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. Author manuscript; available in PMC: 2016 Apr 6.
Published in final edited form as: Dev Cell. 2015 Mar 26;33(1):22–35. doi: 10.1016/j.devcel.2015.01.033

Figure 1.

Figure 1

shRNA screen to identify phosphatases in Shh signaling. (A) ShhLightII cells were infected with virus encoding a single shRNA targeting one of 320 genes; infected cells were selected with puromycin, stimulated with Shh-conditioned media, SAG, or vehicle control then Gli-responsive luciferase activity was measured. (B) Primary screen average Firefly/Renilla luciferase values confirm Shh pathway activation by Shh and SAG (N=1720 wells, *P<0.01, Error bars = SEM). (C) Replicate Firefly/Renilla luciferase values were averaged for each shRNA and converted to their natural log value. (D) Primary screen robust z-score results. Genes with two or more hairpins giving results less than −1.5 or greater than 1.5 qualified as hits. (E) A subset of hits from the primary screen were re-screened. Firefly/Renilla luciferase values were normalized to median Firefly/Renilla luciferase value of negative control shRNAs. Values greater than four-fold of the median response (above the black line) or less than 25% of the median response qualified as hits. Red squares indicate values corresponding to an shRNA targeting Smo and yellow circles indicate values corresponding to an shRNA targeting Eya1. (F) Affymetrix array of medulloblastomas reveal a significant increase in Eya1 expression in the Shh subtype compared to WNT, Group 3 or Group 4 (P<0.0001 by ANOVA). (G) An shRNA targeting Eya1 blocks SAG-mediated induction of Firefly/Renilla luciferase (N=4,*P<0.05, **P<0.01, Error bars = SEM; NS = not significant, P>0.05). (H) Eya1 shRNA blocks SAG-mediated induction of Gli1 mRNA (N=5, *P<0.05, **P<0.01, Error bars = SEM; NS = not significant, P>0.05). (I) Induction of Gli1 protein is blocked by shRNAs targeting Eya1. Actin = loading control. (J) Induction of Gli1 protein is blocked by an Eya1 shRNA (N=8,*P<0.05, **P<0.01, Error bars = SEM; NS = not significant, P>0.05). (K) An shRNA targeting Eya1 blocks induction of Ptch1 mRNA (N=5, *P<0.05, **P<0.01, Error bars = SEM; NS = not significant, P>0.05). Gli1 (L) and Ptch1 (M) mRNA induction are reduced in Eya1−/− MEFs compared to Eya1+/+ littermates (N=3, *P<0.05, Error bars = SEM, NS = not significant, P>0.05). (N) Gli1 mRNA and (O) Ptch1 mRNA in Eya1−/− and Eya1+/+ MEFs, following reintroduction of wild type or D273A Eya1 (N=3, *P<0.05, Error bars = SEM).