shhn cdna  (Addgene inc)

Journal: bioRxiv

Article Title: Scube2 primes Dispatched and ADAM10-mediated Shh release by recruiting HDL acceptors to the plasma membrane

doi: 10.1101/2025.01.20.633902

Figure Lengend Snippet: A-C) Solubilization of non-palmitoylated C25S Shh, non-cholesteroylated ShhN and non-lipidated C25S ShhN in serum-depleted media. C25S Shh release remains associated with A10 function but is less controlled by Scube2. ShhN release is independent of A10 and Scube2, as is the solubilization of the C25S ShhN control. A’-C’) Protein quantification from HEK cells or A10 -/- cells in the presence of Scube2. Shh signals from HEK cells also expressing Scube2 were set to 100%. Unpaired t-test, two-tailed. ns: p>0.05, ****: p<0.0001, n=7 (A’), n=6 (B’), n=8 (C’). A’’-C’’) Quantification of protein solubilization from HEK cells in the presence or absence of Scube2. Shh signals from HEK cells also expressing Scube2 were set to 100%. Unpaired t-test, two-tailed. ns: p>0.05, *: p<0.05, n=7 (A’’), n=3 (B’’), n=8 (C’’). See Supplementary Table 1 for detailed statistical information.

Article Snippet: Unlipidated C25S ShhN cDNA and non-palmitoylated C25S Shh cDNA (amino acids 1-438) were inserted into pcDNA3.1 (Invitrogen).

Techniques: Control, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Conserved cholesterol-related activities of Dispatched drive Sonic hedgehog shedding from the cell membrane

doi: 10.1101/2020.10.19.346395

Figure Lengend Snippet: A) Cap-dependent Shh translation and cap-independent Hhat translation from bicistronic mRNA. IRES: internal ribosomal entry site. The 45 kDa Shh precursor autoprocesses into the cholesteroylated (C) 20 kDa Shh signaling domain. Shh N-palmitoylation (P) is independently catalyzed by Hhat. B,C) Scube2 enhances the conversion of cellular, dual-lipidated Shh (arrowhead) into truncated soluble forms (arrow). Scube2 ΔCUB and Scube2 ΔEGF are less active. Unlipidated C25S ShhN is secreted independent of Scube2. EV: empty vector control. D) Co-expressed Scube2 increases conversion of tagged and untagged Shh variants (arrowheads) into similarly sized truncated proteins (arrows). Truncated Shh lacked α-HA reactivity and showed reduced antibody reactivity against an N-terminal peptide sequence called the Cardin-Weintraub (CW) motif. This indicates proteolytic cleavage upstream or within these sites, resulting in the loss of C-terminal and N-terminal peptides. Emergence of an upper band corresponding in size to the cellular signal (arrowheads) suggests a new processing site downstream of the HA tag (close to the cholesterol, see also ). The same (stripped) blot was used for all incubations. Bottom: Merge of inverted, false-colored α-Shh, α-CW, and α-HA immunoblots. E) All truncated protein variants are bioactive. ***: p<0.001, n=2. One-way analysis of variance (ANOVA), Dunnett’s multiple comparison post hoc test.

Article Snippet: Unlipidated C25S ShhN cDNA and non-palmitoylated C25A Shh cDNA (amino acids 1-438) were inserted into pcDNA3.1 (Invitrogen).

Techniques: Plasmid Preparation, Sequencing, Western Blot

Journal: bioRxiv

Article Title: Conserved cholesterol-related activities of Dispatched drive Sonic hedgehog shedding from the cell membrane

