Additional file 1 of Fluid shear stress induces a shift from glycolytic to amino acid pathway in human trophoblasts

Additional file 1: Figure S1. Validation of cell viability by staining of cleaved caspase 3. First trimester placental villi were stained for cleaved caspase 3 and E-cadherin. A positive control with first trimester villi treated with Staurosporin (2 µM) for 4 h as previously described [64] was used (a, arrowheads indicate caspase 3 positive cells). Placental villi cultured under flow (c) did not show increased caspase 3 activation, when compared to static conditions (b). Figure S2. Syncytiotrophoblast markers in response to fluidic flow. Gene expression patterns (relative rlog values) of differentiated BeWo cells treated either under static conditions (light grey legend) or flow culture (dark grey legend) for 24 h. Figure S3. Key steps in glycolysis. Glycolysis converts glucose to pyruvate (left pathway), while GOT1 and GOT2 convert cysteinesulfinate to pyruvate and taurine (centre path). Cysteine can also be converted to glutathione (right path). Figure S4. Effects of fluidic flow on the glucose uptake and their final processing in trophoblasts. Intracellular uptake of 2-NBDG-Glucose by differentiated BeWo cells either under static or fluidic flow culture (3 ml/min) for 24 h (a). Glucose concentration in supernatant in undifferentiated (DMSO) and differentiated (forskolin) BeWo cells, cultured under static or flow conditions (b). Expression of lactate dehydrogenase subunit B (c, encoded by LDHB) and component X of the pyruvate dehydrogenase (d, PDHX) was analyzed by qPCR. Scale bar in (a) represents 50 µm. Data are presented as mean ± SEM. Experiments were performed with a minimum of three different cell passages. Experiments with placental explants (c and d) were performed with four different placenta samples. Figure S5. Effect of fluidic flow culture on HUVEC. GLUT1 (encoded by SLC2A1) mRNA (a) and protein (b and c) expression, as well as glucose concentration in supernatant (d) and HK2 mRNA expression (e) was analyzed in HUVEC, which were cultured for 24 h either under static or fluidic flow culture conditions. In the same samples, GOT1 mRNA (f) and protein (b, middle panel and g) as well as GOT2 mRNA (h) and protein levels (i and j) were analyzed. Data are presented as mean ± SEM. Static samples were used as controls and set to 1. Experiments were performed with five different cell passages. Figure S6. Flow-dependent change of intracellular amino acid composition and expression of enzymes involved in glutathione biosynthesis. Relative amount of amino acids (a) was determined by mass spectrometry in cell lysates from differentiated BeWo cells cultured for 24 h either under static conditions (white) or flow exposed cells (blue). Additional measured mass spectrometry data were able to detect intracellular (b) and extracellular (c) cysteine. Mass spectrometry data were normalized to total cell protein and static conditions set to 1. Glutathione (GSH) biosynthesis involves two ATP-dependent steps, including initial synthesis of γ-glutamylcysteine (γ-Glu-Cys) from l-glutamate and cysteine, catalysed by γ-glutamate-cysteine synthase (γ-GCS). In a second step, glycine (Gly) is added to the C-terminus of γglutamylcysteine by glutathione synthetase (GSS) (d). Expression of glutathione synthase (e, GSS) and glutamate-cysteine ligase catalytic subunit (f, GCLC). Data of amino acids are presented as mean ± SEM. Experiments were performed with five different cell passages; statistical analysis was done with one sample t-test. ns (not significant) Data from the glutathione pathway are presented as mean ± SEM and were obtained from three different experiments. Statistical analysis was done with an unpaired t-test for c and d. Figure S7. Effect of SLC2A1-silencing and different glucose availability on expression of HK2 and PFKP. HK2 expression was determined by qPCR in differentiated BeWo cells after GLUT1-silencing (a), encoded by SLC2A1. Gene expression of HK2 (b) and phosphofructokinase (c) encoded by PFKP in differentiated BeWo cells cultured under different glucose concentrations for 24 h. Data are presented as mean ± SEM. Experiments were performed with a minimum of three different cell passages. Figure S8. Placental GOT2 mRNA expression. GOT2 expression was determined by qPCR in placental tissue cohorts from Berlin (a; controls, n=9 and IUGR, n=4) and Graz (b; controls, n=5 and IUGR, n=5). Figure S9. Uncropped representative Western blot images, as acquired with the iBrightTM software. Table S1. Differential gene expression between static vs 3 ml/min is provided online in a separate excel sheet (Supplemental Table 1_DGA). Table S2. Baseline characteristics of CTRL and IUGR cases (Cohort Graz, used for protein- and qPCR analysis). Table S3. Baseline characteristics of CTRL and IUGR cases (Cohort Munich, used for immunofluorescence). Table S4. Baseline characteristics of CTRL and IUGR cases (Cohort Berlin, used for qPCR). Table S5. Primer sequences. Table S6. Antibodies used for immunoblotting. Table S7. Primary antibodies for immunofluorescence staining. Table S8. Primers for in situ hybridization. Table S9. Padlock probes for in situ hybridization. Table S10. Detection Oligos in situ hybridization.