Fig 1.
T3 inhibits growth of HCT116 and A2780 cells via different mechanisms.
(A) Viabilities of A2780 and HCT116 cells normalized to the DMSO control after 72 h of T3 treatment. The x axis shows the compound concentration (logM). The y axis shows the percent inhibition of adenosine triphosphate contents, as compared to the DMSO control. Data are presented as the mean ± standard deviation (SD) of three independent experiments. (B) Time-lapse microscopic images of A2780 and HCT116 cells treated with DMSO or 1 μM T3 for 6 or 24 h. The yellow arrowheads show shrunken cells in A2780 and enlarged cytoplasm in HCT116 cells after treatment with T3 for 6 h or 24 h. Scale bar = 100 μm. (C) Flow cytometric analysis of the cell cycle distribution of HCT116 and A2780 cells treated with DMSO or 1 μM T3 for 16 and 24 h. Data are representative of two independent experiments. (D) Flow cytometric analysis of apoptosis of A2780 and HCT116 cells stained with Annexin V/PI. Cells were incubated with T3 for 24 h (A2780 and HCT116 cells) or 48 h (HCT116 cells). Apoptosis was analysed by Annexin V/PI staining and flow cytometry. The percentage of cells within each quadrant are indicated. Q1: Annexin V (-)/PI (+), Q2: Annexin V (+)/PI (+), Q3: Annexin V (+)/PI (-) and Q4: Annexin V (-)/PI (-). Data are representative of two independent experiments. (E) Caspase 3/7 activity was measured in the presence or absence of Z-VAD-FMK and is presented as the fold change, as compared to the DMSO control samples. Data are presented at the mean ± SD of three independent experiments. Statistical analyses were performed using an unpaired Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001).
Fig 2.
T3-induced reduction of anti-apoptotic proteins expression in A2780 and HCT116 cells.
Immunoblot analyses were performed using protein lysates from A2780 and HCT116 cells treated with T3 for 16 h at the indicated concentrations. The positions of Mcl-1L, Mcl-1S, cFLIPL, and cFLIPS are indicated on the right. Uncropped blot images are shown in S2 File.
Fig 3.
Alteration of MCL1 pre-mRNA splicing induced by T3 in A2780 and HCT116 cells.
(A) Cells were treated with T3 for 6 or 16 h at the indicated concentrations in the presence or absence of Z-VAD-FMK. Expression of MCL1 and BCL2L1 splice isoforms were analysed by RT-PCR, and PCR products were analysed by capillary electrophoresis. The quantitative expression for the long and short forms of MCL1 and BCL2L1, respectively, are shown. ACTB mRNA expression was evaluated as an internal control. Data are representative of three independent experiments. (B) Signal intensity was quantified and the percent spliced in (PSI) values for Mcl-1 and BCL2L1 alternative exons after T3 treatment for 6 or 16 h in the presence or absence of Z-VAD-FMK are shown. Data are presented as the mean ± SD of three independent experiments.
Fig 4.
Alteration of CFLAR pre-mRNA splicing induced by T3 in A2780 and HCT116 cells.
Cells were treated with T3 for 6 or 16 h at the indicated concentrations. Expression of CFLAR long and short splice isoforms (CFLAR-L and CFLAR-S) were analysed by RT-PCR, and PCR products were analysed by capillary electrophoresis. Closed triangles indicate the long and short forms of CFLAR-L, respectively. ACTB mRNA expression was evaluated as an internal control. Data are representative of three independent experiments. Raw electropherogram images are shown in the S3 File.
Fig 5.
T3-induced reduction of mTORC2 and involvement of proteasome pathway in A2780 and HCT116 cells.
(A) Immunoblot analyses were performed using protein lysates from A2780 and HCT116 cells treated with T3 for 16 h at the indicated concentrations. (B) HCT116 cells were pre-treated with T3 for 16 h at the indicated concentrations and then co-treated with 2.5 or 10 μM of MG132 for 6 h. Data are representative of two independent experiments. (A) (B) Uncropped blot images are shown in S2 File.
Fig 6.
Synergistic effects on apoptosis induction were observed by T3 in combination with Bcl-2 inhibitor in HCT116 and A2780 cells.
HCT116 and A2780 cells were treated with T3 or T3-1 and ABT-263 at the indicated concentrations for 16 h or 24 h, respectively. Caspase 3/7 activities were measured. Combination index (CI) values [24] are shown above the bar. A CI value of < 1 indicates synergism. Data are presented as the mean ± SD of two independent experiments.