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. 2017 Oct 18;6:e27356. doi: 10.7554/eLife.27356

Figure 3. Pro-commitment phosphatases regulate MAPK signalling and AP1 transcription factors.

(a) Gene Ontology (GO) term enrichment analysis of ranked peptides dephosphorylated at 4 hr. (b) Venn diagram showing intersection of signalling pathways regulated at 4 hr. (c) Top 15 dephosphorylated peptide sites at 4 hr, showing ratio between change in phospho-peptides and change in total protein. Highlighted in orange are phosphorylations on MAPKs. (d, e) Representative western blot (d) and quantification (e) showing phospho-ERK1/2 and total ERK1/2 in cells harvested after 0, 4, 8 and 12 hr in suspension. Cyclophilin B: loading control. (f) Heatmap represents mRNA expression (relative to 18 s RNA) of AP1 transcription factor superfamily members during suspension-induced differentiation post-phosphatase knockdown (n = 3; values plotted are means of three independent transfections). See Supplementary file 6 for p-values generated by two-way ANOVA. (g–h) Representative western blot, with quantitation, of phospho-ERK1/2 and total ERK1/2 in suspended cells following DUSP10 knockdown. siSCR and loading controls are shown. (i) Heatmap showing mRNA expression (relative to 18 s mRNA) of JUN, FOS and MAF family members after DUSP6 and DUSP10 knockdown (values plotted are means of three independent transfections normalised against scrambled control; see Supplementary file 7 for p-values generated by two-way ANOVA). (j) Clonal growth (representative dishes and quantification) following doxycycline-induced over-expression of DUSP10, DUSP6 and mutant DUSP6C293S in primary keratinocytes (n = 3 independent cultures). p-values were calculated using one-way ANOVA with Dunn's multiple comparisons test (*p<0.05; ns = non significant).

Figure 3.

Figure 3—figure supplement 1. Effects of DUSP10 knockdown and DUSP6 and DUSP10 over-expression.

Figure 3—figure supplement 1.

(a, b) Western blot (a) and RT-qPCR measurements (b) of DUSP10 knockdown by SMART pool siRNA. p-values were generated by one-tail t-test. (c, d) Western blot of phospho-p38 and total p38 in suspended cells following DUSP10 knockdown. siSCR, cyclophilin B loading control and quantitation are shown. (e) RT-qPCR quantification of doxycycline-induced over-expression of DUSP6, mutant DUSP6C293S and DUSP10 relative to 18 s mRNA. Cells were treated with 1 µg/ml doxycycline for 24 hr. p-values were generated by a one-tail t-test. (f) RT-qPCR quantification of Cumate-induced over-expression of DUSP6 and mutant DUSP6C293S relative to 18 s mRNA. p-values were generated by one-way ANOVA. (g) Representative dishes showing effects of overexpressing wild type and mutant DUSP6 on clonal growth (representative of n = 3 dishes). (b, e, f) Error bars represent s.d. *p<0.05; ***p<0.0005; ****p<0.0001; ns = non significant.