Mesenchymal Stromal Cells Accelerate Epithelial Tight Junction Assembly via the AMP-Activated Protein Kinase Pathway, Independently of Liver Kinase B1

Figure 1

Role of the AMPK kinases, LKB1 and CaMKK, in AMPK activation and ZO-1 relocation following a Ca²⁺ switch in MDCK cells. Representative immunoblotting (a) and quantifications (b) of phospho-acetyl-Coa carboxylase (pACC), phospho-AMP-activated protein kinase (pAMPK), and total AMPK (AMPKt) in low Ca²⁺ conditions (S-MEM) and following Ca²⁺ switch using MDCK cells or LKB1-shRNA MDCK cells. Compounds STO-609 and C were used as CaMKK and AMPK inhibitors, respectively. Quantifications of immunoreactive signals were performed by stain-free method after normalization to total protein content of each lane. Quantifications of phospho-ACC, phospho-AMPK, and AMPKt signals following Ca²⁺ switch were calculated and expressed by the ratio to the immunoreactive signal of SMEM condition in each individual experiment (a). For the sake of bar-graph clarity (b), SMEM values of all experiments were normalized to 1 in order to represent mean ratios of phospho-ACC/total protein content and phospho-AMPK/AMPKt in different experimental conditions (b). Data are presented as mean ± SD; ns: not significant, , . Representative immunofluorescence (c) and quantifications (d) of ZO-1 deposits at increasing time points following Ca²⁺ switch in similar conditions as in (a) and (b) (scale bar: 16 μm). No statistically significant difference was observed between MDCK and LKB1-shRNA MDCK (ns: not significant). MDCK exposed to compound C () or STO-609 (^§) showed a significant reduction of ZO-1 relocation in comparison to control MDCK. Data are presented as mean ± SD.

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