Figure 1.
FC101 inhibits cell proliferation and reduces cell viability.
COS7 and HEK 293 cells were treated with FC101 (0–5 µM) for 6 days (for COS7) or 4 days (for HEK 293) (A, B), or 48 h (C, D), followed by cell number counting (A), morphological analysis (B), one solution assay (C), and trypan blue exclusion assay (D). For (A), (C), and (D), data represents mean ± SE (n = 6). *P<0.05, **P<0.01, ***P<0.001, difference with the control group (FC101 = 0 µM).
Figure 2.
FC101 arrests cells at G0/G1 phase of the cell cycle.
COS7 cells were treated with FC101 (0–5 µM) for 24 h. The cells were then harvested and processed for cell cycle analysis using Cellular DNA Flow Cytometric Analysis Kit and flow cytometry. (A) Histograms from a representative experiment show the effect of FC101 on cell cycle profile in COS7 cells. (B) Bar graphs show the effect of FC101 on the distribution (%) of COS7 cells in the G0/G1, S and G2/M phases of the cell cycle. Data represents mean ± SE (n = 3). *P<0.05, **P<0.01, ***P<0.001, difference with the control group (FC101 = 0 µM).
Figure 3.
FC101 downregulates protein expression of cyclin D1, Cdc25A, CDK4/6 and upregulates expression of p21Cip1 and p27Kip1, leading to hypophosphorylation of Rb.
COS7 cells were treated with FC101 for 24 h at indicated concentrations (A), or treated with FC101 at 1 µM for indicated time (B), followed by Western blotting with indicated antibodies. β-Tubulin was used for loading control. Representative blots are shown. Similar results were observed in at least 3 independent experiments.
Figure 4.
FC101 induces apoptosis. COS7 cells were treated with FC101 (0–5 µM) for 72 h.
The cells were harvested and processed for apoptosis assay using the Annexin V-FITC Apoptosis Detection Kit. The cell distribution was analyzed by flow cytometry. (A) Histograms from a representative experiment show the apoptotic effect of FC101 on COS7 cells. The percentages of necrotic, late apoptotic, viable, and early apoptotic cells are displayed in Q1, Q2, Q3 and Q4, respectively. (B) Bar graphs show that FC101 induced apoptosis of COS7 cells in a concentration-dependent manner. Quantitative results (Q2+Q4) are displayed as fold change compared with control. Data represents mean ± SE (n = 3). *P<0.05, **P<0.01, ***P<0.001, difference with the control group (FC101 = 0 µM).
Figure 5.
FC101 downregulates expression of the anti-apoptotic proteins (Bcl-2, Mcl-1, Bcl-xL and survivin) and increases expression of the pro-apoptotic protein BAD, increasing cleavages of caspase 3 and PARP.
COS7 cells were treated with FC101 for 24 h at indicated concentrations, followed by Western blotting with indicated antibodies. β-Tubulin served as a loading control. Representative blots are shown. Similar results were observed in at least 3 independent experiments.
Figure 6.
FC101 induces caspase-dependent apoptosis.
(A–D) COS7 cells, pretreated with or without Z-VAD-FMK (10 µM) for 1 h, were incubated with FC101 at indicated concentrations for 24 h, followed by caspase 3/7 activity assay (A), for 48 h, followed by trypan blue exclusion assay (B), or for 72 h, followed by Annexin V-FITC/PI staining and flow cytometry (C, D). (C) Histograms from a representative experiment show the effect of Z-VAD-FMK on FC101-induced apoptosis of COS7 cells. The percentages of necrotic, late apoptotic, viable, and early apoptotic cells are displayed in Q1, Q2, Q3 and Q4, respectively. (D) Bar graphs show that Z-VAD-FMK partially prevented FC101-induced apoptosis of COS7 cells. Quantitative results (Q2+Q4) were displayed as fold change compared with control. For (A), (B), and (D), data represents mean ± SE (n = 3). aP<0.05, bP<0.01, cP<0.001, difference with the control group (FC101 = 0 µM). dP<0.01, difference with Z-VAD-FMK group.