Fig 1: Sesaminol directly binds to ANT2 in MCF7 cells. (a) Scheme of fixation of sesaminol onto magnetic FG beads and the estimated structure of sesaminol-fixed beads. (b) Four sesaminol-binding proteins were purified from whole-cell extracts of MCF7 cells with sesaminol-fixed (+) or not (−) FG beads and detected by silver staining. Mass spectrometric analysis identified phosphate carrier protein (PCP), ANT2, ribosomal protein S5 (RPS5) and ribosomal protein S18 (RPS18) as sesaminol-binding proteins. (c) The amino-acid sequence of ANT2. The residues predicted by mass spectrometry are shown in red. (d) Bound ANT2 were detected by western blotting with an anti-ANT2 antibody. (e) Purified recombinant FLAG-ANT2 was incubated with sesaminol-fixed (+) or not (−) FG beads and bound FLAG-ANT2 was detected by western blotting with an anti-FLAG antibody.
Fig 2: The depletion of ANT2 does not decrease intracellular ATP levels in cancer cells. (a) ANT2 and cyclin D1 were analyzed by western blotting in MCF7 cells treated with 50 μM sesaminol or 50 μM troglitazone for 3 h. α-Tubulin was used as a loading control. (b) MCF7 cells were treated with 50 μM sesaminol or 5 μM valinomycin for 24 h and then lysed to quantify total intracellular ATP levels. Obtained data were normalized to the absorbance previously measured by a Cell Counting Kit-8 assay. Columns, means (n=3); bars, s.d. **P<0.01, significantly different from the dimethyl sulfoxide (DMSO)-treated control. (c) MCF7 cells were treated with 50 μM troglitazone or 5 μM valinomycin for 24 h and then lysed to quantify total intracellular ATP levels. Obtained data were normalized to the absorbance previously measured by a Cell Counting Kit-8 assay. Columns, means (n=3); bars, s.d. **P<0.01, significantly different from the DMSO-treated control. (d) A variety of cancer cells (MCF7 cells, MDA-MB-231 cells, SK-MEL-28 cells, A549 cells and RKO cells) were treated with siANT2 #2 or siNeg for 48 h and then lysed to quantify total intracellular ATP levels. Results were normalized to the absorbance previously measured with a Cell Counting Kit-8 assay. Columns, means (n=6); bars, s.d. **P<0.01, significantly different from the siNeg-treated control; NS, not significant.
Fig 3: The depletion of ANT2 downregulates cyclin D1 at the mRNA and protein levels with mTORC1 inhibition and protein degradation, analogous to the treatment with sesaminol. (a) The expression of cyclin D1 mRNA was measured by real-time RT–PCR in MCF7 cells treated with siANT2 #1, siANT2 #2 or siNeg for 48 h. Cyclin D1 mRNA was normalized to ß2MG mRNA, and the data obtained with siNeg are taken as 1.0. Columns, means (n=3); bars, s.d. The experiments were carried out three times independently. (b) The expression of cyclin D1 mRNA was measured by real-time RT–PCR in MCF7 cells treated with 50 µM sesaminol for 3 h. Cyclin D1 mRNA was normalized to ß2MG mRNA, and the data obtained with dimethyl sulfoxide (DMSO) are taken as 1.0. Columns, means (n=3); bars, s.d. The experiments were carried out three times independently. (c) Phosphorylated S6K at threonine 389, total S6K, phosphorylated 4E-BP1 at serine 65 and total 4E-BP1 were analyzed by western blotting in MCF7 cells treated with siANT2 or siNeg for 48 h. (d) Phosphorylated S6K at threonine 389, total S6K, phosphorylated 4E-BP1 at serine 65 and total 4E-BP1 were analyzed by western blotting in MCF7 cells treated with 50 µM sesaminol for the indicated times. (e) MCF7 cells were treated with siANT2 or siNeg for 24 h, and the medium was then replaced with that containing 10 µM MG132 or DMSO. After being incubated for 24 h, ANT2 and cyclin D1 were analyzed by western blotting. ß-Actin was used as a loading control. (f) Cyclin D1 was analyzed by western blotting in MCF7 cells treated with 50 µM sesaminol with or without 10 µM MG132 for 6 h. ß-Actin was used as a loading control.
Fig 4: Schematic representation of the pleiotropic regulation of cyclin D1 by ANT2. ANT2 regulates cyclin D1 at the mRNA level (for example, promoting transcription or stabilization of cyclin D1 mRNA) and at the protein level with mTORC1 activation (for example, promoting translation) or protein stabilization.
Fig 5: The depletion of ANT2 causes a reduction in cyclin D1 in cancer cells. (a) MCF7 cells were treated with two different siRNAs targeting ANT2 (siANT2 #1 and #2) or non-targeting siRNA (siNeg) for 48 h, and the medium was then replaced with the fresh one. After being incubated for 24 h, cell viability was measured with a Cell Counting Kit-8 assay. The data obtained with siNeg are taken as 100%. Columns, means (n=3); bars, s.d. **P<0.01, significantly different from the siNeg-treated control. (b) MCF7 cells were treated with siANT2 or siNeg for 48 h, and the medium was then replaced with the fresh one. After being incubated for 24 h, the DNA contents of the cells were determined by flow cytometry. The percentages of cells in the G1 and S phases of the cell cycle are shown. Columns, means (n=3); bars, s.d. **P<0.01, significantly different from the siNeg-treated control. (c) ANT2, cyclin D1 and phosphorylated RB at serine 780 and serines 807/811 were analyzed by western blotting in MCF7 cells treated with siANT2 or siNeg for 48 h. ß-Actin was used as a loading control. (d) ANT2 and cyclin D1 were analyzed by western blotting in a variety of human cancer cell lines (MDA-MB-231 cells, SK-MEL-28 cells, A549 cells and RKO cells) treated with siANT2 #2 or siNeg for 48 h. a-Tubulin was used as a loading control.
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