Figure 1
From: AMPK contributes to autophagosome maturation and lysosomal fusion
Generation of AMPK α1 knockout (KO) HEK293T cells. (A) Validation of AMPK α1 KO by T7 endonuclease 1 (T7E1) assay. HEK293T cells were transfected with either pX459/AMPK α1 gRNA #1 or pX459/AMPK α1 gRNA #2, and single colonies were isolated. The genomic PCR products were analyzed by T7E1 assay. Two different clones (KO #1 and KO #2) were used for the further experiments. (B) DNA sequencing analysis revealed the presence of AMPK α1 mutation in KO #1 and KO #2 (Top). The CRISPR-Cas9 system introduced an indel mutation in the target sites of the AMPK α1 gene (bottom). (C) Validation of AMPK α1 KO by Western blotting. Equal amounts of HEK293T wild-type (WT) and AMPK α1 KO cell lysates were subjected to Western blotting with an anti-AMPK α1 antibody. Experiments were repeated three times with similar results. (D) Proliferation of AMPK α1 KO cell lines was analyzed by MTT assay. The MTT assay was performed in triplicate, and the graph shows the average and standard deviation (SD). Control vs. knockout cells. *P < 0.05, **P < 0.01. (E) Phase contrast microscope images (400x) of control cells and AMPK α1 knockout cells.
