Fig. 5. CerS activity-dependence of labeling with RBM5-177. A375 cells stimulated (+Dox) for AC overexpression were incubated for 4 h with RBM5-177 (5 μM, 4 h), prior to treatment with vehicle, SOCLAC (5 μM, 1 h) or FB1 (100 μM, 24 h). The click reaction was carried out by incubation with CO-1 (1 μM, 10 min). Cells were analysed by fluorescence confocal microscopy (scale bar: 10 μm) (A) or the lipid extracts were analyzed by UPLC-HRMS (B). Lysotracker (75 nM) was used to stain lysosomes. White arrowheads point out putative mitochondrial staining. Co-localization coefficients were measured on the different stacks of images (at least n = 4) with each stack containing 3–10 cells. Pearson's (P) and Manders' (M₁ and M₂) coefficients: RBM5-177-Lysotracker with vehicle (P = 0.816; M₁ = 0.791; M₂ = 0.778) and RBM5-177-Lysotracker with FB1 (P = 0.841; M₁ = 0.717; M₂ = 0.761). Data in (B) (mean ± SD) were obtained from two different experiments in triplicate. Data were analysed by one-way ANOVA followed by Dunnett's multiple comparison post-test if ANOVA P < 0.05 (*, P < 0.001 from vehicle). The right panel in (B) corresponds to the acyl chain of reacylated RBM5-019 (C14, tetradecanoyl, etc.).