Fig 2: Maml1 cooperates with Gli1 in a Notch-independent manner. (a) Proteins expression was detected by immunoblot analysis as indicated in WCE derived from NIH3T3 cells transfected with Maml1 siRNA or control siRNA (scr). Anti-tubulin was used as a loading control (left panel). Optical densitometry (OD) analysis of Maml1 and Gli1 protein expression (right panel). (b) qRT-PCR analysis of Shh target genes in NIH3T3 48 h after silencing of Maml1 compared with control (scr). (c) WCE separated by SDS-page from NIH3T3 upon 72 h treatment with DAPT or control vehicle (DMSO). To reveal the activated form of Notch1, an anti-Notch1 Val 1744 antibody was used (left panel). Expression level of Hes1 mRNA was evaluated by qRT-PCR (right panel). (d) Expression levels of Shh target genes evaluated by qRT-PCR in DAPT or DMSO (Ctr) treated NIH3T3 cells. (e) Luciferase assays in HEK293T cells cotransfected with 12xGli-luc (left panel) or Patched1-luc (right panel) and different combinations of expression vectors as indicated. After DAPT or control vehicle treatment, cell lysates were harvested for luciferase assay. Data represent mean±S.D. NS, not significant; **P=0.01; ***P=0.001; ****P=0.0001

Fig 3: Maml1 interacts with Gli proteins and sustains Gli-mediated transcription. (a) Luciferase assay in HEK293T cells transfected with 12xGli-luc (left panel) or Patched1-luc (right panel) reporter and different combinations of plasmids encoding for Maml1, Gli1 and Gli2 as indicated. Luciferase activity is expressed as fold induction relative to pcDNA3 alone (empty control). (b) Endogenous Gli1 mRNA levels evaluated by qRT-PCR in NIH3T3 cells transfected with increasing amounts of Maml1, compared with pcDNA3. (c) Coimmunoprecipitation (co-IP) experiments performed in HEK293T cells, transfected with expression vectors encoding for the indicated tagged proteins and immunoprecipitated with anti-Flag without (IP) or with (Ctr) blocking peptide. The interaction with Gli1 and Gli2 was revealed with anti-HA (left panel) or anti-Myc (right panel), respectively. (d) Reciprocal co-IP was performed by using anti-Gli1 antibody or control mouse antisera (IgG) and the interaction with Maml1 was detected with anti-Flag. Gli1 and Maml1 protein levels in total cell lysates are shown. (e) Ptch1-/- MEFs were lysed and immunoprecipitated with Maml1 antibody or control rabbit antisera (IgG) and the blot was reprobed with Gli1. (f) Representative immunofluorescence images of endogenous Gli1/Maml1 interaction in NIH3T3 cells analyzed by in situ proximity ligation assay (PLA). Negative controls lacking one of the primary antibodies. No significant fluorescent signal was detected in NIH3T3 cells incubated with only one primary antibody (only Gli1; only Maml1). Single plane confocal images were captured using a 60 × oil objective. Protein complexes were visualized in red; nuclei were DAPI labeled (blue). Scale bar: 100 µm. (g) ChIP assay in HEK293T cells. PCR was performed using primers that amplify Gli consensus binding sites on human Ptch1 promoter (upper panel) as shown in schematic representation (lower panel). According to Genomatix, basepairs in bold and underlined are important, since they appear in a position where the matrix exhibits a high conservation profile (ci-value>60); basepairs in capital letters denote the core sequence used by MatInspector. Dark circles represent predict binding sites. Data reported as mean±S.D. **P=0.01; ****P=0.0001

Fig 4: Maml1 COOH-terminus plays a functional role on Gli1 activity. (a) Schematic representation of full-length (FL) and truncated Maml1 constructs. Transcription activation domain (TAD): TAD1 (75–301 aa); TAD2 (303–1016 aa). (b) Whole-cell extract (WCE) from HEK293T cells cotransfected with indicated expressing vectors were immunoprecipitated with anti-Flag (IP) or anti-Flag with blocking peptide (Ctr). Immunoprecipitates and aliquots of cell lysates (Total) were immunoblotted with the indicated antibodies. (c) Luciferase assays of cotransfected HEK293T with 12xGli-luc (upper panel) or Patched1-luc (lower panel) and different combinations of expression vectors, as indicated. Luciferase activity is expressed as fold induction relative to control (pcDNA3). Data represent mean±S.D. ***P=0.001; ****P=0.0001. (d) Representative single plane confocal immunofluorescence images of HEK293T cells cotransfected with indicated expression vectors. Flag- (green) or HA- (red) tags were visualized by confocal microscopy. Confocal images were captured using a 60 × oil objective. Nuclei were DAPI labeled (blue). Scale bar: 10 µm

Fig 5: Decreased binding of the Notch1 transcriptional complex to the Hes1 promoter element in extracts prepared from ?TAD MEFs. (A) B6 E14.5 FL cells were cultured on OP9-DL1 cells for the times indicated (4 h or 10 h) in the presence of vehicle control (DSMO) in the “Notch on” state or GSI in the “Notch off” state. SLAM-LSKs were sorted from the coculture, and RNA obtained from the SLAM-LSKs was used for microarray analysis. Selected genes with decreased expression in the presence of GSI are shown. (*) Genes with decreased expression after both 4 h and 10 h of treatment in the presence of GSI; Q-value (percent) is used to express the false discovery rate. (B) qPCR measurements from E14.5 FL SLAM-LSKs for Hes1, Jag1, and Itgal (Integrin-a/LFA-1) mRNA transcripts. All mRNA values were normalized to Ef1a. (C) +/+ MEFs were treated for 24 h with GSI or DMSO vehicle control. GSI was washed out at 18 h, and cells were cultured in vehicle control for an additional 6 h. Hes1 mRNA was normalized to Ef1a values. Graph is representative of four independent experiments. (D,E) Graph of absolute values of Hes1 mRNA (D) and Jag1 mRNA (E) from ?TAD/?TAD and +/+ MEFs. Values were normalized to Ef1a from four independent experiments for Hes1 and two independent experiments for Jag1. (F) Schematic for oligonucleotide immunoprecipitation. (G) +/?TAD nuclear lysates were incubated with the Hes1 promoter oligonucleotide. (Left panel) Western blot for cleaved Notch1 (Val1744) in +/?TAD nuclear lysates (input) shows increased ?TAD protein relative to wild-type (+) Notch1 protein. At three concentrations (1 µg, 0.75 µg, and 0.5 µg) of oligonucleotide, wild-type protein binding to Hes1 promoter is enriched relative to ?TAD protein binding. (H) Association of Maml1 with Hes1 oligonucleotide in the presence of +/+, +/?TAD, or ?TAD/?TAD MEF nuclear lysates. Western blot for total Maml1 (left panel) and oligonucleotide-bound Maml1 (right panel) from +/+ or ?TAD/?TAD nuclear lysates incubated with 1 µg of Hes1 oligonucleotide. (I) Model of impaired formation and/or stabilization of Notch ternary complex with loss of the Notch1 TAD.