DEV022871 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. IRS1 expressed in ShHH-treated CGNPs is phosphorylated on tyrosine, which occurs when the IGF receptor is activated. Immunoprecipitation of IRS1 followed by immunoblotting for phospho-tyrosine shows that IRS1 in SHH-treated CGNPs is phosphorylated when compared with IgG controls.

• Supplemental Figure S2 -

  Fig. S2. IRS1 is expressed in proliferating CGNPs in vivo. The top panels show that IRS1 (left, red) expression is found in the EGL of PN7 mice colocalizing with the proliferation marker PCNA (green). By contrast, IRS2 (right, red) is localized to Purkinje cells. The colocalization of IRS1 with PCNA persists up to PN15 (lower left, red). BrdU-positive cells in culture are exclusively Zic positive (lower right), confirming that proliferating cells in mixed cultures are CGNPs.

• Supplemental Figure S3 -

  Fig. S3. IRS2-null mice have reduced GLI1 and IRS1 expression in the EGL. Cerebella of postnatal day 7 IRS2 null or wild-type littermates were sectioned and expression of GLI1 and IRS1 was analyzed. In situ hybridization show reduced GLI1 expression in the EGL of IRS1-null (bottom left panel) compared with wild-type littermates (top left panel). Immunostaining for IRS1 (red) showed reduced IRS1 levels in the EGL of IRS2 null mice compared with wild-type littermates (right panels).

• Supplemental Figure S4 -

  Fig. S4. Knock down of IRS1 decreases SHH-mediated proliferation without affecting cell survival. Proliferation of CGNPs was assessed by immunostaining for BrdU (A, green) or Ki67 (B, red) after treatment with IRS1 targeted shRNAs. (C) IRS1 knock down did not affect CGNP survival, as seen by immunostaining for cleaved caspase 3 (red). (D) Infection of CGNPs with a GFP-targeting lentivirus did not affect proliferation.

• Supplemental Figure S5 -

  Fig. S5. IRS1 overexpression is sufficient in maintaining CGNP proliferation in the absence of SHH. CGNPs were treated with SHH for 24 hours prior to infection with retrovirus expressing IRS1. (A) Levels of CGNP proliferation in response to IRS1 expressing retrovirus infection were assessed with Ki67 staining (green). Numbers of Ki67+ cells are increased with in CGNPs treated with Shh, as well as CGNPs infected with pIG-IRS1 from which Shh was withdrawn for 24 hours. (B) To assess the effects of virus treatment on cell death, CGNPs were stained with antibodies to activated caspase 3 (red). Levels of cleaved caspase 3 remain the same for all treatment groups. DAPI staining was used to visualize total cell numbers (blue). There was no change in cell survival regardless of treatment.