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. 2022 Dec 16;11(24):4094. doi: 10.3390/cells11244094

Figure 1.

Figure 1

Metformin activates AMPK in a dose−dependent manner in NIH 3T3 cells. The efficiency of siRNA and shAMPK knockdown of AMPK in NIH 3T3 cells was verified by Western blotting for further experiments. (A,B) Protein expression of pAMPK and AMPK in NIH 3T3 cells with various doses of metformin treatment was detected by Western blotting. GAPDH was used as an internal control. Densitometry analysis of the Western blots demonstrated pAMPK/AMPK ratios. (C,D) Western blotting was used to measure the protein expression levels of pAMPK and AMPK in the Blank, siNC, si−Prkaa1−mus−827, si−Prkaa1−mus−1250, and si−Prkaa1−mus−1337 groups. Densitometry analysis of Western blotting results. (E) AMPK knockdown lentiviruses with enhanced green fluorescent protein were successfully transfected into NIH 3T3 cells. (Above, left) Small hairpin negative control group in bright field. (Above, right) Small hairpin negative control in fluorescent light. (Second row, left) Small hairpin AMPK group 1 in bright field. (Second row, right) Small hairpin AMPK group 1 in fluorescent light. (Third row, left) Small hairpin AMPK group 2 in bright field. (Third row, right) Small hairpin AMPK group 2 in fluorescent light. (Below, left) Small hairpin AMPK group 3 in bright field. (Below, right) Small hairpin AMPK group 3 in fluorescent light. BF, bright field; GFP, green fluorescent protein. Scale bar = 100 μm. (F,G) The protein expression levels of pAMPK and AMPK in the Blank, shNC, shAMPK1, shAMPK2, shAMPK3 groups. Statistical results using relative gray values from the blotting. All data are expressed as the means ± SDs of values from triplicate experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001. Control: NIH 3T3 cells, LPS: NIH 3T3 cells treated with 1 μg/mL LPS for 24 h, Met (2.5 mM): NIH 3T3 cells treated with 2.5 mM metformin for 24 h, Met (5 mM): NIH 3T3 cells treated with 5 mM metformin for 24 h, Met (10 mM): NIH 3T3 cells treated with 10 mM metformin for 24 h, Blank: NIH 3T3 cells, siNC: siAMPK negative control group, si−Prkaa1−mus−827: NIH 3T3 cells transfected with si-Prkaa1-mus-827, si-Prkaa1-mus-1250: NIH 3T3 cells transfected with si−Prkaa1−mus−1250, si-Prkaa1−mus−1337: NIH 3T3 cells transfected with si−Prkaa1−mus−1337, shNC: lentivirus AMPK negative control group, shAMPK1: lentivirus AMPK knockdown group 1, shAMPK2: lentivirus AMPK knockdown group 2, shAMPK3: lentivirus AMPK knockdown group 3.