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 ß-glucosidases GBA1 and GBA2, and analyzed the cell responses using high-throughput RNA sequence. Overall design: 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 ß-glucosidases GBA1 and GBA2 (N=3).