Fig. 3

Deletion of Dusp10 increases IL-5 production in Tpath2 cells. a Intracellular staining profiles of phospho-p38 in Tpath2 cells and ILC2s purified from memory Th2 mice (as shown in Supplementary Fig. 1a) after stimulation with PMA plus Ionomycin (P + I) or IL-7 plus IL-33 (IL-7 + IL-33) for 30 min. The number in the histogram represents MFI. Black line shows non-stimulated cells. Graph shows mean MFI ± SD values of phospho-p38 in stimulated cells (n = 3, separately). b Phosphorylated p38 (p-p38) and Tub.α in Tpath2 cells and ILC2s after stimulation with P + I, or IL-7 + IL-33 for 30 min. Band intensities were measured with a densitometer. The arbitrary ratio of the intensity of p-p38 to that of tubulin is shown. c Deletion patterns of the Dusp10 locus in control Tpath2 cells and sgDusp10-treated Tpath2 cells. Reference (REF) sequence is shown on top of clonal sequences from each population with sgRNA target (blue) and PAM (green) sequences indicated. Red dashes denote deleted bases. d DUSP10 in sgDusp10-treated Tpath2 cells. C refers to Cas9 Ctrl Tpath2 cells. An antibody to tubulin (Tub) was used as a loading control. e) Intracellular staining profiles of IL-5 and IL-13 in sgDusp10-treated Tpath2 cells (sgDusp10) and non-targeted Tpath2 cells (Control) are shown. The cells were stimulated with indicated stimuli for 6 h. The percentages of IL-5 positive Tpath2 cells are shown (mean ± SD; n = 3 for each group). f Quantitative RT-PCR analysis of Il5 and Il13 in edited cells and control cells after indicated stimulation for 4 h. Relative expression (normalized to Hprt) is shown with standard deviation. g ELISA analysis of the indicated cytokines from Tpath2 cells. 1 × 10⁴ cells were stimulated with indicated stimuli for 48 h. h Intracellular staining of phospho-p38 in Dusp10 edited cells and control after stimulation with IL-7 plus IL-33 for 30 min. Gray filled histogram shows isotype control staining. Graph shows mean MFI ± SD values of phospho-p38 in each edited cells (n = 5, separately). Three independent experiments were performed and showed similar results (a–h) (**p < 0.01, *p < 0.05). Three technical replicates were included in f and g