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. 2015 Dec 24;6(1):74–84. doi: 10.1016/j.stemcr.2015.12.002

Figure 4.

Figure 4

Signaling via the MEK-ERK Pathway Is Necessary for Basal and Drug-Induced SKPs Self-Renewal

(A–C) SKPs were treated with alprostadil (Alp), TM, or vehicle (Control) for 10 min, and lysates probed on western blots with anti-phosphorylated ERK1/2 T202/Y204 (pERK) (A) or anti-phosphorylated STAT3 (C) and reprobed for total ERK1/2 or total STAT3. (B) shows quantification by scanning densitometry of three experiments as in (A), where pERK was normalized to total ERK1/2.

(D) Western blot of SKPs treated with varying concentrations of trametinib (MEKi) or DMSO (Control) for 30 min, probed for pERK1/2 and reprobed for total ERK1/2.

(E and F) Number (E) and size (F) of SKP spheres generated in clonal methylcellulose assays from secondary neonatal rat SKPs grown 14 days in 100 nM trametinib or DMSO.

(G and H) Secondary rat SKPs were cultured for 4 days, and 10 or 100 nM trametinib added for 2 more days. (G) shows spheres immunostained for Ki67 (top) and counterstained with DAPI (bottom). (H) shows the percentage of Ki67-positive cells. Scale bar represents 100 μm.

(I) Western blot of SKPs treated with 100 nM trametinib for 30 min and coincidently stimulated with 100 nM alprostadil or TM for the final 10 min, probed for pERK1/2, and reprobed for total ERK1/2.

(J) Number of rat SKP spheres generated over 7 days in 100 nM alprostadil or TM with or without 100 nM trametinib.

(K) Heatmap comparing microarray data for three independent preparations of dissociated rat SKPs cultured for 24 hr in TM with or without trametinib, focusing on 29 of the top 50 genes that were altered by TM relative to control (see Figure 3), and that were significantly normalized by MEK inhibition. The heatmap shows the average of the raw expression (log2) data for probesets that differed by p < 0.01 (unadjusted). In all cases, results are pooled from at least three independent experiments.

Error bars indicate SEM, and ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA with multiple comparison post hoc tests, except (E and F), which were analyzed by Student's t test.