Additional file 1 of Oxidant therapy improves adipogenic differentiation of adipose-derived stem cells in human wound healing

Additional file 1: Supplement Figure 1. Detection of adipocytes in unfixed granulation tissue. Intra-vital confocal microscopy single channel images of unfixed granulation tissue compared with subcutaneous fat tissue (merge displayed in Fig. 1b) stained with BODIPY-493/503 to highlight lipid droplets, wheat germ agglutinin (CF®555 WGA) to highlight ECM substrates and cell morphology (N-acetyl-D-glucosamine and sialic acid present in hyaluronan and other heterogeneous polysaccharides), and Hoechst 33342 staining for nuclei. Supplement Figure 2. PPARG overexpression in day 3 differentiated cells. Representative immunoblotting of PPARG- or puromycin overexpressing human ASC subjected to in vitro differentiation for 3 days in the presence of macrophage-CM. Supplement Figure 3. Numbers of NCT-regulated cytokines. Comparison of cytokines with greater than two-fold change in response to NCT-treatment in various CM. Shown are the total number of regulated cytokines per CM (Set Size), and the number of regulated cytokines found exclusively in one CM or shared between different CM types (Intersection Size). Intersections between different CM types are indicated with connected black dots, single dots indicate a single CM. As shown, 22, 7, 6 and 2 cytokines were found exclusively regulated by M(IFNG/LPS), Mo, M0 and M(IL4/IL13) respectively. Four cytokines were similarly regulated in M(IFNG/LPS) and Mo, 3 in M(IFNG/LPS) and M0, 2 in M0 and M(IL4/IL13), 1 in M(IFNG/LPS) and M(IL4/IL13), and 2 in M(IFNG/LPS), M0 and M(IL4/IL13). Supplement Figure 4. Efficacy of IL1RA to inhibit anti-adipogenic effect of IL1B. IL1B receptor mediated signaling was inhibited by the addition of 100ng/ml recombinant IL1RA. Effect on adipocyte differentiation was evaluated by flow cytometry assessing numbers of lipid-laden cells. Values depict the percentage of lipid-laden adipocytes as a percentage of untreated control cells (0ng IL1B) that emit green fluorescence after staining with BODIPY-493/503, n=3. Statistical significance was determined by using non parametric unpaired Student’s t-test. Data are shown as mean ± SEM. Asterisks indicate p-values < 0.05 (*). Supplement Table 1. Table with log2 cytokine protein expression levels used to generate the heatmap in Fig. 2c. (1) and (2) depict values obtained from two independent measurements. Supplement Table 2. Regulation of cytokines by NCT co-treatment in various CM. Contains log2 fold change (M) and log2 average (A) values representing the extent of regulation, and average expression, of cytokines following NCT co-treatment of CM (e.g. a comparison of NCT co-treated Mo cells against untreated Mo cells). Supplement Table 3. List of Primers used for quantitative RT-PCR.