doi: 10.1101/2020.10.19.346395

Figure Lengend Snippet: Schematics of expressed Shh constructs (before their release) are shown. A) As previously published ( ; ), dual-lipidated Hh (cellular lipidated) migrates faster in SDS-PAGE than does E. coli -expressed unlipidated Hh (no lipids), although mass spectrometry determined molecular weights of 20167 Da for the former form and 19560 Da for the latter. Increased electrophoretic cellular lipidated Shh mobility, despite its higher molecular weight, was explained by SDS association with the large hydrophobic sterol backbone of cholesterol and the C 16 hydrocarbon tail of the palmitate. Consistent with this, chemical hydrolysis of the ester bond that attaches cholesterol to Shh was described to decrease electrophoretic mobility of the delipidated product . B) Increased electrophoretic mobility of soluble Shh over the dual-lipidated cellular protein (P+C) results from the loss of both lipidated terminal peptides during release: Shh delipidation decreases its electrophoretic mobility (see A), but the additional loss of associated terminal peptides (trunc) compensates for this decrease. As a consequence, proteolytic Shh processing results in a small net increase in electrophoretic mobility. Note that coupled Shh release and processing depends on Scube2. Bottom: schematic of Shh release. C) Insertion of a C-terminal HA tag supports this hypothesis: removal of terminal peptides, including the 1 kDa tag, increases the net electrophoretic mobility gain of solubilized proteins compared with the dual-lipidated (tagged) cellular precursor ( ; ). D) Genetic deletion of the N-terminal palmitate (C25S) impairs C25S Shh HA conversion into the fully processed protein. Instead, a C-processed but N-terminally unprocessed intermediate (asterisk) is released ( ; ), suggesting that N-palmitate (by its continued membrane association) controls quantitative Shh N-processing . An additional decreased electrophoretic mobility fraction that still carries the C-terminal HA tag (top band, compare with A) is also detected, suggesting generation of a second cleavage site proximal to the cholesterol as a consequence of HA insertion. E) ShhN co-expression with Hhat (resulting in N-palmitoylated proteins lacking the cholesterol moiety) also results in the solubilization of truncated proteins (compare with Scube2 independent secretion of non-palmitoylated C25S ShhN, ). F) Reverse-phase HPLC confirms Shh delipidation during release. Non-lipidated soluble control C25A ShhN (gray solid line) and cholesterol-modified C25A Shh (dotted line) were released from Disp-expressing Bosc23 cells in the presence of Scube2. Both soluble proteins bound to and eluted from a hydrophobic C4 column in a similar manner, but soluble C25A Shh was less hydrophobic than its cholesteroylated precursor in Bosc23 cell lysates (black solid line). This demonstrates loss of the cholesteroylated C-terminus during release. Elution profiles are expressed relative to the highest protein amount in a given fraction (set to 100%). fr#: fraction number.

Article Snippet: Unlipidated C25S ShhN cDNA and non-palmitoylated C25A Shh cDNA (amino acids 1-438) were inserted into pcDNA3.1 (Invitrogen).

Techniques: Construct, SDS Page, Mass Spectrometry, Molecular Weight, Expressing, Modification

Journal: bioRxiv

Article Title: Conserved cholesterol-related activities of Dispatched drive Sonic hedgehog shedding from the cell membrane

doi: 10.1101/2020.10.19.346395

Figure Lengend Snippet: A) Alignment of targeted disp gene sequences from Disp −/− cells and from non-targeted (nt Ctrl) cells. A’) Schematic representation of the Disp protein structure. An asterisk indicates the CRISPR/Cas9-generated stop codon introduced at position 323. This deleted 11 of 12 transmembrane domains (TM), representing ~80% of the protein sequence. L1/L2: extracellular loops. TM2-6 (red): sterol-sensing domain (SSD). B-E) Immunoblots of cellular (c) and released (into the media, m) Shh and unlipidated control C25S ShhN in nt Ctrl and Disp −/− cells. Scube2 co-expression is indicated; arrows indicate solubilized Shh and the arrowhead indicates accumulated cellular material in Disp −/− cells. D, E: The absence of Scube2 in serum-free media or the presence of 10% serum reduced Shh processing and rendered Shh release independent of Disp. B’-E’) Quantifications of relative Shh release from nt Ctrl and Disp −/− cells. Ratios of solubilized versus cellular Shh were determined and expressed relative to Shh release from nt Ctrl cells (set to 100%, black bars). Unpaired t-tests. B’: n=22 datasets from 11 independent experiments. C’: n=9 datasets from 4 independent experiments. D’: n=7 datasets from 3 independent experiments. E’: n=18 datasets from 5 independent experiments.

Article Snippet: Unlipidated C25S ShhN cDNA and non-palmitoylated C25A Shh cDNA (amino acids 1-438) were inserted into pcDNA3.1 (Invitrogen).

Techniques: CRISPR, Generated, Sequencing, Western Blot, Expressing

Journal: bioRxiv

Article Title: Conserved cholesterol-related activities of Dispatched drive Sonic hedgehog shedding from the cell membrane

doi: 10.1101/2020.10.19.346395

Figure Lengend Snippet: A) Reduced Shh shedding from A10 −/− cells compared with Shh release from HEK wild-type (WT) cells. Arrows indicate solubilized truncated Shh. A’) Quantification of relative amounts of processed Shh as shown in A. n=5 datasets from 5 independent experiments, unpaired t-test. B) Similar unlipidated C25S ShhN release from WT and A10 −/− cells (arrows). C) Increased release of processed Shh from A10 −/− cells that co-express Disp tg . Note the reduced release and only partial processing of Shh from A10 −/− cells (arrow and arrowhead) compared with empty vector (EV) transfected WT cells (asterisk). C’) Quantification of relative processed Shh release as shown in C. n=9 datasets from 4 independent experiments, one-way ANOVA, Sidak’s multiple comparison post hoc test. D) A10 C with its C-terminal transmembrane domain replaced by C-cholesterol increases Shh shedding from the surface of Disp −/− cells (arrows). D’) Quantification of relative processed Shh release as shown in D. n=3 datasets from 3 independent experiments, one-way ANOVA, Dunnett’s multiple comparison post hoc test.

