Journal: Scientific Reports

Article Title: fMRI indicates cortical activation through TRPV1 modulation during acute gouty attacks

doi: 10.1038/s41598-019-48656-6

Figure Lengend Snippet: Cortical TRPV1 expression was enhanced by gouty arthritis. ( A ) ROI selection. ( B ) Cortical TRPV1 expression on the MSU-affected and control sides. ( C ) TRPV1 immunoreactivity intensity. ( D ) TRPV1 immunoreactivity coverage. ( E ) Immunoprecipitation of beta-tubulin with TRPV1 immunoblotting, which demonstrates a strong interaction between TRPV1 and neuronal filament proteins. The cropped images of the gels are used in the figure, and images of the full-length gels are presented in Supplementary Fig. S2. (***Indicates statistically significant difference with p < 0.001 after ANOVA). Scale bar = 50 µm.

Article Snippet: The primary antibodies used were: anti-TRPV1 (1:2000; PC420; Merck Millipore, Darmstadt, Germany); anti-COX-2 (1:1000; Thermo Fisher Scientific); β-tubulin (1:2000; MCA2703; Bio-Rad Laboratories); β-actin (1:4000; sc-47778; Santa Cruz Biotechnology).

Techniques: Expressing, Selection, Control, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A) Representative wild-type, Ttbk2 null/null , and Ttbk2 sca11/sca11 E10.5 embryos, as indicated. Scale bar = 1mm. Arrowheads point out the forebrain, which in Ttbk2 null/nul and Ttbk2 sca11/sca11 fails to form two distinct hemispheres (holoprosencephaly). * Indicates the midbrain flexure which in both mutants is narrowed and takes on a pointed appearance, similar to other mutants with disrupted cilia and SHH signaling. (B-C, E-F) Transverse sections through neural tubes of wild-type (B-C) and Ttbk2 sca11/sca11 (E-F) E10.5 embryos. Sections were taken at the level of the forelimbs and immunostained for ISL1 (B, E) to label differentiated motor neurons or NKX2.2 and OLIG2 (C, F) to label V3 interneuron progenitors and motor neuron progenitors, respectively. Scale bar = 100μm. (D, G) Mesenchymal cells surrounding neural tube of E10.5 WT and Ttbk2 sca11/sca11 embryos, immunostained for ARL13B to label cilia (green) and γ-Tubulin (red). Scale bar = 13μm.

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques:

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A,B) Representative Wild type (A) and Ttbk2 sca11/null (B) E10.5 embryos. Scale bar = 1mm. (C,D) Mesenchymal cells surrounding neural tube of E10.5 Wild-type (C) and Ttbk2 sca11/null (D) embryos. Sections are immunostained for cilia using ARL13b (red) and γ-Tubulin (green). Scale bar = 20μm. (E,F) Transverse sections of E10.5 neural tubes of Wild-type (E) and Ttbk2 sca11/null (F). Sections are immunostained for NKX2.2 to label V3 interneuron progenitors and OLIG2 to label motor neuron progenitors. Scale bar =100μm.

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques:

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A,B) MEFs of the indicated genotype were serum starved for 48 hours and immunostained for IFT88 (green) as well as γ-Tubulin (red) to label centrosomes and Acetylated α-Tubulin (magenta) to label the axonemes of cilia. Ttbk2 sca11/sca11 cells lack cilia and also lack IFT88 at the mother centriole. (C,D) . Serum starved MEFs were treated as above and stained for CP110 (green) and γ- Tubulin (red). Ttbk2 sca11/sca11 cells retain CP110 on both centrosomes in the absence of serum. Scale bar = 5μm.

