Skip to main content
. 2022 Oct 9;12(16):7158–7179. doi: 10.7150/thno.78376

Figure 2.

Figure 2

PA-S14 is a novel LKB1 activator through binding with D176 residue in kinase domain. (A) The detailed structural formula of PA-S14. (B) MTT assay in HKC‐8 cells treated with PA or PA-S14 at different concentrations. *P < 0.05, #P < 0.01, †P < 0.001 versus control group. (C) The ATP production in PA or PA-S14-treated HKC‐8 cells. *P < 0.05, #P < 0.01, †P < 0.001 versus control group. (D) Complex I activity was measured by spectrophotometrical assay in PA or PA-S14-treated HKC‐8 cells. *P < 0.05, #P < 0.01, †P < 0.001 versus control group. (E) The interaction binding mode diagram of LKB1 and PA-S14 was established based on the homology modeling technique in Discovery Studio 2021. The electrostatic surface of LKB1 is shown in blue, positively charged; red, negatively charged; white, neural residues. (F, G) Sequence analyses showed a high conservation sequence among human, mouse, and rat species. (H) The affinity of LKB1 to PA-S14 was shown. SPR analyses showed dose-dependent (0.5-50 µM) binding of PA-S14 to LKB1 on an immobilized sensor chip. The fitted constants are ka = 54.9M-1 S-1; kd = 6.57 × 10-6 S-1; KD = 1.2× 10-7 M. (I-J) Representative western blot and quantitative data showing the ratio of p-LKB1/LKB1. LKB1 kinase domain was mutated at five sites, i.e. E130, C132, D176, S193 and V197. ###P < 0.001 versus pFlag-LKB1 group. (K) Representative graphs showed D176 mutation (▲176) blocked the binding of LKB1 with MO25 or STRAD. Hela cells were transfected with pFlag-LKB1 or D176 mutated plasmid, and then treated with PA-S14 (0.5 µM) for 6 h. Co-immunoprecipitation was performed. Whole cell lysates were immunoprecipitated with an antibody against LKB1 and blotted with antibodies against MO25 and STRAD. (L) Representative graphs showing the binding of LKB1 with MO25 or STRAD. HKC-8 cells were treated with or without PA-S14 (0.5 µM) for 6 h. Whole cell lysates were immunoprecipitated with an antibody against LKB1 and blotted with antibodies against MO25 and STRAD. (M) HKC‐8 cells were treated with PA-S14 (0.5 µM) for indicated time period (0, 0.25, 0.5, 1, 6, 12, 24, 48 h). The expression levels of p-LKB1, LKB1, p-AMPKα, and AMPKα were then assessed by western blotting. (N) Representative western blot showing the expression of p-AMPKα. HKC-8 cells were pretreated with PA-S14 (0.5 µM) for 1 h, followed by the stimulation of PP2Cα for 24 h. (O) Representative western blot showing protein expression of p-LKB1, LKB1, p-AMPKα, and AMPKα in different groups. HKC-8 cells were pretreated with PA-S14 (0.5 µM) for 1 h, followed by the stimulation of TGF-β1 (5 ng/ml) for 24 h. Numbers (1-3) indicate each individual culture in a group.