Fig. 6

Spatial oxidation of LPL at the cell periphery. a Confocal microscopy images of MV3 LPL eGFP cells upon PLA staining. Cells were kept untreated or were treated with 100 µM H₂O₂. Then, the cells were fixed with PFA in the presence of 5 mM dimedone and stained for LPL and dimedone with specific antibodies and subjected to PLA. Ten xyz optical sections/sample were imaged in each experiment using confocal microscopy. Representative images of eGFP-, wt LPL eGFP-, and C101A LPL-expressing MV3 cells in the absence and presence of H₂O₂ are shown. Shown are nuclei (white), LPL eGFP (green) and PLA puncta (red). Each red punctum represents sulfenylation on LPL. Scale bar = 5 µm; ×100 magnification. b, c Quantification of the PLA puncta/cell in the presence or absence of H₂O₂. b Fifty cells were imaged in each of three independent experiments. Each dot in the histograms represents the average PLA puncta/cell for a different experiment. c Cumulative comparison of the PLA puncta/cell values in eGFP-, wt LPL eGFP-, and C101A LPL eGFP-expressing MV3 cells in the absence or presence of H₂O₂. A total of 150 cells/sample from three independent experiments were imaged. Each dot represents a cell from one of three experiments. d Quantification of the PLA puncta/cell in untreated versus DPI- or H₂O₂-treated MV3 cells expressing eGFP, wt LPL eGFP, or C101A LPL eGFP. P-values were calculated by one-way ANOVA. The data are presented as the mean ± SEM (n ≥ 3; *p < 0.05, **p < 0.01, ***p < 0.001, ns nonsignificant)