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques: Staining

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A, B) A comparison of rescue of cilia formation by WT TTBK2-GFP in MEFs derived from Ttbk2 null/null embryos vs Ttbk2 sca11/sca11 embryos. A representative field of cells from each condition is shown in (A) immunostained for ARL13B (green) and γ-Tubulin (red). Scale bar = 20μm. The mean percent of ciliated cells in Ttbk2 null/null MEFs versus Ttbk2 sca11/sca11 MEFs after rescue with TTBK2 WT is shown in (B). The graph represents the average of 6 fields of cells across two independent experiments (null+TTBK2 WT n= 172; sca11+Ttbk2 WT n=190). Error bars represent SEM. p=0.0002 by Student T-test. (C-E) A comparison of cilia morphology in WT cells, Ttbk2 null/null MEFs rescued with TTBK2-GFP, and Ttbk2 sca11/sca11 MEFs rescued with TTBK2-GFP. (C) Depicts two representative images of cilia of each condition immunostained for ARL13B (green) and γ-Tubulin (red). Scale bar = 2μm. ARL13B fluorescence intensity is shown in (D). ARL13b intensity is lower in Ttbk2 sca11/sca11 +TTBK2-GFP compared to both WT and Ttbk2 null/null +TTBK2-GFP (1-way ANOVA, p<0.0001), however the mean cilia length is not different between these conditions (E).

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques: Derivative Assay, Fluorescence

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A) Schematic of the Ttbk2 gene trap ( Ttbk2 gt ) targeting design (schematic adapted from International Mouse Phenotyping Consortium). (B) RT-PCR analysis of WT splicing in Ttbk2 gt/gt . RNA from 3 biological replicate brains per genotype was used, and primers targeting the exon 4-5 boundary show that WT transcript is still produced in Ttbk2 gt/gt mice. (C) P30 mice showing phenotypic differences between Ttbk2 gt/+ and Ttbk2 gt/gt . (D) H&E staining of neural cortex and kidney tissue from 6mo old Ttbk2 gt/gt mice showing hydrocephaly and polycyctic kidneys. Scale bar = 1mm. (E) Western blot showing decreased TTBK2 protein levels (150kDa) in lysates from Ttbk2 WT , Ttbk2 gt/+ , and Ttbk2 gt/gt brains. γ-Tubulin is a loading control. (F) Quantification of the relative intensity of the Western blot bands for TTBK2. TTBK2 protein levels in Ttbk2 gt/gt brain lysate is about 8.2% of the amount in Ttbk2 WT brain lysate.

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques: Reverse Transcription Polymerase Chain Reaction, Produced, Staining, Western Blot

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A-D) Representative images of MEFs taken from the embryos of the indicated and serum starved for 48-hours to induce ciliogenesis. Cilia were immunostained for ARL13b (green) to label cilia and γ-Tubulin (red) to label centrosomes. Scale bar = 20μm. (E-I) Localization of endogenous TTBK2 (green) in MEFs of each indicated genotype. Cells were counterstained with γ-Tubulin (red) to label centrosomes and Acetylated α-Tubulin (magenta) to label cilia. Staining of Ttbk2 null/null cells is shown as a negative control. Lower panels show zoomed images of from individual cells seen in the top panel. Scale bar = 10μm, 2μm for zoomed panels. (J) Quantification of the percentage of ciliated cells from MEFs of each genotype following 48 hours of serum starvation. Cilia frequency is reduced in all mutants compared with heterozygous cells, and is further reduced in Ttbk2 sca11/gt cells relative to Ttbk2 null/gt . Bars represent the mean percentage of ciliated cells across 3 experiments with 3 replicates each (n= 289, Ttbk2 gt/+ ; n= 252, Ttbk2 gt/gt ; n= 584, Ttbk2 null/gt ; n= 340 Ttbk2 sca11/gt ). Error bars denote SEM. (K) Quantification of cilia length in MEFS of each genotype following 48 hours of serum starvation. Cilia in each of the mutant MEFs were significantly shorter than heterozygous cells, but did not differ significantly from each other. (L) Quantification of the percentage of cells with endogenous TTBK2 localized to the centrosome following 48 hours of serum withdrawal. Ttbk2 mutant cells, particularly Ttbk2 null/gt and Ttbk2 sca11/gt have reduced TTBK2 at the centrosome, however these two genotypes did not differ from one another with respect to TTBK2 localization. For J-L, stars denote statistical comparisons between the indicated groups (1- way ANOVA with Tukey-Kramer post hoc test, **** denotes p< 0.0001, *** denotes p<0.001, ** denotes p=0.0017, * denotes p= 0.014.)

