Skip to main content
. 2022 Apr 7;56(2):185–197. doi: 10.2478/raon-2022-0012

Figure 4.

Figure 4

Vactosertib weakens radiation – induced breast cancer stem cell properties. (A) Fluorescence immunohistochemistry assay of NANOG in irradiated primary tumors of 4T1-Luc allografted BALB/c syngeneic mice. In confocal images, green fluorescence indicates NANOG (magnification: 20×, scale bar: 50 μm). Representative images are shown from three independent experiments. (B) Protein expression of cancer stem cell markers (NANOG, OCT4, KLF4, SCA1) in irradiated primary tumors of 4T1-Luc allografted BALB/c syngeneic mice with or without co-treatment with vactosertib. For quantification, protein expression was normalized by that of β-ACTIN. The graph shows the protein expression of each marker as a measure of fold change from the relative value of the control group. (C) Relative mRNA expression of pluripotent stem cell regulators (Nanog, Oct4, Sox2, c-Myc and Klf4) in 4T1-Luc cells. The mRNA expression level was normalized by that of Ppia mRNA. (D) Protein expression of pluripotent stem cell regulators (NANOG, OCT4, SOX2, C-MYC and KLF4) in 4T1-Luc cells was determined by western blot analysis. Protein expression was normalized by that of GAPDH. Data represent means of three independent experiments performed in triplicate. Significance evaluation was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test (*, **, and *** indicate p < 0.05, p < 0.01, and p < 0.005, respectively). (E) Radiation-induced mammosphere formation ability in breast cancer cell lines (4T1-Luc and MDA-MB-231). Breast cancer cells were irradiated with 10 Gy with or without 30-min pretreatment with vactosertib (100 nM). After 1, 2, 6, and 24 h of incubation, cells were reseeded in ultra-low attachment dishes and cultured for seven days. The left panels show the phase contrast microscopic images of spheres after seven days of culture in the ultra-low attachment condition (magnification: 5x, scale bar: 200 μm). Representative images are shown from three independent experiments. The right graph shows the mammosphere forming efficiency (MSFE) based on the number of spheres (> 50 μm) from 24 h–incubated cells.