Figure 5

Impairment of lysosomal dysfunction causes the accumulation of LD. (A) Endothelial cells were pre-treated with either AICAR (200 μM) or metformin (100 μM) 30 min prior to PA (200 μM). The acidic lysosome was stained with a lysotracker (red), and the nucleus was stained with Hoechst 33342 (blue). Treatment with PA elevated pH of the lysosome and pre-treatment with AICAR or metformin restored the acidic PH of the lysosome. (B) Endothelial cells were transfected with scrambled or siRNA for LAL. siRNA for LAL reduced the expression of LAL. (C) The knock-down of LAL blunted the effect of metformin in LD reduction. Human endothelial cells were transfected with siRNA for scrambled or LAL. Endothelial cells were pre-treated without or with metformin (100 μM) 30 min prior to PA (200 μM). LD was stained with BODIPY (green). The number of LD was quantified by using Image J and is shown in bar graphs (mean ± SEM). ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 indicate that the samples are statistically different between the indicated samples. (D) The knock-down of LAL increased the co-localization of lipid droplet (BODIPY, green), with LAMP-1 (lysosome marker, red). Six microscopic fields per condition were obtained and quantified by using Image J. The quantification of colocalization of LD with LAMP-1 is quantified with Image J and is shown in bar graphs (mean ± SEM). **p < 0.01 and *p < 0.05, indicates that the samples are statistically different between the indicated samples. The full-length agarose gel is presented in Supplemental Figure S5A, and the inverted image is presented in Supplemental Figure S5B. The dotted lines in the Supplemental figures indicate the cropped area.