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. 2018 Mar 1;4(2):e00544. doi: 10.1016/j.heliyon.2018.e00544

Fig. 1.

Fig. 1

BCAAs prevent ATP decrease and cell death in cultured cells. (A–C) ATP decrease and cell death are prevented by BCAA administration but not by supplemental glucose. HeLa cells were cultured under an amino acid deficit and tunicamycin (TM) (3 μg/mL) with different concentrations of glucose (G, 1, 2 and 4.5 g/L), with or without branched chain amino acids (BCAA, B) (40 mM) for 16 hours. (A) Relative ATP levels determined by luciferase activity. *p < 0.05, **p < 0.01 and ***p < 0.001, Tukey HSD. N = 6, (B) Live cell numbers counted after trypsinization. ***p < 0.001, Tukey HSD. N = 6, (C) Representative photographs of HeLa cells. Scale bar: 20 μm. (D and E) Dose dependency of cell protective effect by BCAAs. HeLa cells were cultured with TM (3 μg/mL) and with different concentrations of BCAAs (0, 0.04, 4 or 40 mM) for 16 hours. (D) Relative ATP levels determined by luciferase activity. *p < 0.05, **p < 0.01 and ***p < 0.001, vs. TM without BCAAs, N = 6, Tukey HSD. (E) Live cell numbers counted after trypsinization. **p < 0.01 and ***p < 0.001, vs. TM without BCAAs, Tukey HSD. (F and G) Prevention of decrease of ATP and cell death by each BCAA and by the formulation of BCAAs. HeLa cells were cultured under an amino acid deficit and TM (3 μg/mL). A formulation of BCAAs, or sole isoleucine, leucine, or valine (40 mM each) were added, and cells were cultured for 16 hours. (F) Relative ATP levels determined by luciferase activity. *p < 0.05, **p < 0.01 and ***p < 0.001, vs. TM without BCAA, N = 6, Tamhane. (G) Live cell numbers counted after trypsinization. *p < 0.05 and ***p < 0.001, vs. TM without BCAA, N = 6, Tukey HSD. B, the formulation of BCAAs; I, isoleucine; L, leucine; V, valine. (H and I) Attenuation of decrease of ATP and cell death by BCAAs. HeLa cells were cultured under an amino acid deficit with antimycin (A, 30 μM) or oligomycin (O, 1μg/mL), with or without BCAAs (B, 40 mM) for 24 hours. (H) Relative ATP levels determined by luciferase activity. *p < 0.05, Tukey HSD, N = 6. (I) Live cell numbers counted after trypsinization. *p < 0.05, Tamhane. N = 6. (J and K) Effect of BCAAs on the decrease of ATP and cell death induced by 2-deoxy-D-glucose. HeLa cells were cultured with or without 2-deoxy-D-glucose (2DG, 50 or 100 mM), and with or without BCAAs (B, 40 mM) for 24 hours. (J) Relative ATP levels determined by luciferase activity. *p < 0.05, Tamhane, N = 6. (K) Live cell numbers counted after trypsinization. *p < 0.05, **p < 0.01 and ***p < 0.001, Tamhane, N = 6. (L and M) Inhibition of glycolysis abrogated the effect of BCAAs on the decrease of ATP and cell death under ER stress conditions. HeLa cells were cultured with or without 2DG (50 mM) and with or without BCAAs (B, 40 mM) in the presence of TM (3 μg/mL) for 16 hours. (L) Relative ATP levels determined by luciferase activity. *p < 0.05. **p < 0.01 and ***p < 0.001, vs. TM with neither BCAA nor 2DG, Tamhane, N = 6. (M) Live cell numbers counted after trypsinization. **p < 0.01 and ***p < 0.001, vs. TM with neither BCAAs nor 2DG, Tamhane, N = 6. (N) Calculated amount of consumed glucose/cell. HeLa cells were cultured with or without BCAAs (B, 40 mM) in the presence of TM (1 μg/mL) for 7 hours. **p < 0.01, vs. TM without BCAAs, Tukey HSD, N = 3.