Fig. 6.

Knockdown of CAMKK1 in MSC-ex inactivates the AMPK signalling pathway and increases lipid accumulation.

(A) CAMKK1 expression in MSC-ex^shCtr and MSC-ex^shCAMKK1 was examined by immunoblotting. (B) Representative confocal microscopy images of CD63 (red) and CAMKK1 (green) colocalisation in L02 cells treated with OPA in combination with PBS, MSC-ex^shCtr, or MSC-ex^shCAMKK1; nuclei are labelled with DAPI (blue). Scale bars, 20 μm. (C and D) The expression of AMPK signalling proteins in L02 cells stimulated with OPA (2.0 mM, 2:1 ratio) and PBS, MSC-ex^shCtr (800 μg/ml), or MSC-ex^shCAMKK1 (800 μg/ml) for 24 h was detected by immunoblotting (C) and quantified (D). (E and F) Intracellular lipid droplets in L02 cells were visualised by Nile red staining (E) and quantified by ImageJ for three random areas (F). Scale bars, 100 μm. Data are represented as the mean ± SEM. Statistical analyses by a one-way ANOVA (D and F). ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 vs. PBS group; ^#p <0.05, ^##p <0.01 vs. MSC-ex (800 μg/ml) group. AMPK, AMP-activated protein kinase; CAMKK1, calcium/calmodulin-dependent protein kinase 1; CPT-1A, carnitine palmitoyltransferase 1A; FASn, fatty acid synthase; MSC-ex, MSC-derived exosomes; MSC, mesenchymal stem cell; OPA, oleate and palmitate; p-AMPK, phosphorylated AMPK; PPARα, peroxisome proliferator-activated receptor alpha; SREBP-1C, sterol regulatory element-binding protein-1C.