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques: Staining, Negative Control, Mutagenesis

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A) Representative images of mesenchymal cells surrounding the neural tube of E10.5 embryos of the indicated genotype. Cilia were immunostained for ARL13b (red) to label cilia and γ- Tubulin (green) to label centrosomes. Scale bar = 20μm. (B) Quantification of the percentage of ciliated cells in the mesenchyme of the indicated genotype. Cilia are less abundant in Ttbk2 sca11/gt embryos. Statistical comparison was performed by 1-way ANOVA with Tukey-Kramer post-hoc test. (p<0.0001 vs Ttbk2 gt/+ ; p<0.0001 vs Ttbk2 gt/gt ; p=0.0279 vs Ttbk2 null/gt ). n=two fields of view, three biological replicates, over 1000 total cells counted per genotype. (C) Quantification of cilia length in the mesenchyme of the indicated genotype. Cilia length is not statistically significantly different across the Ttbk2 allelic series in the embryonic mesenchyme. n=50 cilia pooled from 3 biological replicates.

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques:

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A, B) SMO localization in heterozygous and Ttbk2 mutant MEFs. Immunostaining for SMO (magenta) is shown for each of the indicated genotypes in (A), with centrosomes stained for γ- Tubulin (red), and the ciliary axoneme labeled with Acetylated α-Tubulin (green). Cells were serum starved for 24 hours before being treated with 200nm SAG for an additional 24 hours. Two representative images are shown. (B) depicts a quantification of pixel intensity of SMO within the ciliary axoneme of each genotype upon stimulation with SAG. Bars represent the mean intensity of 50 measurements across 3 replicates for each genotype. Error bars depict SEM. Statistical comparison was performed by 1-way ANOVA with Tukey-Kramer post-hoc test. Ttbk2 sca11/gt cells display reduced SMO intensity within the cilium compared with each of the other genotypes (p<0.0001 vs Ttbk2 gt/+ and Ttbk2 gt/gt ; p= 0.0003 vs Ttbk2 null/gt ). (C,D) Localization of GLI2 (magenta) to the ciliary tip in MEFs of each indicated genotype. In (C) cells are counterstained for γ-Tubulin (red) to label centrosomes and Acetylated α-Tubulin (green) to label the axonemes. Three representative images are shown for each genotype. Quantification of pixel intensity of GLI2 at the ciliary tip of each genotype is shown in (D). (E,F) Localization of KIF7 (magenta) to the ciliary tip in MEFs of each indicated genotype. In (E) cells are counterstained for γ-Tubulin (red) to label centrosomes and Acetylated α-Tubulin (green) to label the axonemes. Three representative images are shown for each genotype. The percentage of cilia with KIF7 localized to the ciliary tip upon treatment with SAG is shown in (F). We find that the frequency of KIF7 localization is reduced in Ttbk2 sca11/gt cells compared with other genotypes (1-way ANOVA, p= 0.0003 vs Ttbk2 gt/+ ; p=0.0009 vs Ttbk2 gt/gt ; p=0.012 vs Ttbk2null/gt .) n=50 cilia pooled from 3 biological replicates. Scale bar = 5μm

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques: Mutagenesis, Immunostaining, Staining, Labeling

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A-C) Immunostaining in MEFs of each indicated genotype for IFT81 (magenta), γ-Tubulin (red) to label centrosomes and Acetylated α-Tubulin (green) to label the cilia (A). Quantification of IFT81 pixel intensity throughout ciliary axoneme (B) or the basal body pool only (C). Quantification of pixel intensity throughout the axoneme is shown in (B) but was not statistically significant between indicated genotypes. IFT81 pools are depleted in Ttbk2 sca11/gt basal bodies compared to other genotypes (C). Statistical comparison was performed by 1-way ANOVA with Tukey-Kramer post-hoc test. (p<0.0001 vs Ttbk2 gt/+ ; p=0.0106 vs Ttbk2 gt/gt ; p=0.0150 vs Ttbk2 null/gt ). Scale bar = 5μm. (D-F) Immunostaining of IFT88 (magenta) shows depleted staining throughout the axoneme and at the basal body for Ttbk2 sca11/gt cells (D). Quantification of pixel intensity throughout the axoneme is shown in (E). Statistical comparison was performed by 1-way ANOVA with Tukey-Kramer post-hoc test. (p=0.0282 vs Ttbk2 gt/+ ; p=0.0052 vs Ttbk2 null/gt ).Quantification of pixel intensity at the basal body is shown in (F). Statistical comparison was performed by 1-way ANOVA with Tukey-Kramer post-hoc test. (p<0.0001 vs Ttbk2 gt/+ ; p<0.0001 vs Ttbk2 gt/gt ; p=0.0647 vs Ttbk2 null/gt ). (G-I) Immunostaining of IFT140 (magenta) shows depleted staining at the basal body of the axonemes for Ttbk2 sca11/gt cells(G). Quantification of pixel intensity throughout the axoneme is shown in (H). Ttbk2 sca11/gt axonemes display less IFT140 compared to other other genotypes. Statistical comparison was performed by 1-way ANOVA with Tukey-Kramer post-hoc test. (p=0.0022 vs Ttbk2 gt/gt ; p<0.0001 vs Ttbk2 null/gt ). Quantification of pixel intensity at the basal body is shown in (I). Statistical comparison was performed by 1-way ANOVA with Tukey-Kramer post-hoc test. (p<0.0001 vs Ttbk2 gt/+ ; p<0.0001 vs Ttbk2 gt/gt ; p=0.0484 vs Ttbk2 null/gt ). n=50 or more basal bodies and cilia pooled from 3 biological replicates.

