Schematic of the proposed signaling in MSC spheres activating the production of TSG6, STC1, and PGE2. amount. Supplemental Number 2. Phases of MSC sphere formation. MSC compact into spheres when cultured in hanging drops. During the culture, the average size of the MSC decreases to approximately one fourth in 3 days. Supplemental Number 3. MSC in spheres up-regulate the manifestation of genes for IL1 signaling molecules. (A-D) The manifestation of IL1A, IL1B, IL1R1, and IRAK2 are increased when MSC are cultured in hanging drops. Adh Low sample was used like a baseline. Ideals are mean SD (= 3). (E-G) Dynamic changes in the manifestation of IL1A, IL1B, and IL1R1 during compaction Cambinol of MSC into spheres. Ideals are mean (assay triplicates) RQs using time 0 like a baseline. Abbreviations: Adh Large, adherent monolayer MSC plated at high denseness (5000 cells/cm2) and incubated for 3 days; Adh Low, adherent monolayer MSC plated at low denseness (100-150 cells/cm2) and incubated for 7 days; Adh VH, adherent monolayer MSC plated at very high denseness (200000 cells/cm2) and incubated for 3 days; RQ, relative amount; Sph, sphere MSC Cambinol from 3 day time hanging drop cultures. Supplemental Number 4. MSC in spheres up-regulate the manifestation of genes for Notch signaling molecules. (A-C) The manifestation of DLL1, NOTCH2, and HEY1 are improved when MSC are cultured in hanging drops. Adh Low sample was used like a baseline. Ideals are mean SD (= 3). (D,E) Dynamic changes in the manifestation of JAG1 and HEY1 during compaction of MSC into spheres. Ideals are mean (assay triplicates) RQs using time 0 like a baseline. Abbreviations: Adh Large, adherent monolayer MSC plated at high denseness (5000 cells/cm2) and incubated for 3 days; Adh Low, adherent monolayer MSC plated at low denseness (100-150 cells/cm2) and incubated for 7 days; Adh VH, adherent monolayer MSC plated at Cambinol very high denseness (200000 cells/cm2) and incubated for 3 days; RQ, relative amount; Sph, sphere MSC from 3 day time hanging drop cultures. Supplemental Number 5. Effects of caspase and NFB activation inhibition on secretion of IL1 and IL1. (A,B) Caspase inhibition (10 M Q-VD-OPh) in MSC spheres reduces the secretion of IL1 and IL1. (C,D) NFB inhibition (1 M QNZ) in MSC spheres reduces the secretion of IL1 and IL1. Ideals are mean SD (= 3). *, .05; ***, .001 compared to vehicle control MSC spheres (DMSO). Abbreviations: Casp, caspase; CM, conditioned medium; DMSO, dimethyl sulfoxide; inh, inhibitor; ND, not detected; RQ, relative amount; Sph, sphere MSC from 3 day time hanging drop cultures. Supplemental Rabbit Polyclonal to SNAP25 Number 6. Effects of IL1ra on manifestation of genes for enzymes required for PGE2 synthesis in MSC. (A-D) Effects of blocking IL1 signaling with IL1ra (500 ng/ml) on COX2, PTGES, PLA2G4A, and PLA2G4C manifestation in MSC spheres. Adh Low sample was used like a baseline. Ideals are mean SD (= 3). ns, .05; *, .05; ***, .001 compared to vehicle control MSC spheres (0). Abbreviations: Adh Low, adherent monolayer MSC plated at low denseness (100-150 cells/cm2) and incubated for 7 days; RQ, relative amount; Sph, sphere MSC from 3 day time hanging drop cultures. Supplemental Number 7. Effects of IL1 antibody combination on manifestation of genes for enzymes required for PGE2 synthesis in MSC. Cambinol (A-D) Mixture of antibodies to IL1, IL1, and IL1R1 (0.33, 1, 3, or 6 g/ml) decreases the manifestation of COX2, PTGES, PLA2G4A, and PLA2G4C in MSC spheres. Adh Low sample was used like a baseline. IgG antibody was used at 18 g/ml. In (C) and (D) IL1 antibody dose was 18 g/ml. Ideals.