Fig 5. SBDS mutation results in increased lysomome trafficking and activity, and a decrease in ATP and lactate levels predicted by changes in the steady-state of the mRNAs identified by RNA-seq.

(A) Changes in the steady-state levels (total, bona fide for transcriptional) of genes with peptidase activity as detected both on polysomes and total. Heat maps representing absolute gene expression levels in Sbds^MOCK and Sbds^RESCUE samples for the subset of gene sets with peptidase activity by Gene Ontology analysis. (B) qPCR validation of selected genes associated to lysosome activity. Real time analysis of selected genes associated to lysosome trafficking and activity. Data shown for n≥3; mean±s.d., T-test, paired, two-tailed. (C) Sbds^MOCK cells have a decrease in intracellular pH value. Representative graph showing increased lysotracker fluorescence intensitity in Sbds^MOCK cells respect to Sbds^RESCUE cells. This result suggests more lysosome trafficking and activity in Sbds^R126T/R126T cells. Data shown for n≥3; mean±s.d., T-test, paired, two-tailed. (D-E). Lamp1 immunostaining (representative cells, D) and quantitation (E). Scale bar indicates 8.3μm. Globally, A-E show increase in the proteolitic potential of Sbds^MOCK respect to Sbds^RESCUE cells. (F-G) SBDS levels positively regulate ATP accumulation (F) and lactate/pyruvate production, an index of glycolysis (G). All graphs represent mean ± s.d. Statistic applied was T-test, paired, two-tailed, n≥4.