Fig. 4

CD98hc regulates sphingolipid biosynthesis and integrin mechanosensing. a Schematic strategy to identify CD98hc mutants regulating simultaneously integrin mechanosensing and cell metabolism. b Re-expression of C109S CD98hc mutant in CD98hc-null cells but not of C330S mutant rescues mechanically coupled RhoA activation, one experiment representative of n = 2. c Quantification of RhoA activation in control, C109S or C330S-expressing CD98hc-null cells. Means are plotted with s.e.m. as error bars from n = 2 experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 in a two-way ANOVA. d Volcano plot depicting metabolites fold changes versus p value in C330S versus CD98hc re-expressing CD98hc-null cells. Pink, long chain based sphingoids, ceramides and phytoceramides; magenta, glycosphingolipids and sphingomyelins; green, amino acid metabolism. Numbered metabolites are analogous to the numbering in Fig. 5i. 1, 3-ketosphinganine; 2, sphinganine; 3, N-palmitoyl-sphinganine; 4, N-palmitoyl-sphingosine; 5, palmitoyl dihydrosphingomyelin; 6, sphingosine. P values were calculated using a Student’s t test, values are reported in Supplementary Data 1. e Inhibition of S1P synthesis with dimethyl-sphingosine (DMS) treatment at 10 µM for 1 h does not impair mechanically coupled RhoA activation triggered by FN-coated paramagnetic beads, one experiment representative of n = 2. f Sphingolipid and cholesterol depletion impairs mechanically coupled RhoA activation, one experiment representative of n = 2. g, h Exogenous supply of C12 sphingomyelin to C330S-expressing cells restores mechanically coupled RhoA activation. H is representative of n = 3 experiments. Means are plotted with s.e.m. as error bars in I from n = 3 experiments. ^*P < 0.05 in a Student’s t test