Journal: eLife

Article Title: ARL13B regulates Sonic hedgehog signaling from outside primary cilia

doi: 10.7554/eLife.50434

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Gli3 (Goat Polyclonal) , R and D , R and D Systems Cat# AF3690, RRID: AB_2232499 , 1:200 , .

Techniques: CRISPR, Mutagenesis, Staining, Sequencing, Recombinant, Purification, Software

Journal: bioRxiv

Article Title: Cholesterol activates the G-protein coupled receptor Smoothened to promote morphogenetic signaling

doi: 10.1101/070623

Figure Lengend Snippet: ( A ) Gli1 mRNA, encoded by a direct Hh target gene, was measured by quantitative real-time reverse-transcription PCR (qRT-PCR) and normalized to mRNA levels of the housekeeping gene GAPDH after treatment (12 hours) with various doses of naked MβCD or a saturated MβCD:cholesterol (8.8:1 molar ratio) complex. In both cases, the concentration of MβCD is plotted on the abscissa. ( B ) Immunoblotting was used to measure protein levels of GLI1, full-length GLI3 and the repressor fragment of GLI3 after treatment (12 hours) with various concentrations (in mM) of MβCD:cholesterol. Dotted lines demarcate non-contiguous regions of the same immunoblot that were juxtaposed for clarity. ( C ) Gli1 induction in response to various doses of SHH in the presence or absence of a low dose of MβCD:cholesterol. Inset shows non-linear curve fits to the data after a normalization in which the Gli1 mRNA level in the absence of SHH was set to 0% and at the maximum dose of SHH was set to 100%. ( D ) Time course of Gli1 induction (left y-axis) after treatment with SHH (265 nM) or the MβCD:cholesterol complex (2.5 mM). The gray circles (right y-axis) show the kinetics of increase in unesterified cholesterol (normalized to total protein) after the addition of MβCD:cholesterol. In all graphs, circles depict mean values from 3 replicates and error bars show the SD.

Article Snippet: Antibodies against GLI3 and GLI1 were from R&D Systems (AF3690) and Cell Signaling Technologies (Cat#L42B10) respectively.

Techniques: Reverse Transcription, Quantitative RT-PCR, Concentration Assay, Western Blot

Journal: Developmental biology

Article Title: Desert hedgehog-primary cilia cross talk shapes mitral valve tissue by organizing smooth muscle actin.

doi: 10.1016/j.ydbio.2020.03.003

Figure Lengend Snippet: Fig. 1. DHH ligand is expressed by the endocardium and activates Smoothened ciliary translocation in interstitial cells (A) RT-PCR analyses of cultured murine VICs showing expression of Smo, Gli1, Gli2, Gli3 and Patched 1 (P1) but not Shh, Ihh, or Dhh. Hprt was used as a control for the RT reaction. (B) mRNA reads from RNAseq datasets of isolated anterior mitral valve leaflets at P0 showing Dhh is the only hedgehog ligand present within the mitral valves. (C) Section in situ for Dhh mRNA at E15.5 and P0 showing expression in valve endocardium (arrows), primary atrial septum (PAS), epicardium (epi), ventricular endothelium (blue ar- rowheads) and endothelium of forming chordae tendineae (black arrowheads). (D) IHC shows the expression of DHH protein by endocardial cells of the developing mitral valve (white arrows), epicardium (epi) and ventricular endothelium (white arrows). Expression within the valve is more concentrated along the atrialis. No discernible expression is observed at adult timepoint (**). (E) High resolution confocal microscopy and 3D reconstruction of IHC of the anterior leaflet of P0 mitral valves show Smoothened (red) translocation onto ciliary axoneme (green-acetylated tubulin) indicating active HH signaling (arrowheads). AVC ¼ atrioventricular cushion, AL ¼ anterior leaflet, PL ¼ posterior leaflet.

Article Snippet: Primary antibodies and their dilutions included: GLI3 (Origene, #TA337186, 1:1000), RAC1 monoclonal antibody (Cytoskeleton Inc., #ARC03, 1:500) and Smoothened (GeneTex, #GTX60154, 1:1000).

Techniques: Translocation Assay, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Expressing, Control, Isolation, In Situ, Confocal Microscopy

Journal: Science Advances

Article Title: A Shh/Gli-driven three-node timer motif controls temporal identity and fate of neural stem cells

doi: 10.1126/sciadv.aba8196

Figure Lengend Snippet: ( A ) Scheme of mESC differentiation. ( B ) Scheme of Shh signaling pathway. ( C ) Heatmap of expression levels of genes associated with pluripotency, neuralization, and ventralization. ( D ) Gene expression levels fragments per kilobase of transcript per million mapped read (FPKM) in NSCs over time. Shaded area, SEM ( E ) Genes with biphasic expression profile, repressed at 3.5DDC by CyC treatment (0DDC to 3.5DDC) and bound by Gli1 or Gli3, and their relative temporal expression profile. ( F and G ) Western blot for Gli1-3 proteins in NSCs at different DDC and corresponding quantification. ( H ) Immunofluorescence of Arl13b, Nkx2.2, Gli2, and Smo at 3DDC and 5.5DDC. ( I ) Effect of treatment of cultures with CyC from 2.5DDC on Nkx2.2, Phox2b, and Nkx2.9 expression at 4.5DDC and number of Gata3 + 5HTNs at 5DDC. ( J to L ) Box plot of Gli2, Gli1, and Nkx2.9 expression levels in Nkx2.2 + nuclei at different DDC. Whiskers, 5th to 95th percentile; outliers were omitted. ( M ) Quantification of Nkx2.2 + NSCs expressing Phox2b over time. ( N and O ) Scatter plot of nuclear protein levels of Gli1 with Nkx2.9 or Phox2b in Nkx2.2 + nuclei at 3.5DDC. Trend line in red; r , correlation coefficient. (F, I, and M) Error bars, means ± SEM; asterisks, Student’s t test, *** P ≤ 0.001. MNw, motor-neuron window; 5HTNw, serotonergic-neuron window; A.U., arbitrary units.

