Fluorescence ALP substrate (0.2 mM, 4-methylumbelliferyl phosphate) [59] in EPPS buffer was added into the wells. or without GFX (5 M). (C) Effect of BMP-4 or activin A on phosphorylation level of AKT, GSK-3, and ERK-1/2. After starvation of FGF-2 and insulin over night, 201B7 hiPS cells were stimulated with with FGF-2 (100 ng/ml), BMP-4 (10 ng/ml) or activin A (100 ng/ml). (D) Effect of addition of activin A with and without inhibitors on phosphorylation level of AKT, GSK-3, and ERK-1/2. After starvation of FGF-2 and insulin over night, H9 hES cells were stimulated with FGF-2 (10 ng/ml) and activin A (10 or 100 ng/ml) together with U0126 (5 M) and GFX (5 M) or G?6976 (5 M) for quarter-hour. (E) Effect of GFX concentration on phosphorylation level of AKT, CGRP 8-37 (human) GSK-3, and ERK-1/2. After starvation of FGF-2 and insulin over night, H9 hES cells were stimulated with FGF-2 (100 ng/ml) with GFX at 110 M. The phosphorylation levels in the cells were measured by AlphaScreen? SureFire? assay kit. The values of the y-axis are the ratio of each phosphorylation to each total signal protein. The data are displayed as means SD (n?=?3). *P 0.05.(TIF) pone.0054122.s001.tif (620K) GUID:?D8B16CAB-AA9B-45EF-9584-6D7A1EC76D5F Number S2: Summary of the result of the effect of PI3K, MEK-1/2, or PKCs inhibitor about FGF-2-induced phosphorylation of AKT, GSK-3, and ERK-1/2. (TIF) pone.0054122.s002.tif (335K) GUID:?7D12F422-ECCF-47B2-A330-876CF8709C33 Figure S3: Knockdown efficacy and effect of siRNA targeting PKC, , and . (A) Total RNAs were extracted for analysis 72 hours after the fast transfected to 201B7 iPS cells. The effectiveness of siRNA was evaluated by quantitative RT-PCR. siRNAs and primers were outlined as Table S4. (B) Summary of the result of the PKC-, PKC-, PKC-knockdown effect on CGRP 8-37 (human) phosphorylation of GSK-3 and AKT in FGF-2 signaling.(TIF) pone.0054122.s003.tif (225K) GUID:?13585995-17F8-4CB8-8176-3CBECB15432B Number S4: Effect of inhibitory peptides for PKCs about phosphorylation level of ERK-1/2. After starvation of FGF-2 and insulin, the H9 hES cells (right panel) or the 201B7 iPS cells (remaining panel) were stimulated with FGF-2 (100 ng/ml) for 15 mins with indicated combination of membrane-permeable specific inhibitory peptides for PKC isoforms; PKC, , and inhibitory peptide (50 M), PKC inhibitory peptide (50 M), PKC inhibitory peptide (50 M), or PKC inhibitory peptide (20 M). The phosphorylation levels in CGRP 8-37 (human) the cells were measured by AlphaScreen? SureFire? assay kit. The values of the y-axis are the ratio of each phosphorylation to each total signal protein. The data are displayed as means SD (n?=?3). *P 0.05.(TIF) pone.0054122.s004.tif (91K) GUID:?648F3703-A5E3-45C8-98C4-1A8E92AF39A0 Figure S5: Tradition of hiPS cells in the hESF9 + activin A + 2i or CGRP 8-37 (human) the hESF9 + activin A + GFX conditions. (A) Phase-contrast image of H9 hES cells serially cultured in hESF9 + activin A + 2i (hESF9a2i) or hESF9 + activin A + GFX mediums at three passages, as explained in Number 5A and 5B. Level bars, 200 m. (B) Immunocytochemical staining for OCT3/4 manifestation of H9 cells cultured as explained (A). The H9 hES cells stained with anti-OCT3/4 antibody were visualized with Alexa Fluor 488 (green). Nuclei were stained with Hoechst 33342 (blue). Level bars, 50 m. (C) Anti-OCT3/4 staining intensity profiles in the cell human population cultivated in the hESF9 + activin A + 2i or the hESF9 + activin A + GFX conditions were analyzed by IN Cell image analyzer (lower panels). Antigen histogram (reddish); control histogram (green); Y axis is definitely cell figures and X axis is definitely fluorescence intensity for anti-OCT3/4 antibody.(TIF) pone.0054122.s005.tif (4.5M) GUID:?90EACC3E-5140-4531-9A94-Abdominal3FE62C126B Number S6: Immunocytochemical staining of H9, KhES-4, 201B7, and Tic hPS cells for TRA-1-60. The cells cultivated on FN in hESF9a2i as explained in Number 5C were stained with TRA-1-60 antibody and Alexa Fluor 647-conjugated secondary antibody. Nuclei were stained with Hoechst 33342 (blue). Level bars, 200 m.(TIF) pone.0054122.s006.tif (2.6M) GUID:?20515A3F-348F-4AD7-8E67-8AF0391F5CC7 Figure S7: Hoxa2 Long-term culture of hiPS cells in the hESF9a2i medium. Human being iPS 201B7 cells were cultured on FN in hESF9a2i medium serially for more than 30 passages. The cells were split at a percentage of 13C15 every five days. (A) Phase-contrast image of 201B7 hiPS cells cultured on FN in hESF9a2i medium..