Article Snippet: Unlipidated C25S ShhN cDNA and non-palmitoylated C25A Shh cDNA (amino acids 1-438) were inserted into pcDNA3.1 (Invitrogen).

Techniques: Plasmid Preparation, Transfection

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet: (A) Control pCAG-GFP plasmids (control-IUE) or pCAG-ShhN-ires-GFP plasmids (ShhN-IUE) were electroporated into the cortical VZ on E13.5. The E18.5 brains were analyzed. The distribution patterns of electroporated cells (GFP + ) in the cortex are shown. Note that the mRNA levels of Gli1 , Ptch1 , Gad1 , Tshz1 , and Prokr2 were dramatically increased in the ShhN-IUE cortex. (B) The expressions of GSX2, ASCL1, DLX2, SP8, SP9, and OLIG2 were greatly increased in the ShhN- IUE cortex. (C) RNA-seq analysis revealed increased expression levels for SHH pathway target genes, OB interneuron lineage and oligodendrocyte lineage genes in the ShhN-IUE cortices at P0. Data are presented as means ± SEM; n = 3. ***p < 0.001, *p < 0.05; n.s., non-significant; Student’s t test in (C). Scale bars, 200 mm in (A) and (B).

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques: Control, RNA Sequencing, Expressing

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet: (A and B) Scatterplot of cells after principal-component analysis and t-SNE visualization, colored according to Seurat clustering and annotated by major cell types for all the cells in the wild-type sample (A) and the ShhN -IUE sample (B). (C and D) t-SNE of cells colored by mean expression of Gsx2 and Olig2 in wild-type (C) and ShhN -IUE (D) samples. (E) The eight Gsx2 + cells in the E16.5 wild-type sample consisted of four tri-IPCs and four OB-IPCs, based on the expressions of specific genes.

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques: Expressing

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet: (A) Seurat clustering was performed on all the progenitor cells in the ShhN-IUE sample. Seven clusters were identified and annotated to six cell types based on gene expression features. (B) Heatmap showing marker gene expressions in the seven cell clusters. Each column represents expressions in one cell, and each row represents expressions of one gene. (C) The t-SNE plots of cells colored by mean expression of specific marker genes. (D) Monocle analysis of all the progenitors in the ShhN-IUE samples revealed differentiation trajectories and pseudo-timelines along the cell differentiation axis. Each point represents a cell, colored by cluster identity (top) or pseudo-timeline (bottom). (E) Seurat clusters shown along the predicted pseudo-timeline differentiation trajectory.

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques: Gene Expression, Marker, Expressing, Cell Differentiation

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet: (A and B) The expression of GSX2, OLIG2, and DLX2 in the cortical VZ/SVZ of wild-type (A) and ShhN-IUE (B) mice at E17. Note that very few GSX2 + cells (green) were present in the cortical SVZ. Arrows indicate GSX2 + OLIG2 + DLX2 tri-IPCs, and arrowheads indicate GSX2 + DLX2 + OB-IPCs. (C and D) The expression of SP9 and SP8 in the cortical VZ/SVZ of wild-type (C) and ShhN-IUE (D) mice at E17. (E and F) More OB interneuron lineage cells (E) and more tri-IPCs and OB-IPCs (F) were observed in the ShhN-IUE cortices than in the controls. (G) The sequential expression of GSX2/DLX2/SP9/SP8 is linked to lineage differentiation from tri-IPCs/OB-IPCs/OB neuroblasts, indicating the core transcriptional network for OB interneuron generation. Data are presented as means ± SEM; n = 3 mice for each condition. ***p < 0.001, **p < 0.01, *p < 0.05; Student’s t test in (E) and (F). Scale bars, 50 mm in (A)–(D) and (G).

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques: Expressing

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet:

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques:

Journal: Cell reports

Article Title: Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

doi: 10.1016/j.celrep.2020.03.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The ShhN cDNA was cloned from pcDNA3.1-ShhN plasmid (Addgene # 37680) and inserted into pCAGGS-ires-EGFP vector, using NotI and XhoI restriction sites.

Techniques: Recombinant, Imaging, Software