Fig 1: Truncated EPHA7 Is Expressed and Secreted from Pre-reprogramming Cells and Supports SSEA-1-Negative Cells to Progress toward a Pluripotent State(A) Immunoblotting analysis for pERK1/2 levels of the sorted samples (shown in Figure S4A).(B) qRT-PCR analysis of truncated EphA7 in the sorted samples.(C) A scheme for the puromycin selection experiment. OSKM were introduced into MEFs, derived from Nanog-GFP-IRES-PuroR mice, and the cells were treated with puromycin (1 μg/ml) from day 7 to day 9 for selection.(D) qRT-PCR analysis of Nanog, EphA7 and truncated EphA7 from cells treated with puromycin (NANOG+ cells) or without puromycin (NANOG− cells/NANOG+ cells). In (B) and (D), data are shown as mean ± SEM (n = 3 independent experiments).(E) Immunoblotting analysis of conditioned media from cells treated with puromycin (NANOG+ cells) or without puromycin (NANOG− cells/NANOG+ cells).(F) Effect of truncated EPHA7-expressing pre-reprogrammed cells on reprogramming of SSEA-1+ and SSEA-1− cells. Left: a scheme for the experiment. OSKM-introduced Nanog-GFP MEFs were sorted into SSEA-1+ and SSEA-1− cells at day 8 and plated on control shRNA-treated, OSKM-introduced WT MEFs (sh Luc) or on EphA7 shRNA-treated, OSKM-introduced WT MEFs (sh EphA7 #1). Right: the number of NANOG-positive colonies after 4 days of incubation.(G) Effect of truncated EPHA7 on reprogramming of SSEA-1+ and SSEA-1− cells. Left: a scheme for the experiments. OSKM-introduced Nanog-GFP MEFs were sorted into SSEA-1+ and SSEA-1− cells at day 8 and plated on control WT MEFs or OCT3/4-overexpressing WT MEFs. EPHA7FC (5 μg/ml) or PBS was added to the culture medium. Right: the number of NANOG-positive colonies after 4 days of incubation. In (F) and (G), data are shown as mean ± SEM (n = 3 independent experiments; ∗p < 0.05). The p values were calculated using Student’s unpaired two-tailed t tests.
Fig 2: Truncated EPHA7 Promotes MEF Reprogramming through Inducing ERK Activity Reduction(A) Immunoblotting analysis for pERK1/2 in mCherry (Ctrl) or OSKM-introduced MEFs. The samples here were the same as those used in Figure 1C.(B) Immunoblotting analysis for pERK1/2 levels in EphA7 shRNA-treated cells.(C) Effect of the addition of truncated EPHA7 on pERK1/2 levels. EPHA7FC (5 μg/ml) was added to the culture medium in OSKM-introduced MEFs treated with EphA7 shRNA #4 or sh Luc at day 8.(D) EphA7 shRNA (#1)-treated, OSKM-introduced MEFs were treated with the MEK inhibitor PD0325901 (0.5 μM) for 2 days as indicated and examined for the number of Nanog-positive colonies at day 12. Left: representative images; right: quantification. The scale bars represent 1 mm.(E) Effect of the addition of truncated EPHA7 and/or the MEK inhibitor (PD0325901) treatment on reprogramming efficiency. EPHA7FC was added to the culture medium from day 6 to day 10. PD0325901 treatment was from day 7 to day 8. The number of NANOG-positive colonies at day 12 was examined.In (D) and (E), data are shown as mean ± SEM (n = 3 independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). The p values were calculated using Student’s unpaired two-tailed t tests.
Fig 3: Truncated EPHA7 (EPHA7FC), but Not Full-Length EPHA7, Enhances Reprogramming Efficiency(A) Knockdown efficiencies of EphA7 shRNAs. EphA7 mRNA levels at day 6 were determined. Luciferase shRNA (sh Luc), control.(B) Effect of EphA7 shRNAs on Nanog mRNA levels during reprogramming.(C) Effect of EphA7 shRNAs on the number of NANOG-positive colonies. Left: representative images at day 12 (left). Top: NANOG-positive colonies; bottom: Hoechst. The scale bars represent 2 mm. Right: quantification.(D) Effect of exogenous expression of full-length EPHA7 on reprogramming efficiency. Full-length EphA7 or mCherry (Ctrl) was expressed in control shRNA-treated, OSKM-introduced MEFs (sh Luc) or in EphA7 shRNA-treated, OSKM-introduced MEFs (sh EphA7 #1 and #4). In (D), (E), and (G), quantification of the NANOG-positive colonies is shown.(E) Effect of the addition of truncated EPHA7 protein to the culture medium on reprogramming efficiency. EPHA7FC (a truncated form of EPHA7; 5 μg/ml) was added to the culture medium at day 6 and incubated until day 10.(F) Effect of the exogenous expression of full-length EphA7 or the addition of truncated EPHA7 protein to the culture medium on Nanog mRNA levels during reprogramming.(G) Effect of the addition of truncated EPHA7 protein (EPHA7FC) to the culture medium at the different time points.In (A)–(G), data are shown as mean ± SEM (n = 3 independent experiments; n = 4 independent experiments in [C]; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). The p values were calculated using Student’s unpaired two-tailed t tests.
Fig 4: EPHA7 Is Upregulated during MEF Reprogramming(A) qRT-PCR analysis of EphA7 (full-length plus truncated EphA7) and truncated EphA7. Ctrl (control), mCherry instead of OSKM.(B) qRT-PCR analysis of Nanog, endogenous Oct3/4, and E-cadherin.(C) Immunoblotting analysis for EPHA7, NANOG, E-CADHERIN, and α-TUBULIN (as a loading control). Triangles indicate full-length EPHA7 (top) and truncated EPHA7 (bottom).(D) Immunoblotting analysis of conditioned media. A triangle indicates truncated EPHA7.(E) ELISA of the conditioned medium for EPHA7.(F) qRT-PCR analysis of EphA7 and truncated EphA7. mCherry (Ctrl), Oct3/4, Sox2, Klf4, c-Myc, or OSKM was introduced to MEFs at day 0.(G) qRT-PCR analysis of both full-length EphA7 and truncated EphA7. Oct3/4 was introduced to MEFs at time 0. Ctrl, mCherry.(H) Potential OCT3/4 biding sites in the upstream region of EphA7.(I) Chromatin fragments of the OCT3/4 introduced MEFs were immunoprecipitated with control immunoglobulin G (IgG) or anti-OCT3/4 antibody. Each site was quantified by qRT-PCR. The values with control IgG were set to 1. Data are shown as mean ± SEM (sites B and D, n = 4 [∗p < 0.05]; site E, n = 3; site A, n = 2 independent experiments; site C, not detected). The p values were calculated using Student’s unpaired two-tailed t tests.In (A), (B), (F), and (G), data are shown as mean ± SEM (n = 3 independent experiments).
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Mouse EphA7 Fc Chimera Protein, CF