Figure 3. Jak2 phosphorylates Atoh1 Y78.

(a) IP of Jak2 or Atoh1 in tumor and P5 cerebellar tissue shows that Atoh1 and Jak2 interact in tumor tissue. (b) Upon inhibition of Jak2, Y78 phosphorylation levels fall dramatically in ex vivo culture (n = 3, Two-way ANOVA with Sidak's multiple comparisons test). (c) Transcriptional activity of Atoh1 is increased upon Jak2 introduction, independent of canonical Jak2 pathways (n = 46 in duplicates, One-way ANOVA with Dunnett's multiple comparisons test compared to Atoh1-GFP). Values are mean ± s.e.m.; ****p<0.0001, ns = not significant.

Figure 3—figure supplement 1. Jak2 phosphorylates tyrosine 78.

(a) FACS analysis of GFP signal intensity generated by BiFC complementation assay using DAOY cells transfected with the indicated plasmids. n = 3. (b) In vitro, non-radioactive kinase assay of in vitro transcribed kinase/substrates. n = 3. (c) qRT-PCR analysis of Atoh1 and Jak2 expression in P5, adult cerebellum, and Nd2::SmoA1 tumors, relative to P5 expression, normalized to the housekeeping gene GAPDH. n = 3. (d–e) Dual luciferase reporter assay using UW228 (d, orange) or ONS-76 (e, purple) cell lines transfected with the indicated plasmids and treated with the indicated inhibitors of the canonical Jak2 cascades. n = 46. ****p<0.0001, ns denotes p>0.05.

Figure 3—figure supplement 2. Jak2 increases Atoh1 stability.

Stability assay of stable cell lines infected with Jak2. in DAOY (a, blue), UW228 (b, orange), or ONS-76 (c, purple) parental cell lines. n = 3. Y78 = Atoh1-GFP WT, Y78F = Atoh1-GFP Y78F, Y78D = Atoh1-GFP Y78D; *p<0.05, ns denotes p>0.05.

Figure 3—figure supplement 3. Jak2 increases proliferation via tyrosine 78.

In vitro proliferation assay using medullospheres of DAOY (a, blue), UW228 (b, orange), or ONS-76 (c, purple) cell lines stably expressing indicated Atoh1 constructs and infected with Jak2 viral particles as indicated. n = 3 in triplicates. Y78 = Atoh1-GFP WT, Y78F = Atoh1-GFP Y78F; ****p<0.0001.