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. 2022 Jun 3;10(6):1316. doi: 10.3390/biomedicines10061316

Figure 4.

Figure 4

Effect of β-glucosidase inhibitors on rottlerin effects in a model of impaired intracellular lipid trafficking. (A) The graph represents β-glucosidase activity. C6 cells were incubated in LPDS medium in the presence of AMP-deoxynojirimycin (AMP-DNM) or isofagomine D-tartrate (IFG) for 2 h, then treated with or without (Control) rottlerin (Rot), and the reaction substrates for β-galactosidase (C12FDG) or β-glucosidase (FDGlu) were added for the last 30 min and finally analyzed by flow cytometry. (A) β-glucosidase activity. (B) β-galactosidase activity. Results are the mean ± SEM of three independent experiments. The results with rottlerin are normalized to 1. MIF, median intensity of fluorescence. (C) Total number of particles per experiment analyzed by nanoparticle tracking. The experiments started with the same number of cells in all the conditions. Means ± SEM of three measures. (D) Sphingolipid cell contents. Cells were incubated without (Control) or with 5 μM rottlerin (Rot) in combination with or without AMP-DNM or IFG for 4 h. Ceramides, hexosylceramides, and sphingomyelin were measured by mass spectrometry as described in Methods. Results are the mean ± SEM of three independent experiments. The control is normalized to 1. Statistical comparisons shown are rottlerin versus control (* p < 0.05, ** p < 0.01, *** p < 0.001) or β-glucosidase inhibitor plus rottlerin versus rottlerin alone (+ p < 0.05, ++ p < 0.01, +++ p < 0.001).