For cells that are exposed to compound C and metformin (Fig 5C and 5D) why is th

Question

For cells that are exposed to compound C and metformin (Fig 5C and 5D) why is the apoptotic rate decreased?

Figure 5. The AMPK inhibitor reverses the metformin-regulated apoptosis and cytotoxicity of CD19-CAR T cells but does not affect mTOR and HIF1? 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 HIF1? 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.

AMPK pathway is involved in metforminregulated CD19-CAR T-cell apoptosis and cytotoxicity

To further delineate whether the role of metformin on CD19-CAR T cells was AMPK-dependent, compound C, a potent AMPK inhibitor, was applied to block AMPK phosphorylation (Figure 5A). As shown in Figure 5B–D, inhibition of AMPK phosphorylation with compound C reversed the metformin-modulated cytotoxicity and apoptosis in CD19-CAR T cells, whereas the metforminsuppressed mTOR and HIF1? expression was not affected by compound C (Figure 5E and F). Taken together, these results strengthen the opinion that AMPK pathway is involved in metformin-modulated biological functions of CD19-CAR T cells.

A Metformin++ Comp.C p-AMPK 80 AMPK o 60 Control Metformin Mt comp.C Comp.C Metformin O 40 + comp.Co o 20 2 E:T Met (10 mM) Met + comp.C 104 103 a102 101 100 104 103 102 101 100 30 O 20 14.0% 6.63% Metformin t + comp.C 10 13.8% 5.66% 100 10 102 103 104 10 101 102 103 104 Annexin V Metformin (10 mM) Time (min) 05 10 30 60 c 1.5 p-AMPK p-mTOR 0.5 mTOR HIF1? ?-Actin- ick 0.0 p-AMPK p-mTORHIF1a

Explanation / Answer

It is given in the information that -

Figure 5D is the statistical analysis of the flow cytometery results of figure 5C. Now keeping in mind the facts given above, lets look at figure 5D. Metformin has a higher apoptotic rate because it works along with a fully activated AMPK pathway which causes apoptosis. The black bar graph in figure 5D (Met. + Comp C) shows a reduced apoptotic rate since compound C is an AMPK inhibitor and inhibits the same. This stops the synergistic effect of metformin and the AMPK pathway which was causing higher % ofapoptosis and cytotoxicity. As a result lower apoptotic rate % can be seen in figure 5D(Met. + Comp C).

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