Article Snippet: Primary antibodies used are as follows: mouse anti-Gli1 (Cell Signaling Technology), goat anti-Gli2, Gli3 (R&D Systems), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Thermo Fisher Scientific), and mouse anti-actin (Seven Hills Bioreagents).

Techniques: Expressing, Gene Expression, Western Blot, Immunofluorescence

Journal: Science Advances

Article Title: A Shh/Gli-driven three-node timer motif controls temporal identity and fate of neural stem cells

doi: 10.1126/sciadv.aba8196

Figure Lengend Snippet: ( A and B ) Western blot of immunoprecipitated (IP) Gli2 or Gli3 protein from NSCs isolated at different DDC and quantification of protein ratios. ( C ) Western blot of Gli1-3 in WT and Ptch1 −/− NSCs at 3.5DDC and 5.5DDC. ( D ) Immunofluorescence of Nkx2.2, Phox2b, and Nkx2.9 in Ptch1 −/− ESC cultures at 3.5DDC and 6DDC. ( E and F ) Western blot of IP Gli2 or Gli3 protein from WT or Ptch1 −/− NSCs isolated at 3.5DDC and 5.5DDC and differentiated in the presence or absence of SAG as indicated. Quantification of protein ratios of Gli2 and Gli3 bands in WT (+SAG) and Ptch1 −/− (−SAG) NSCs at 3.5DDC. ( G ) Down-regulation of Gli genes produces parallel temporal GliA and GliR gradients. (B and F) Error bars, means ± SEM; shaded area, 95% confidence intervals (CIs).

Article Snippet: Primary antibodies used are as follows: mouse anti-Gli1 (Cell Signaling Technology), goat anti-Gli2, Gli3 (R&D Systems), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Thermo Fisher Scientific), and mouse anti-actin (Seven Hills Bioreagents).

Techniques: Western Blot, Immunoprecipitation, Isolation, Immunofluorescence

Journal: Science Advances

Article Title: A Shh/Gli-driven three-node timer motif controls temporal identity and fate of neural stem cells

doi: 10.1126/sciadv.aba8196

Figure Lengend Snippet: ( A and C ) Transverse sections at r2/3 ventral hindbrain level of mouse embryos. (A and B ) Expression of Gli1-3 , Ptch1 , Nkx2.9 , Phox2b , and Tgf β 2 in WT and Gli1 ON embryos at E9.5 and E10.5 in Nkx2.2 progenitor domain (A) and plots of relative transcript expression levels along the ventral-dorsal extent of the Nkx2.2 progenitor domain at E10.5 (B). Blue dashed lines delimit Nkx2.2 domain; red dashed line, approximate ventral limit of Gli2/Gli3 expression. (C and D ) Expression of Smo in Arl13b + cilia within Nkx2.2 domain at E10.5 and box plots of Smo protein levels in individual Arl13 + cilia at E9.5 and E10.5 in WT and Gli1 ON embryos. ( E ) Expression of Smo, Arl13b, and Nkx2.2 in WT and Gli1 ON ESC cultures at 5.5DDC and box plots of Smo levels in Arl13 + cilia at 4.5DDC and 5.5DDC. ( F ) Western blot of Gli1 and of IP Gli2 or Gli3 proteins from WT and Gli1 ON NSCs isolated at 4.5DDC. ( G ) Electroporation of CAGG constructs expressing Nkx2.2, Gli3A, Nkx2.2 + Gli3A, Nkx2.2 + Gli3A + GliR at HH12 and analysis of TGF β 2 expression in the neural tube (boxed region) after 40 hours. ( H ) Summary of the temporal network. (D and E) Box plots, whiskers define 5th to 95th percentile, outliers are omitted. Asterisks, pairwise Wilcoxon test, *** P ≤ 0.001.

Article Snippet: Primary antibodies used are as follows: mouse anti-Gli1 (Cell Signaling Technology), goat anti-Gli2, Gli3 (R&D Systems), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Thermo Fisher Scientific), and mouse anti-actin (Seven Hills Bioreagents).

Techniques: Expressing, Western Blot, Isolation, Electroporation, Construct

Journal: Bioengineered

Article Title: All-trans-retinoic acid suppresses rat embryo hindlimb bud mesenchymal chondrogenesis by modulating HoxD9 expression.

doi: 10.1080/21655979.2021.1940613

Figure Lengend Snippet: Figure 5. ATRA suppresses Shh-Gli3 in rEHBMCs. rEHBMCs were treated with ATRA as noted above. (a) mRNA levels of Shh and Gli3 were quantified by RT-PCR and qPCR assays. Protein expression of Shh (b) and Gli3 (c) was quantified by immunoblotting. Gene expression was normalized to GAPDH. Data are shown as means ± SD of at least three independent experiments. aP < 0.05 vs control group, bP < 0.01 vs control group.

Article Snippet: Primary antibodies against the following proteins were used: Sox9 and HoxD9 (both from Abcam, Cambridge, UK), as well as Shh, Gli3, and GAPDH (all from Proteintech, Chicago, IL, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Gene Expression, Control