Inhibition of MEK1 prevents the formation of EPL cells in response to MEDII.

<p>A. mES cells were pre-treated with 1 μM PD0325901 for 60 minutes. 200 μM l-proline was added, as denoted, and the cells incubated for a further 60 minutes. Cells were collected and analysed by western blot for the presence of phosphorylated ERK1 or ERK2. Total ERK1/2 was used as a loading control. The intensity of the pERK2 band was measured using Quantity One software (BioRad) and represented as a proportion of total ERK1/2. Error bars represent SEM; n = 4; * <i>p</i> ≤ 0.05 when compared to mES cells. B-C. mES cells were cultured in MEDII- and DMSO-containing medium (B) and MEDII- and 1μM PD0325901-contianing medium (C) for 3 days. Scale bar = 200 μm. D. MEDII- and DMSO-containing medium (■) and MEDII- and 1μM PD0325901-contianing medium (□) for 3 days. RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog</i>, <i>Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i>, <i>Otx2</i> and <i>Fgf5</i> by real-time PCR. Expression was normalized to <i>β-actin</i> and expressed relative to mES cells (<i>Fgf5</i> has been expressed relative to MEDII + DMSO). Error bars represent SEM; n = 3. mES cells + MEDII + PD0325901 were compared to mES cells + MEDII + DMSO (** p ≤ 0.05) or mES cells (# p ≤ 0.05).</p

The role of ERK1/2, Src Family Kinases and p38 MAPK in the maintenance of EPL cells.

<p><b>A-D.</b> Photomicrographs of mES cells cultured in MEDII for 2 days and subsequently in MEDII containing medium supplemented with DMSO (A), 1 μM PD0325901 (B) 10 μM PP2 (C) and 10 μM SB203580 (D). Cells were stained for alkaline phosphatase activity (red/purple stain). Scale bar = 200 μm. <b>E, F.</b> mES cells were cultured in MEDII for 2 days and subsequently in MEDII containing medium supplemented with DMSO (■), DMSO with 1 μM PD0325901 (E, □), DMSO with 10 μM PP2 (F, □) or DMSO with 10 μM SB203580 (G, □). RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog</i>, <i>Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i> and <i>Otx2</i> by real-time PCR. Expression was normalized to <i>β-actin</i>. Error bars represent SEM; n = 3. EPL cells cultured in MEDII with inhibitor were compared to EPL cells in MEDII with DMSO, ** <i>p</i> ≤ 0.05, or mES cells, # <i>p</i>≤ 0.05.</p

The role of p38 MAPK in the formation of EPL cells in response to MEDII.

<p><b>A.</b> mES cells were pre-treated with 10 μM SB203580 for 60 minutes. 200 μM l-proline was added, as denoted and the cells incubated for a further 60 minutes. Cells were collected and analysed by western blot for the presence of phosphorylated pHspb2. Total Hspb2 was used as a loading control. <b>B, C.</b> mES cells were cultured medium supplemented with MEDII and DMSO (A) or MEDII and 10 μM SB203580 for 3 days. Scale bar = 200 μm. <b>D.</b> mES cells were cultured in medium supplemented with MEDII and DMSO (■) or MEDII and 10 μM SB203580 (□) for 3 days. RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i>, <i>Otx2</i> and <i>Fgf5</i> by real-time PCR. Expression was normalized to <i>β-actin</i> and expressed relative to mES cells (<i>Fgf5</i> has been expressed relative to MEDII + DMSO). Error bars represent SEM; n = 4. mES cells + MEDII + SB203580 were compared to mES cells + MEDII + DMSO (** p ≤ 0.05) or mES cells (# p ≤ 0.05). <b>E.</b> mES cells were cultured in ES cell medium supplemented with MEDII, DMSO or MEDII and 10 μM 10 μM SB203580 for 3 days and formed into EBs. EBs were collected on days 2, 3 and 4, RNA was isolated and analyzed for expression of <i>T</i> and <i>Gapdh</i> by RT-PCR. n = 3.</p

The regulation of progression of the pluripotent lineage in culture.

<p>The cell states represented in vitro, naïve mES cells, primed mES cells and EPL cells have been aligned with the expression of Nanog and Otx2 and with their deduced intracellular signaling activity. Inducers of lineage progression are shown in orange; Calcineurin exerts its effects through dephosphorylation of NFAT and promotes NFAT translocation to the nucleus.</p

Summary of inhibitors used in this study.

<p>Summary of inhibitors used in this study.</p

The addition of l-proline to ES cells increases ROS.

<p><b>A-C</b>. MitoSox Red staining of mES incubated with 200 μM l-proline (B) or l-proline and ascorbic acid (C) and compared to untreated cells (A). Hoechst staining of the same fields of view are shown in Ai, Bi and Ci. D. ROS levels were measured as fluorescence using ROS-Glo™ H₂O₂ Assay (Promega) in cells that had been incubated with l-proline, 150 μM DHP or 1 mM GSH, as denoted. Results are shown as arbitrary fluorescent units. n = 4, *p≤0.05 when compared with mES cells. # p≤0.05 when compared with mES cells incubated with l-proline. E. The expression of <i>Dnmt3b</i> and <i>Otx2</i> was analysed in mES cells cultured in medium supplemented 200 μM l-proline or 200 μM l-proline and 150 μM DHP. Expression was normalized to β-actin and expressed relative to mES cells. Error bars represent SEM; n = 4. Comparisons were made to mES cells (*p ≤ 0.05) or mES cells cultured with 200 μM l-proline (# p ≤ 0.05).</p