Fig 2: SCD inhibition alters cellular cardiolipin composition leading to cytochrome C release and sensitisation towards apoptosis. a DU145 cells were treated with different concentrations of SCD inhibitor (SCDi II) and proliferation was determined by BrdU labelling. b Cells treated as in a were used to determine apoptosis using Annexin V staining. Data represent mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t test (*p = 0.05). c Quantitative lipid profiling of cardiolipin (CL) content of DU145 cells grown for 48 h in medium containing full (FS) or low (LS) serum using LC-MS/MS. Concentrations of mono-unsaturated (=4 double bonds) and poly-unsaturated (>4 double bonds) CL species are displayed. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t test (***p = 0.001). d DU145 cells were treated with SCD inhibitors (100 nM) or solvent (DMSO) for 48 h in medium containing low serum or medium supplemented with BSA or BSA-coupled oleic acid (BSA-Oleate). Quantitative lipid profiling of CL content was determined using LC-MS/MS. Concentrations of mono- and poly-unsaturated CL species are displayed. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t tests (*p = 0.05). e DU145 cells were treated with SCD inhibitor (SCDi I) or solvent (DMSO) for 48 h in medium containing low serum or medium supplemented with BSA or BSA-oleate. Cells were lysed by digitonin, and the presence of cytoplasmic cytochrome C was determined. UV treatment was used as positive control. Vinculin is shown as loading control. Levels of phosphorylated Akt (S473) and total Akt were detected in total lysates. f DU145 cells were treated as in e but 3 µM of Akt inhibitor was added prior to addition of BSA-oleate. Cytoplasmic cytochrome C was detected in digitonin lysates. g DU145 cells were treated with SCD inhibitor (SCDi I) or solvent (DMSO) in medium containing full (FS) or low (LS) serum or medium supplemented with BSA or BSA-oleate. The presence of full-length and cleaved (cl.) PARP was determined. Actin is shown as loading control. h DU145 cells were transfected with siRNA targeting SCD (siSCD) or non-targeting controls (siCtrl) and cultured as in e. The presence of full-length and cleaved (cl.) PARP was determined. Actin is shown as loading control. i DU145 or MDA-MB-468 cells were treated with SCD inhibitor (100 nM) for 48 h, and oxygen consumption rate (OCR) before and after addition of oligomycin, FCCP and rotenone was determined using a Seahorse Bioanalyzer. j–m DU145 cells were treated with the indicated doses of SCD inhibitor (SCDi II) either alone or in combination with different doses of metformin (Metf, j), rotenone (RN, k), paclitaxel (PTX, l) or staurosporin (STP, m) for 72 h in medium containing low serum. Cell viability was determined by crystal violet staining. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t tests (*p = 0.05, **p = 0.01 compared to SCDi alone; #p = 0.05, ##p = 0.01 compared to no SCDi)

Fig 3: pAktSer473/Akt ratios are not altered in different compartments of stimulated motoneurons. After 5 DIV in the presence of CNTF (5 ng/ml), the cells were serum as well as neurotrophic factor deprived for 6–7 h and stimulated for 5 min with BDNF (20 ng/ml), followed by fixation and staining against pAktSer473, Akt, and GFP. Compartment-specific quantification of the pAktSer473/Akt ratios is shown in Fig. S9. pAktSer473/Akt ratios strongly increase upon BDNF stimulation, but comparable pAktSer473/Akt values were achieved after Syap1 knockdown or mock treatment in all the tested compartments of the motoneurons. Scale bars Soma and axon: 10 µm; growth cone: 5 µm

Fig 4: Representation of experimental and model-derived CVs. The average of the kinetics of mCherry-AKT membrane association in 10 individual cells for (A) the clone Hepa1_6-D8 and (B) clone Hepa1_6-E2 are shown with the standard error of the mean indicated for each time point. (C + D) The calculated dynamics for 10 simulated cells with extrinsic noise contribution as given by the parameters are shown. For the clones (E) Hepa1_6 D8 and (F) E2 the CV's for the experimental fluctuations in mCherry-pAKT (blue line), theoretical intrinsic fluctuations in mCherry-pAKT (green line), and the corresponding combination of extrinsic and intrinsic fluctuation (red line) are plotted.

Fig 5: The SeP-neutralizing Ab improved glucose metabolism impaired by excess SeP. a, b Neutralizing mAb AE2 improved glucose tolerance impaired by human SeP administration in vivo. After the administration of AE2 and hSeP following the scheme of Supplementary Fig. 4a, glucose (1.5 g/kg) was administered, and then blood glucose was determined (a, n = 6–8, means ± s.e.m.). Open triangle, PBS/control IgG; green square, PBS/AE2; red circle, hSeP(+)/control IgG; blue circle, hSeP(+)/AE2. **P < 0.01, when compared hSeP(+)/control IgG group with other groups, Tukey-ANOVA. Area under the curve for blood glucose levels is shown in b (n = 6–8, means ± s.e.m.). *P < 0.05, Student's t-test. c–e Neutralizing mAb AE2 improved insulin resistance impaired by SeP administration in vivo. After the administration of AE2 and hSeP following the scheme shown in Supplementary Fig. 4a, insulin (0.5 U/kg) was administered, and blood glucose was determined (c, n = 5–8, means ± s.e.m.). **P < 0.01, ***P < 0.001, Tukey-ANOVA. Area under the curve for blood glucose levels is shown in d (n = 5–8, means ± s.e.m.). ***P < 0.001, Student's t-test. Effect of AE2 mAb administration on phosphorylation of insulin receptor (IR) and Akt in skeletal muscle (e). In AE2- and hSeP-treated mice, muscle tissues were excised 15 min after insulin administration (0.5 U/kg), and analysed by western blotting (n = 5–8, means ± s.e.m.). *P < 0.05, Student's t-test. f Blood insulin levels were significantly decreased by human SeP administration. Blood insulin levels were determined 2 h after second hSeP or PBS administration under feeding conditions (n = 5, means ± s.e.m.). **P < 0.01, Student's t-test. g Blood insulin levels were significantly increased by AE2 administration under feeding conditions (n = 5, means ± s.e.m.). *P < 0.05, **P < 0.01, Tukey-ANOVA. h Blood insulin levels were significantly increased during a glucose-tolerance test. At each designated time, blood insulin was determined (n = 7–8, means ± s.e.m.). *P < 0.05, **P < 0.01, Tukey-ANOVA (c, h). Closed circle, hSeP(+)/control IgG; open circle, hSeP(+)/AE2