Figure 5: Changes in expression of genes involved in G1 phase regulation. (a)–(c) mRNA expressions of cell-cycle regulators for G1 phase, PCNA (a), cyclin D1 (b), and p21cip1(c), were examined using real-time PCR assays in MDA-MB231 cells after treatment with α-mangostin, as compared to controls. White bars indicate the results for controls and black bars indicate the results of analysis at 6 h, 12 h, and 24 h after treatment with α-mangostin (* 𝑃 < 0 . 0 5 , ** 𝑃 < 0 . 0 1 ). PCNA and cyclin D1 expressions were significantly lower and p21cip1 was significantly higher in cells treated with α-mangostin for 24 h, as compared to controls. Relative target gene levels were calculated using the ΔΔCT method. GAPDH was used as a reference gene. Data are presented as means ± SD of triplicate independent measurements. (d) Western blots of PCNA (36 kDa) and cyclin D1 (36 kDa) showed significantly lower levels, and p21cip1 (21 kDa) showed significantly higher levels in cells treated with α-mangostin for 24 h, as compared to controls (left lane). These results were similar to those for PCR analysis, as shown in (a)–(c). β-Actin served as an internal control (lowest panel). Three samples from control and α-mangostin-treated cells were used for measurement.