Table 1.
The compositions of branched-chain amino acids and aromatic amino acids in medium.
Figure 1.
DNA damage-inducing drugs cause premature senescence.
(A) HepG2 cells were cultured in RPMI medium with 0.1% DMSO or 10 µM etoposide for 0, 12, 24, 36 and 48 hours. (B) U2OS cells were cultured in RPMI medium with 0.1% DMSO, 2 µM etoposide, or 2 µM bleomycin for 0, 3, 5 and 7 days. For the assay of SA-β-Gal activity, cells stained with blue color were counted as described in Materials and Methods. The data (mean ± S.D.) were obtained from at least three independent experiments. Significant test results (P values) are shown.
Figure 2.
Cells cultured in BCAA_3 medium have higher activities to induce premature senescence.
(A) HepG2 cells cultured in BCAA_1, 3, 5 and BCAA_5 with 100 nM rapamycin were treated with 10 µM etoposide for 2 days, and observed with microscope after SA-β-Gal staining assay. (B, C) HepG2 cells cultured in BCAA medium with or without 100 nM rapamycin as indicated were treated with 10 µM etoposide (B) or 2 µM bleomycin (C) for 2 days. (D) U2OS cells cultured in RPMI-based medium with or without 100 nM rapamycin as indicated were treated with 2 µM etoposide for 7 days. For the assay of SA-β-Gal activity, cells stained with blue color were counted as described in Materials and Methods. The data (mean ± S.D.) were obtained from at least three independent experiments. Significant test results (P values) are shown.
Figure 3.
BCAA medium does not affect cell proliferation.
U2OS cells cultured in BCAA medium for 7 days were labeled with 10 µM BrdU for 3 h. BrdU-labeled cells were observed with microscope after immunostaining for BrdU and Heochst staining (left), and the percentage of BrdU-positive cells was quantified (right). The data (mean ± S.D.) were obtained from at least three independent experiments. Significant test results are shown.
Figure 4.
Cells cultured in BCAA_3 medium have higher activities of mTOR and higher protein levels of p21.
(A) HepG2 cells cultured in BCAA medium with or without 100 nM rapamycin as indicated were treated with 10 µM etoposide for 48 hours. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated. The intensities of the bands corresponding to phosphorylated S6K at Thr389 and S6K were quantified by ImageJ, and the ratio of the phosphorylated S6K at Thr389 to S6K was shown as mTORC1 activities. (B) HepG2 cells cultured in BCAA medium with or without 100 nM rapamycin as indicated were treated with 10 µM etoposide for 48 hours. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated. The intensities of the bands corresponding to p21 and α-tubulin were quantified by ImageJ, and the ratio of p21 to α-tubulin was shown. (C) HepG2 cells cultured in BCAA medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 48 hours. The mRNA expressions of p21 and GAPDH were examined by RT-PCR using specific primers against p21 and GAPDH. The intensities of the bands corresponding to p21 and GAPDH were quantified by ImageJ, and the ratio of p21 to GAPDH was shown.
Figure 5.
BCAAs enhance the execution of premature senescence induced by DNA damage-inducing drugs.
(A) HepG2 cells cultured in BCAA medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 48 hours, and observed with microscope after SA-β-Gal staining assay. (B) HepG2 cells were cultured in BCAA as described in A. For the assay of SA-β-Gal activity, cells stained with blue color were counted as described in Materials and Methods. The data (mean ± S.D.) were obtained from at least three independent experiments. Significant test results (P values) are shown. (C) U2OS cells cultured in BCAA medium were treated with or without 2 µM etoposide and 100 nM rapamycin as indicated for 7 days, and observed with microscope after SA-β-Gal staining assay. (D) U2OS cells were cultured in BCAA medium as described in C. The assay of SA-β-Gal activity was carried out as described in B. (E) U2OS cells cultured in BCAA medium were treated with or without 100 nM rapamycin as indicated for 24 hours and cells were harvested at each time point. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated.
Figure 6.
BCAAs upregulate p21 protein level mediated through the mTORC1 pathway.
(A) HepG2 cells cultured in RPMI medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 1 or 2 days. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated. (B) HepG2 cells cultured in BCAA medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 2 days. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated.