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. 2023 Oct 10;14(10):664. doi: 10.1038/s41419-023-06202-3

Fig. 3. Increased GYS1 expression in Drp1 knockdown cells is transcriptionally regulated by AMPK.

Fig. 3

A Cell lysates of sh-Ctrl, sh-Drp1 PT130 cells were analyzed for the expression of Drp1, GYS1, phospho-AMPK (p-AMPK), total AMPK and β-actin using western blot. B Representative western blot as shown in (A) were quantified to determine the relative p-AMPK levels by normalizing p-AMPK to total AMPK. Data were presented as mean ± SD (n = 3, *p < 0.05 and **p < 0.01). C Sh-Ctrl and sh-Drp1 PT130 cells were treated with DMSO or AMPK inhibitor compound C (AMPKi, 10 μM) for 24 h in low glucose media. Cell lysates were analyzed for the expression of Drp1, GYS1, p-AMPK, phospho-ACC (p-ACC), total ACC, total AMPK, and β-actin using western blot. D Representative western blots as shown in (C) were quantified to determine the relative GYS1 levels by normalizing GYS1 to β-actin. Data were presented as mean ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). E The relative expression of GYS1 mRNA was determined using RT-qPCR in sh-Ctrl and sh-Drp1 PT130 cells treated with DMSO or AMPKi. Data were presented as mean ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). F Sh-Ctrl and sh-Drp1 PT130 cells were treated with DMSO or AMPKi in low glucose media for 24 h. Representative confocal images were obtained from cells stained with the anti-glycogen antibody. Scale Bar, 10 μm. G The relative fluorescence intensity of glycogen staining was quantified using ImageJ fluorescence analyzer. Data were presented as mean ± SD (n = 20, *p < 0.05 and ****p < 0.0001).