights (Figure 6J) were measured and quantified (n = 5 for Control group; n = 5 for Z734 group; n = 5 Lapatinib group; n = 5 for Z734 + Lapatinib group). Data shown in (B–G,I,J) were used one–way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 2: Z734 causes apoptosis and inhibits MCF–7 cells proliferation and migration. (A) The chemical structure of Z734. (B) Apoptosis levels were detected via flow cytometry. WT MCF–7 cells were treated with Z734 (0, 5, 10, and 15 µM) for 12 h. Flow cytometric analysis of apoptotic cells was conducted using AnnexinV–FITC. (C) Measurement of Caspase3 activity after Z734 intervention in MCF–7 cells. (D) Apoptosis–related protein levels after treatment with Z734 were determined via Western blot analysis. (E) MCF–7 cells were treated with different doses of Z734 and incubated for 12 h. Cell proliferation was assessed via CCK–8 assays. (F) Ki67 distribution after treatment with Z734 was determined via immunofluorescence. Quantification of fluorescence was performed on the average optical density using the ImageJ software. Scale bars = 10 µm. (G) The percentage of colonies of MCF–7 cell lines was determined after treatment with Z734. (H) MCF–7 cells were treated with Z734 for 12 h and then analyzed for migration by transwell analysis. Scale bars = 20 µm. Data shown in (B,C,E,G,H) were used one–way ANOVA with Tukey’s post hoc test. Data shown in (F) was used two–tailed Student’s t–test. All assays were performed in triplicate, and the data are expressed as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., no significant difference.
Supplier Page from Abcam for Human Ki67 ELISA Kit