SUPPLEMENTARY INFORMATION. Prolyl isomerase Pin1 and protein kinase HIPK2 cooperate to promote

Transcription

1 SUPPLEMENTARY INFORMATION Prolyl isomerase Pin1 and protein kinase HIPK2 cooperate to promote cortical neurogenesis by suppressing Groucho/TLE:Hes1-mediated inhibition of neuronal differentiation Running Title: Pin1 and HIPK2 promote cortical neurogenesis Roberta Ciarapica 1,2, Laurent Methot 1, Yeman Tang 1, Rita Lo 1, Rola Dali 1, Manuel Buscarlet 1, Franco Locatelli 2,3, Giannino del Sal 4,5, Rossella Rota 2, and Stefano Stifani *,1 1 Montreal Neurological Institute, McGill University, 3801 rue University, Montreal, Quebec H3A2B4, Canada 2 Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy 3 University of Pavia, Pavia, Italy 4 Laboratorio Nazionale CIB, Area Science Park, Trieste, Italy 5 Dipartimento di Scienze della Vita, Università degli Studi di Trieste, Trieste, Italy * Corresponding author: S Stifani, Montreal Neurological Institute, 3801 rue University, Montreal, Quebec, H3A2B4 Canada. Tel: ; Fax: ; stefano.stifani@mcgill.ca

2 SUPPLEMENTARY FIGURES Figure S1 Expression of endogenous Gro/TLE1, Pin1, and HIPK2 in cortical neural progenitor cells. (a) Double-label immunofluorescence analysis of Gro/TLE (detected using a pangro/tle antibody) and Pin1 expression in cultured cortical neural progenitor cells. (b) Western blot analysis of lysates from dissected E13.5 mouse dorsal telencephalon using antibodies against Gro/TLE1, HIPK2, Pin1, or β-actin. (c, d) Double-label immunofluorescence analysis of the indicated proteins in cultured cortical neural progenitor cells. Where shown in (a, c, d), arrows point to examples of double-labeled cells. Objectives: (a) 40X; (c, d): 20X.

3 Figure S2 Interaction of Gro/TLE1:Hes1 complex with Pin1 and HIPK2. Coimmunoprecipitation studies using HEK293 cells transfected with the indicated combinations of proteins. Immunoprecipitation (IP) of HA-tagged Pin1 using anti-ha antibody (lanes 2-8) results in the coprecipitation of both HIPK2(WT) and HIPK2(K221R), as revealed by Western blot (WB) analysis. Gro/TLE1 and Hes1 coprecipitate efficiently with Pin1 and HIPK2, but not when HIPK2(K221R) is used, nor in the presence of Hes1(ΔWRPW). IgG HC and IgG LC, immunoglobulin G heavy or light chain, respectively. Control immunoprecipitation with preimmune serum is shown in lane 1.

4 Figure S3 Control chromatin immunoprecipitation experiments. (a) Analysis of ASCL1 promoter occupancy in HEK293 cells transfected with empty FLAG vector alone using mouse anti-flag antibody (Ab) (lane 3) or two irrelevant (Control) antibodies (ie, anti-ha and anti-gst; lanes 4 and 5), followed by PCR with primers specific for the human ASCL1 promoter. A specific PCR product is detected only when input genomic DNA (Input) was used. DNA size markers are indicated in base pairs. (b) Analysis of ASCL1 promoter occupancy in HEK293 cells transfected with Hes1 without a FLAG epitope (using plasmid prc-cmv-hes1) together with HA-Pin1 and FLAG- HIPK2(WT), as indicated, using mouse anti-flag antibody (lane 2) or two irrelevant (Control) antibodies (lanes 3 and 4), followed by PCR with primers specific for the human ASCL1 promoter. A specific PCR product is detected only when input genomic DNA (Input) was used. DNA size markers are indicated in base pairs.

5 Figure S4 Inhibition of the anti-neurogenic function of Gro/TLE1:Hes1 complex by Pin1 and HIPK2. (a) Primary cultures of E13.5 mouse cortical neural progenitor cells were transfected with GFP alone (Control) or together with the indicated combinations of proteins, followed by doublelabel immunofluorescence analysis of the expression of GFP and either nestin and MAP2 (a) or Ki67 and type III β-tubulin (not shown) 72 hours after transfection. Arrowheads point to examples of double-labeled cells. (b) Quantification of the fractions of GFP+ cells coexpressing Ki67 or type III β-tubulin (mean±standard deviation of 5 separate experiments performed in duplicates; p values, two-tailed Student t-test). Pin1 and HIPK2(WT) (bar 3), but not HIPK2(K221R) (bar 4), block the anti-neurogenic effect of Gro/TLE1 and Hes1 (bar 2).