At 12?hours after 5zox removal, more than 90% of the cells were still maintained at the G0/G1 stage

At 12?hours after 5zox removal, more than 90% of the cells were still maintained at the G0/G1 stage. the scrambled control RNA or siRNA against Tak1 (siTak1). Asterisks imply significant differences between control and the 5zox treated group at P? ?.05 (N = 6). STEM-37-1595-s004.tiff (9.3M) GUID:?76C2BB11-C026-49A0-985C-259A098D0FDF Supplementary figure S4 Effect of Tak1 inhibition on cell cycle status and cell viability. A, FACS analysis of cell cycle status in the BMMSCs treated with vehicle (same amount of DMSO) or 5zox by the fucci G1\orange/S/G2M\green system. NTC means nontreatment control. 5zox treatment was performed at 20?nM for 6?days. E3330 B, western blot (WB) based caspase 3 assay for the BMMSCs treated with 5zox. Full means uncleaved caspase 3 with full molecular weight managed. Cleaved means caspase 3 treated with digestion as a result of apoptosis. C, observation of apoptosis/necrosis by PI/Annexin V (AnV) staining. PI/AnV double positive populace means cell populace undergoing necrosis or late apoptosis. PInegative/AnV positive portion means cell populace undergoing necrosis or early apoptosis. 5zox treatments were performed for 6?days. STEM-37-1595-s005.tiff (9.3M) GUID:?6D87496A-C77A-42B2-9D60-CC174EDBA73D Supplementary figure S5 Effect of 5zox treatment around the cell cycle status of BMMSC in vivo. FACS analysis of cell cycle status of the BMMSCs collected from juvenile male mice (day14 after birth) treated with the vehicle or 5zox three times each other day. E3330 The BMMSC populations were collected as PS at 48?hours after final injection E3330 and the cell cycle status was analyzed with CACNA2D4 the Vybrant Dye Cycle Violet (Thermo). Asterisks imply significant differences between control and the 5zox treated group at P? ?.05 (N = 6). STEM-37-1595-s006.tiff (9.3M) GUID:?B6F44B69-1D38-4A1E-AB39-17A86D12C693 Supplementary figure S6 Colony formation efficiency under TGF 1 and the inhibitor treatments. Asterisks imply significant differences between control and the TGFb or inhibitor treated groups at P? ?.05 (N = 3). STEM-37-1595-s007.tiff (9.3M) GUID:?52D5402C-EC03-4829-A3FD-D7EA773DDBC8 Supplementary figure S7 Effect of siRNA against Smad2,3, Erk1, and Erk2 on cell proliferation of BMMSCs. A, Expression switch of mRNA by siRNA (siSmad2) treatment. Asterisk means significant differences (P? ?.05, N = 3). B, Cell proliferation of BMMSCs treated with scrabbled RNA (SCR) or siSmad2. NTC means non\treatment control. C, Expression switch of mRNA E3330 by siSmad3 treatment. D, Cell proliferation of BMMSCs treated with SCR or siSmad3. E, Expression switch of mRNA by siErk1 treatment. F, Cell proliferation of BMMSCs treated with SCR or siErk1. G, Expression switch of mRNA by siErk2 treatment. H, Cell proliferation of BMMSCs treated with SCR or siErk2. STEM-37-1595-s008.tiff (9.3M) GUID:?9544392D-B6A6-4818-A6B5-414A08B6DDA0 Supplementary figure S8 ROS accumulation detected by CellROX DeepRed dye and FACS in the transplanted BMMSCs A, detection of the transplanted BMMSCs in the grafted site using the EGFP signaling. B, CellRox\based observation of ROS accumulation in transplanted cells. 5zox represents the transplanted EGFP+ cells that were pre\treated with 5zox before transplantation. The CellROX analysis was performed as follows; total BM cells were collected from your recipients, then reacted with CellROX\DeepRed dye following manufacturer’s instructions. Then the cells were analyzed with FACS Aria II. Transplanted cells were recognized with EGFP fluorescence and then observed ROS accumulation with Ex lover644nm/Em665nm fluorescence of the CellROX dye. STEM-37-1595-s009.tiff (9.3M) GUID:?AC1C6EF7-AE20-404D-B1D6-1400F434D84A Supplementary figure S9 Increased expression of the genes contribute wound healing and immunomodulation. A, increased expression status of the wound healing\ and immunomodulation\related cytokines were detected by microarray analysis. Scores show fold change in expression (vehicle control/5zox treatment), and scores under 0 imply upregulation in the 5zox E3330 treated BMMSCs. 5zox treatment was performed at 20?nM for 6?days. B, qRT\PCR based validation of.