What method/assay was used to make/generate Fig. 4? Please explain/summarize the

Question

What method/assay was used to make/generate Fig. 4? Please explain/summarize the results/findings in Fig 4B. Thank you!

Figure 4. Metformin increases the expression of p-AMPK and reduces the expression of p-mTOR and HIF1? inCD19-CAR T cells. (A) The CD19-CAR T cells were treated by 10 mM metformin for different times (0, 5, 10, 30, and 60 minutes), and the expression of p-AMPK, p-mTOR, and HIF1? was detected by Western blotting analysis. (B) Statistical analysis of relative p-AMPK, p-mTOR, and HIF1? protein expression results. *P,0.05, **P,0.01 versus the control group. Data are represented as mean ± SD from three independent experiments. Abbreviations: CAR, chimeric antigen receptor; p-AMPK, phosphor AMP-activated protein kinase.

Metformin promotes AMPK phosphorylation and suppresses mTOR and HIF1? expression in CD19-CAR T cells

It is well documented that AMPK/mTOR/HIF1? signal pathway play a critical role in metformin-modulated lymphocytes energetic metabolism. To further explore the latent mechanism about metformin on CD19-CAR T cells, the AMPK, mTOR, and HIF1? signaling pathway was examined. As shown in Figure 4A, the AMPK phosphorylation was upregulated when treated with 10 mM metformin, whereas mTOR phosphorylation was downregulated. Meanwhile, metformin also downregulated the expression of HIF1?. The regulation was statistically significant, as quantified by densitometry (Figure 4B). These results indicated that AMPK/mTOR/HIF1? signal pathway may take part in the metformin-modulated glucose metabolism of CD19-CAR T cells.

Explanation / Answer

In figure 4A western blot was done to check the phosphorylation of AMPK and mTOR expression level of HIF1 alpha in presence 10 mM concentration for different time point.

In case of Figure 4B densitometric analysis was done from the 4A figure from three independent set of experiments for statistical analysis. In case of p-AMPK, all the time point were showing the significant increase in the phosphorylation at the different time point. In case of p-mTOR, the phosphorylation was decreased upon the treatment with the drug and all the time all the time point were showing the significant decrease in the phosphorylation at the different time point.

In case of HIF1alpha, only the time with point of 60 minutes was showing the significant decrease in the levels

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