A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest

We used human fibroblasts loaded with sucrose as a simple model of lysosomal accumulation, and we verified by high-throughput RNA sequencing which processes were altered by the treatment at two different time points. Moreover, we hypothesized that ceramide production due to the ectopic catabolism of plasma-membrane glycosphingolipids is involved in cell cycle arrest upon sucrose treatment. To test this hypothesis, we treated sucrose-loaded fibroblasts with two specific inhibitors for the β-glucosidases GBA1 and GBA2, and analyzed the cell responses using high-throughput RNA sequence. Poly(A) RNA capture followed by multiparallel sequencing performed in human skin fibroblasts cultured in presence or absence of sucrose. Three series of experiments have been performed. Series nr 1 includes samples cultured in presence or absence of sucrose for 14 days, and was performed on 3 different fibroblast cell lines (F1, L37 and L40, biological replicates), each in duplicate or triplicate, N=14. Series nr 2 includes samples cultured in presence or absence of sucrose for 2 days, and was performed on one fibroblast cell lines (L40) in triplicate, N=6. Series nr 3 includes samples cultured in presence of sucrose for 14 days, and treated for the last 48 hours with CBE and AMP-DNM, which specifically inhibits β-glucosidases GBA1 and GBA2 (N=3).