Fig. 8

BRD4 inhibitory natural compound 3',4',7,8-tetrahydroxyflavone (THF) regulated the formation of tumorspheres derived from MC38 cells. A The inhibitory effect on the cell proliferation of THF in mouse colon cells (MC38 cells). The cells were cultured with the indicated increasing concentration range of THF for 24 h. Cell viability was measured using the MTS assay. B The inhibitory effect of tumorsphere formation by THF. Tumorspheres derived from MC38 cells were treated with 200 µM THF for 7 days. Tumorsphere images (right) were taken at ×10 magnification. C CSC marker, ALDH expression of MC38 cells. The cells were treated with 200 µM THF for 1 day. ALDH expression was measured using the ALDEFLUOR assay kit and a flow cytometer, as described in the “Materials and methods” section. D THF induced apoptosis in tumorspheres derived from MC38 cells. The tumorspheres were treated with 200 µM THF for 1 days. Apoptosis was analyzed using Annexin V/PI staining, as described in the “Material and methods” section. E Protein expression regulation of THF in tumorspheres. The protein expressions of c-Myc, PD-L1, and RelB were detected by Western blot. The cells were treated with 200 µM THF for 24 h. F Protein expression regulation of THF in the nuclear and cytosolic fractions of tumorspheres. The protein expressions of c-Myc, PD-L1, and RelB were detected by Western blot. The cells were treated with 200 µM THF for 24 h. G IL-6 level of tumorspheres under THF treatment. The level of IL-6 was examined in the THF (100 µM)-treated tumorspheres culture medium. The amount of IL-6 was quantified using a flow cytometer. Experiment values are represented as the mean ± SD of triplicates. Compared with the control as determined by student’s t-test or one-way ANOVA with Dunnett’s multiple comparisons tests, *p < 0.05