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. 2013 Nov 6;8(11):e80411. doi: 10.1371/journal.pone.0080411

Figure 4. Cells cultured in BCAA_3 medium have higher activities of mTOR and higher protein levels of p21.

Figure 4

(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.