Figure 4. Effects of RA-XII on cell proliferation.

(A) Effect of RA-XII on cell proliferation was investigated by [methyl-³H]-thymidine incorporation assay. 4T1 tumour cells were treated with RA-XII (50 and 100 nM) for 18 hours. They were subsequently incubated with [methyl-³H]-thymidine for 6 hours. Cell proliferation was revealed by the amount of [methyl-³H]-thymidine uptaken by cells, and expressed as the percentage of the control (mean + SD of 3 independent experiments). (B) Effect of RA-XII on cell cycle distribution. 4T1 tumour cells, treated with RA-XII (50 and 100 nM) for 24 hours, were analyzed by flow cytometry. Bar charts summarized the percentages of total viable 4T1 cells in G1 and G2/M phases (mean + SD of 3–4 independent experiments). (C) Representative western blot showed the effects on the expression of cyclins and CDKs when 4T1 cells were treated with RA-XII at 50 and 100 nM for 24 hours. The cropped blots have been obtained under the same experimental conditions and the full-length blots were shown in Supplementary information. The histograms summarized the expressions of the target proteins which were normalized with corresponding β-actin protein expression and expressed as fold of untreated controls (mean + SD of 3–4 independent experiments). Statistical differences were determined by One-way ANOVA, followed by Dunnett Test, with *p < 0.05, **p < 0.01, ***p < 0.001 against untreated controls.