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques: Immunostaining, Staining

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A-B) Polyglutamylated tubulin localization in cilia of MEFs derived from embryos of the indicated genotypes. Immunostaining for GT335, which recognizes polyglutamylated tubulin (magenta) as well as PCNT (red) to label centrosomes and Acetylated α-Tubulin (green) to label cilia is shown in (A). A quantification of the pixel intensity of GT335 within cilia is shown in (B). Polyglutamylated tubulin levels are reduced in Ttbk2 sca11/gt relative to the other genotypes of the allelic series (p<0.0001 vs vs Ttbk2 gt/+ ; p<0.0001 vs vs Ttbk2 gt/gt ; p= 0.0327 vs Ttbk2 null/gt ). Scale bar = 5μm. (C-D) Wild type, Ttbk2 null/gt , and Ttbk2 sca11/gt MEFs were treated with 10μM Nocodazole for 10-30 minutes and percentage of ciliated cells was determined. While in WT and Ttbk2 null/gt cells the percentage of ciliated cells reduced only slightly within 30 minutes, cilia were rapidly lost in Ttbk2 sca11/gt MEFs upon treatment with nocodazole (p= 0.0112) (C). Cilia length was determined throughout nocodazole treatment for Wild type, Ttbk2 null/gt , and Ttbk2 sca11/gt . As expected, cilia shortened over time, though not significantly between genotypes over time. n=50 cilia pooled from 3 biological replicates. (E-H) Representative Transmission Electron Microscopy (TEM) images of cilia in the embryonic neural tube from e10.5 embryos of the indicated genotypes. Additionally, vesicles can be seen in Ttbk2 sca11/gt axonemes (arrow heads, H) which were not present in other genotypes. Scale bar = 0.5μm

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques: Derivative Assay, Immunostaining, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

doi: 10.1101/274266

Figure Lengend Snippet: (A,B) Immunostaining for KIF2A (magenta) in MEFs derived from embryos of the indicated genotypes of the Ttbk2 allelic series shown in (A). MEFs were serum starved for 48 hours. Centrosomes are labeled with γ-Tubulin (red) and cilia are stained for Acetylated α-Tubulin (green). Pixel intensity of KIF2A at the centrosome is quantified in (B). While we detected no significant difference between Ttbk2 gt/+ and Ttbk2 gt/gt , Ttbk2 null/gt and Ttbk2 sca11/gt cells have increased levels of KIF2A at the centrosome, and KIF2A is further increased in Ttbk2 sca11/gt . (1- way ANOVA, p<0.0001 for all). n=50 cilia pooled from 3 biological replicates. Scale bar = 5μm (C,D) Ttbk2 gt/+ cells were stably expressing either KIF2A WT or a phospho-mutant variant of KIF2A, KIF2A S135A . Immunostaining is shown for the Kif2a construct (magenta), centrosomes stained for PCNT (red), and the ciliary axoneme labeled with Acetylated α-Tubulin (green). Three representative cilia are shown per condition. Cilia were shorter in Ttbk2 gt/+ cells when expressing KIFT2A S135A . Cilia length was determined and quantified in (D) (1-way ANOVA p<0.0001). Error bars represent SEM.

