Figure 4

ERK suppression is an early signal of cellular reprogramming and increases its efficiency. (a) Phospho-kinase antibody array with GC5 cells incubated in MEF medium containing Dox (4 μg ml⁻¹) for 3 days. (b) Relative intensities of the phospho-kinase spots. Membranes were analyzed using ImageJ software (NIH). (c) Western blot analysis. Cells were incubated in MEF medium containing Dox (4 μg ml⁻¹) for 3 days. Alternatively, cells were cultured in fresh mESC medium containing LIF (J1 mESCs and iPSCs) or in fresh MEF medium (siOG-MEFs and cells differentiated from iPSCs). i4F-MEF, Dox-inducible OSKM-expressing normal MEFs; P-2, passage no. 2; iPS-DC, cells differentiated from iPSCs. (d) Western blot analysis with GC5 cells incubated in MEF medium containing Dox (4 μg ml⁻¹) for the indicated number of days. (e) Reprogramming assay with chemical inhibitors against MAP kinases and some signaling molecules. Sub-lethal concentrations of the chemicals were determined by testing various concentrations (0.01, 0.1, 1 and 10 μM) using Option I (μM). MEK inhibitors: PD03, PD98 (PD98059), U0126 and Tra (trametinib). P38 inhibitors: SB203 (SB203580) and SB202 (SB202190). JNK inhibitors: SP600 (SP600125) and JNKi-1 (JNK inhibitor I). STAT3 inhibitor: WP1066. mTOR inhibitor: Benz235. PI3K inhibitor: LY294002. (f) Western blot analysis of pERK expression in GC5 cells incubated with the indicated chemicals and medium for 24 h. (g) Reprogramming assay of GC5 cells with cytokines (Option III; concentration, 10 ng ml⁻¹) or chemicals (Option I; μM). PD17 (PD173074, FGFR inhibitor), CP67 (CP673451, STAT3 inhibitor) and SC (SC144, PDGFR inhibitor). (h) Western blot analysis of pERK expression and reprogramming assay in GC5 cells infected with a retrovirus encoding MEK-WT, MEK-K101A or MEK-DD. After 3 days of infection, cells were subjected to Western blot analysis or were detached and seeded onto 12-well plates for the Option III assay. The results are the means±s.d. of three independent experiments (two-tailed, unpaired t-test; n=2 (e) or n=3 (g,h)). *P<0.05; **P<0.01. ERK, extracellular signal-regulated kinase.