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. 2018 Apr 3;11:1767–1776. doi: 10.2147/OTT.S154853

Figure 5.

Figure 5

The AMPK inhibitor reverses the metformin-regulated apoptosis and cytotoxicity of CD19-CAR T cells but does not affect mTOR and HIF1α expression. (A) CD19-CAR T cells were treated with 10 mM metformin and/or compound C for 24 hours. The expression of p-AMPK was analyzed by Western blotting (up) and the statistical analysis of relative p-AMPK protein expression results is shown (down). (B) CD19-CAR T cells were cocultured with Raji cells with 10 mM metformin and/or compound C at various effector to target cell ratios (1:1, 5:1, 10:1, 20:1, and 40:1) for 24 hours, and the apoptotic rate was determined by flow cytometry. (C) CD19-CAR T cells were treated with 10 mM metformin and/or compound C for 24 hours, and the apoptotic rate of CD19-CAR T cells was detected by flow cytometry. (D) Statistical analysis of the flow cytometry results. (E) CD19-CAR T cells were treated with 10 mM metformin and/or compound C at different times (0, 5, 10, 30, and 60 minutes), and the expression of p-AMPK, p-mTOR, and HIF1α was analyzed by Western blotting analysis. (F) Statistical analysis of the Western blot results. *P<0.05, **P<0.01 versus the control group. Data are represented as mean ± SD from three independent experiments.

Abbreviations: p-AMPK, phosphor AMP-activated protein kinase; CAR, chimeric antigen receptor; Met, metformin; Comp, compound; E:T, effector to target cell ratio; PI, propidium iodide.