Article Snippet: Commercially available antibodies used in these studies were: mouse anti-NKX2.2, ISL1 (Developmental Studies Hybridoma Bank, each 1:10); mouse anti-Pericentrin, (BD Biosciences #611814, 1:500) γ-Tubulin (Sigma SAB4600239, 1:1000), Acetylated α-Tubulin (Sigma T6793, 1:1000), polyglutamylated Tubulin (Adipogen AG-20B-0020, 1:2000); rabbit anti-IFT88 (Proteintech 13967-1-AP, 1:500), rabbit anti-IFT81 (Proteintech 1174-1-AP, 1:1000), rabbit anti-IFT140 (Proteintech 17460-1-AP 1:500), mouse anti-FLAG (Sigma F1804, 1:1000), rabbit anti-KIF2A (Abcam ab37005, 1:500), TTBK2 (Proteintech 15072-1-AP, 1:1000), Calbindin (Cell Signaling Technology 13176-S, 1:250), VGLUT2 (EMD Millipore AB2251, 1:2500).

Techniques: Immunostaining, Derivative Assay, Labeling, Staining, Stable Transfection, Expressing, Mutagenesis, Variant Assay, Construct

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: Ventral midbrain NSPC isolated from E12 rat embryos are multipotent. (A) VM NSPC cultured in the presence of the mitogenic factor FGF-2 express the markers of undifferentiated cells: Sox2, Vimentin and Nestin in proliferation stage. (B) After 6 days without FGF-2 (differentiation stage), VM NSPC differentiate into the three lineages of Central Nervous System: neurons (β-III Tubulin + and MAP2+), astrocytes (GFAP+) and oligodendrocytes (O4+), confirming the multipotency of these cultures. Nuclei were stained with Hoechst. Scale bars = 100 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Isolation, Cell Culture, Staining

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA promotes neuronal differentiation but decreases dopaminergic neurons in cultured VM NSPC. After proliferation, cells were kept on differentiating conditions for 6 days (N2 medium without FGF-2) and treated daily with different concentrations of HA (from 1 μM to 1 mM HA). VM NSPCs were analyzed after differentiation. (A) Micrographs showing the neuronal marker β-III Tubulin in control and 10 μM HA-treated cultures. Nuclei were stained with Hoechst. (B) Quantification of β-III Tubulin labeled cells relative to the total number of cells, showing a significant increase in the proportion of β-III Tubulin + cells caused by treatment with 10 μM HA. (C) Micrographs of double immunocytochemistry to detect the neuronal marker β-tubulin III and the dopaminergic marker Tyrosine Hydroxylase (TH) in control and 1 mM HA-treated cultures. Nuclei were stained with Hoechst. Scale bar = 100 μm. (D) Graph showing the percentage of TH-positive neurons in control and after HA treatments, relative to the total number of β-Tubulin III-positive cells. TH + neurons were significantly decreased after treatment with 1 mM HA. **p < 0.01. (E) Micrographs showing the dopaminergic marker TH in control cells, and the decrease caused by 1 mM HA. The H1R antagonist chlorpheniramine was tested either with or without HA. Nuclei were stained with Hoechst. (F) HA-induced decrease of TH-positive numbers was antagonized by chlorpheniramine. Note that the percentage of TH-positive cells is lower than in Figure D because it is normalized by the total number of cells. *p < 0.05 relative to control; ## p < 0.01 compared to 1 mM HA. Scale bars = 100 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Cell Culture, Marker, Control, Staining, Labeling, Immunocytochemistry

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA injection decreases dopaminergic neurons without affecting β-III Tubulin. (A) Coronal sections of the ventral midbrain from E14 vehicle- and HA-injected rat embryos stained to detect neurons (β-III Tubulin+) and dopaminergic phenotype (TH+); a representative section of a vehicle-injected embryo showing many TH-positive neurons while in a similar midbrain section from a representative HA-injected embryo less TH-positive cells are present. Nuclei were detected with Hoechst. Inner white squares in the merge images represent the higher magnifications shown at the bottom. Scale bars: 150 μm and 50 μm for the low-power and high-power pictures, respectively. (B) β-III Tubulin and TH expression by qRT-PCR from E14 vehicle- and HA-injected embryos, with β-III Tubulin presenting no change, and a significant decrease in TH caused by HA. **p < 0.01. (C) Densitometric analyses of the effect of HA administration on protein levels of β-III Tubulin and diverse factors involved in dopaminergic specification and phenotype. HA injection decreased the protein level of TH, Lmx1a, Lmx1b and Pitx3 in midbrain tissue from E14 HA-injected embryos without affecting the generation of neurons, compared to the vehicle-injected condition. Values were normalized to GAPDH signal. *p < 0.05.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining, Expressing, Quantitative RT-PCR

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA does not alter GABAergic or serotoninergic neurons. (A) Coronal sections of the ventral midbrain from E14 vehicle- and HA-injected rat embryos stained to identify neurons (β-III Tubulin+) and GABA-synthesizing cells (anti-GAD65/67+ antibody); vehicle-injected embryos had the same proportion of GAD65/67+ cells when compared to HA-injected embryos. Nuclei were stained with Hoechst. Scale bar: 150 μm. (B) HA did not modify the level of GAD65/67 protein in E14 midbrain tissue compared to vehicle-injected embryos by Western blot. The graph represents the densitometric analysis of GAD65/67 protein content where no significant effects were found; values were normalized to GAPDH. (C) Coronal sections of the ventral midbrain from vehicle- and HA-injected E14 embryos stained with β-III Tubulin and serotonin antibodies, where no differences in serotoninergic neurons are observed. Scale bar: 150 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining, Western Blot

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA acts on early dopamine neural precursors but differentiated dopamine neurons are resistant. Sagittal sections of the VM from vehicle- and HA-treated rat embryos that were injected at different developmental stages, and analyzed two days later. Sections were stained to detect neurons (β-III Tubulin+) and dopaminergic phenotype (TH+); nuclei were detected with Hoechst. The decreasing effect of HA injection on dopaminergic phenotype is evident only when injected at early developmental stages (E10-E12 or E12-E14) but not at later developmental stages (E14-E16 or E16-E18). For the vehicle-injected E10-E12 embryos, only a few TH + neurons were found in the isthmic region and these neurons were absent in HA-injected organisms. Scale bars are indicated in the figure.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: High doses of chlorpheniramine affect normal embryonic development but neither chlorpheniramine nor cimetidine affects dopaminergic neurons. (A) Micrographs from E14 rat embryos injected at E12 with different doses of the H 1 R antagonist, chlorpheniramine. Fifty and twenty-five micrograms interfered with normal development, while 15 μg allows normal development of the embryo. (B) TH and β-III Tubulin staining in coronal sections of the VM from E14 rat embryos injected with 15 μg of the H 1 R antagonist, chlorpheniramine or 50 μg of the H 2 R antagonist, cimetidine. The staining patterns were not modified by these antagonists. Scale bar: 150 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Injection, Staining, Modification

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: The H 1 R antagonist chlorpheniramine, but not the H 2 R antagonist cimetidine, abolished the deleterious effect of HA administration on dopaminergic differentiation. (A-D) TH and β-III Tubulin staining in coronal sections of the VM from E14 rat embryos with the indicated treatments. (A) Normal pattern of dopaminergic neuron staining in a vehicle-injected embryo. (B) Decrease in TH immunoreactivity due to HA administration. (C) Protective effect of the H 1 R antagonist chlorpheniramine on HA-induced decrease of TH immunoreactivity. (D) Administration of HA and the H 2 R antagonist cimetidine did not modify dopaminergic neurons compared to HA-injected embryos. Scale bar: 150 μm.

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: Staining, Injection

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: Primer sequences for detection of transcripts by qRT-PCR

Article Snippet: Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 10% NGS: rabbit polyclonal anti-β-III Tubulin (1:2000, Covance); rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:2000, DAKO); mouse monoclonal antibody anti-microtubule associated protein 2 (MAP2; 1:500, Chemicon); mouse monoclonal anti-Nestin (1:100, Developmental Studies Hybridoma Bank); rabbit anti-Nestin (1:1000, Covance); rabbit polyclonal anti-TH (1:1000; Pel-freez); rabbit polyclonal anti-Sox2 (1:200; Millipore).

Techniques: