Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 1. DNA hypomethylation results in the activation of L1TD1 expression and loss of L1TD1 affects cell viability in HAP1 cells. (A) Quantification of DNA methylation levels at the L1TD1 promoter in HAP1 wildtype (WT), DNMT1 KO, and DNMT1/L1TD1 DKO cells using the MethyLight assay. DNA methylation is shown as percentage of methylation ratio (PMR). (B) qRT-PCR analysis of L1TD1 mRNA expression in HAP1 WT, DNMT1 KO, and DNMT1/ L1TD1 DKO cells. GAPDH was used as a normalization control and relative L1TD1 mRNA levels in DNMT1 KO cells were set to 1. Data are shown as a

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Activation Assay, Expressing, DNA Methylation Assay, Methylation, Quantitative RT-PCR, Control

Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 3. L1TD1 cross-talk with its ancestor L1 ORF1p. (A) Volcano plot displaying the comparison of the proteomes of HAP1 DNMT1 KO and DNMT1/ L1TD1 DKO cells determined by mass spectrometry. Differentially abundant proteins were plotted as DNMT1/L1TD1 DKO over DNMT1 KO (log2FC ≥1, adj. p-value<0.05 [red] and log2FC ≤ –1, adj. p-value<0.05 [blue]). (B) Volcano plot illustrating the DESeq2 analysis of RNA-seq performed with HAP1 DNMT1 KO and DNMT1/L1TD1 DKO cells. Differentially expressed genes are plotted as DNMT1/L1TD1 DKO over DNMT1 KO (log2FC ≥1,

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Comparison, Mass Spectrometry, RNA Sequencing

Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 4. L1TD1 promotes L1 retrotransposition. (A) Schematic representation of plasmids used for retrotransposition (figure modified from Kopera et al., 2016 and generated with BioRender.com). The pJJ101/L1.3 construct contains the full-length human L1.3 element with a blasticidin deaminase gene (mblast) inserted in antisense within the 3’UTR. The mblast gene is disrupted by an intron and mblast expression occurs only when L1 transcript is expressed, reverse transcribed, and inserted into the genome. The pJJ105/L1.3 plasmid contains a mutation in the reverse transcriptase (RT), resulting in defective retrotransposition. The backbone plasmid pCEP4 was used as additional negative control. The blasticidin deaminase gene containing plasmid pLenti6.2 was used as transfection/selection control. (B) Workflow of retrotransposition assay. DNMT1 KO and DNMT1/L1TD1 DKO cells were separately transfected with pJJ101 and control plasmids. Equal number of cells were seeded for each condition. Blasticidin selection (10 µg/ml) was started at day 4 and resistant colonies were counted on day 13. This panel was created using BioRender.com. (C) Bar graph showing the average number of retrotransposition events per 106 cells seeded in three independent experiments. Blasticidin-resistant colonies in pLenti6.2 transfected cells were used for normalization. Statistical significance was determined using unpaired t-test. All data in the figure are shown as a mean of ± SD of three independent experiments, ****p≤0.0001. (D) Representative pictures of bromophenol blue stainings of blasticidin-resistant colonies for each genotype and each transfection.

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Modification, Generated, Construct, Expressing, Reverse Transcription, Plasmid Preparation, Mutagenesis, Negative Control, Transfection, Selection, Control

Journal: Cardiovascular Research

Article Title: Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

doi: 10.1093/cvr/cvab263

Figure Lengend Snippet: Effects of TMAO exposure on TF and VCAM1 expression in human endothelial cells. HMEC-1 was left untreated or exposed to 200 µM TMAO for 2, 4, and 6 h and mRNA expression for (A) flTF, (B) asTF, and (C) VCAM1 analysed. In addition, HMEC-1 was treated with vehicle or TMAO at different concentrations as indicated for 2 h and mRNA expression of (D) flTF, (E) asTF, and (F) VCAM1 assessed. (G) Protein amounts of flTF, asTF, and VCAM1 in HMEC treated with 200 µM TMAO for 6 h quantified via western blot. Human monocytic THP-1 cells were treated with TMAO or vehicle for 2 h and mRNA expression of (H) flTF and (I) asTF analysed. Results are presented as mean±SEM. Global P-values shown were obtained by non-parametric Kruskal–Wallis test with Dunn’s multiple comparisons post hoc test to compare different treatments. Differences between two groups were assessed using a Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Gene expression was determined using TaqMan Fast Universal PCR Master Mix (Catalogue #4366072, Thermofisher, Waltham, MA, USA) or TaqManTM RNA-to-CTTM 1-Step Kit (Catalogue #4392653, Thermofisher, Waltham, MA, USA) using the following FAM- or VIC-tagged TaqMan ® gene expression assays (all from Life Technologies, Carlsbad, CA, USA): Human GAPDH (Assay ID Hs99999905_m1), human asTF and flTF (Custom probes, for details see Reference 57 ) human VCAM1 (Assay ID Hs01003372_m1), mouse GAPDH (Assay ID Mm99999915_g1), mouse flTF (Assay ID Mm00438856_m1), mouse asTF (custom probe, Clone ID AP7DRF4), mouse VCAM1 (Assay ID Mm00449197_m1).

Techniques: Expressing, Western Blot, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

doi: 10.1093/cvr/cvab263

Figure Lengend Snippet: TMAO acutely raises TF and VCAM1 expression in aortic tissue in vivo. C57/BL6 mice were injected with TMAO or vehicle intraperitoneally for 1.5 h. Next, (A) plasma TMAO levels were quantified by LC/MS/MS and arotic mRNA expression of (B) flTF, (C) asTF, and (D) VCAM1 quantified via TaqMan rtPCR. To assess protein expression, the animals were injected with vehicle or TMAO for 6 h. Subsequently, (E) TMAO plasma levels and (F) protein amounts of TF and VCAM1 were quantified via western blot and (G) density of the protein bands quantified. (H) To analyse localization within the vessel wall, aortic tissue of TMAO-injected mice was probed for TF and VCAM1 expression 6 h post injection using immunofluorescence staining (TF red, upper panel, VCAM1 red, lower panel). The tissue was counterstained with DAPI (blue) and an f-actin probe (green). Results are presented as mean±SEM. Pairwise comparison was performed using a Mann–Whitney test.

Article Snippet: Gene expression was determined using TaqMan Fast Universal PCR Master Mix (Catalogue #4366072, Thermofisher, Waltham, MA, USA) or TaqManTM RNA-to-CTTM 1-Step Kit (Catalogue #4392653, Thermofisher, Waltham, MA, USA) using the following FAM- or VIC-tagged TaqMan ® gene expression assays (all from Life Technologies, Carlsbad, CA, USA): Human GAPDH (Assay ID Hs99999905_m1), human asTF and flTF (Custom probes, for details see Reference 57 ) human VCAM1 (Assay ID Hs01003372_m1), mouse GAPDH (Assay ID Mm99999915_g1), mouse flTF (Assay ID Mm00438856_m1), mouse asTF (custom probe, Clone ID AP7DRF4), mouse VCAM1 (Assay ID Mm00449197_m1).

Techniques: Expressing, In Vivo, Injection, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Comparison, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

doi: 10.1093/cvr/cvab263

Figure Lengend Snippet: TMAO chronically induces endothelial TF and VCAM1 expression in mouse aortas in vivo. C57/BL6 mice were either fed a control chow diet or a choline diet. After 10 days of diet, (A) plasma levels of TMAO were quantified and aortic mRNA expression for (B) flTF, (C)asTF, and (D) VCAM1 analysed. (E) Protein amounts of TF and VCAM1 in aortic tissue were measured via western blot and related to the corresponding plasma levels of TMAO. Aortas of LPS-injected mice (15 mg/kg for 6 h) as well as recombinant mouse TF were used as positive controls. (F) Density of the detected bands was quantified using an imaging software. (G) Plasma of the same animals was analysed with respect to TAT complexes via ELISA. LPS-injected animals served as a known positive control for TAT induction (H) Immunohistochemistry experiments using specific antibodies were used to assess protein expression of TF, VCAM1, and the endothelial marker CD31 in aortic tissue. (I) Mean OD of endothelial protein expression from three different anatomical sides was quantified using an imaging software and three data points for each animal were plotted. Results are presented as mean±SEM. Pairwise comparison was performed using a Mann–Whitney test.

Article Snippet: Gene expression was determined using TaqMan Fast Universal PCR Master Mix (Catalogue #4366072, Thermofisher, Waltham, MA, USA) or TaqManTM RNA-to-CTTM 1-Step Kit (Catalogue #4392653, Thermofisher, Waltham, MA, USA) using the following FAM- or VIC-tagged TaqMan ® gene expression assays (all from Life Technologies, Carlsbad, CA, USA): Human GAPDH (Assay ID Hs99999905_m1), human asTF and flTF (Custom probes, for details see Reference 57 ) human VCAM1 (Assay ID Hs01003372_m1), mouse GAPDH (Assay ID Mm99999915_g1), mouse flTF (Assay ID Mm00438856_m1), mouse asTF (custom probe, Clone ID AP7DRF4), mouse VCAM1 (Assay ID Mm00449197_m1).

Techniques: Expressing, In Vivo, Control, Clinical Proteomics, Western Blot, Injection, Recombinant, Imaging, Software, Enzyme-linked Immunosorbent Assay, Positive Control, Immunohistochemistry, Marker, Comparison, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

doi: 10.1093/cvr/cvab263

Figure Lengend Snippet: A small molecule TMA-lyase inhibitor reverses TMAO-stimulated increase in aortic TF and VCAM1. C57/BL6 mice were put on a choline diet with and without FMC in the drinking water. (A) Aortic tissue was subjected to immunohistochemistry using antibodies against TF, VCAM1, and the endothelial marker CD31. (B) Mean OD was quantified from three different anatomical sides and three data points are plotted for each animal. (B, lower panel) Levels of plasma TMAO, TMA, and choline were quantified via LC/MS/MS and (C) correlated with endothelial TF and VCAM1 expression. Results are presented as mean±SEM. Pairwise comparison was performed using a Mann–Whitney test. Correlation of TMA and TMAO levels with TF or VCAM1 mean endothelial OD was performed using non-parametric Spearman correlation.

Article Snippet: Gene expression was determined using TaqMan Fast Universal PCR Master Mix (Catalogue #4366072, Thermofisher, Waltham, MA, USA) or TaqManTM RNA-to-CTTM 1-Step Kit (Catalogue #4392653, Thermofisher, Waltham, MA, USA) using the following FAM- or VIC-tagged TaqMan ® gene expression assays (all from Life Technologies, Carlsbad, CA, USA): Human GAPDH (Assay ID Hs99999905_m1), human asTF and flTF (Custom probes, for details see Reference 57 ) human VCAM1 (Assay ID Hs01003372_m1), mouse GAPDH (Assay ID Mm99999915_g1), mouse flTF (Assay ID Mm00438856_m1), mouse asTF (custom probe, Clone ID AP7DRF4), mouse VCAM1 (Assay ID Mm00449197_m1).

Techniques: Immunohistochemistry, Marker, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Expressing, Comparison, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis

doi: 10.1093/cvr/cvab263

Figure Lengend Snippet: The gut microbiota choline TMA-lyase inhibitor FMC shifts the choline diet-induced changes in caecal microbial community associated with vascular TF and VCAM1. (A) Shannon diversity indices distinguishing chow, choline, and choline+FMC samples. Statistical analysis was performed using ANOVA. (B) NMDS based on Bray–Curtis index between the caecal microbiota recovered from mice that were on indicated diets. Statistical analysis was performed using permutational multivariate ANOVA with R2 values for % variance explained by diet being the variable of interest. (C, upper panel) Statistically significant (Benjamini–Hochberg false discovery rate; P < 0.05) genera differentiating three groups (chow, choline, and choline+FMC). Plotted are interquartile ranges (IQRs) (boxes). The dark line in the box is the median, lower whiskers represent smallest observation (≥25% quantile—1.5×IQR), upper whiskers largest observation (≤75% quantile—1.5×IQR) with outliers as dots outside of the box. (C, second panel) Scatter plots based on linear regression showing correlation between abundance of indicated genera with plasma TMAO (μM) levels, (C, third panel) endothelial TF protein and (C, fourth panel) endothelial VCAM1 protein in mouse aortas on the indicated diets, expressed as OD within the annotated endothelial layer quantified by immunofluorescence (as described in Section 2). R2 and P-values are indicated in each panel. For all panels, the same colour scheme was used for data to indicate animal diet: chow (green), choline (purple), and choline+FMC (red). The grey area shows the 95% CI.

Article Snippet: Gene expression was determined using TaqMan Fast Universal PCR Master Mix (Catalogue #4366072, Thermofisher, Waltham, MA, USA) or TaqManTM RNA-to-CTTM 1-Step Kit (Catalogue #4392653, Thermofisher, Waltham, MA, USA) using the following FAM- or VIC-tagged TaqMan ® gene expression assays (all from Life Technologies, Carlsbad, CA, USA): Human GAPDH (Assay ID Hs99999905_m1), human asTF and flTF (Custom probes, for details see Reference 57 ) human VCAM1 (Assay ID Hs01003372_m1), mouse GAPDH (Assay ID Mm99999915_g1), mouse flTF (Assay ID Mm00438856_m1), mouse asTF (custom probe, Clone ID AP7DRF4), mouse VCAM1 (Assay ID Mm00449197_m1).

Techniques: Clinical Proteomics, Immunofluorescence

Journal: Molecular Biology of the Cell

Article Title: Epithelial contribution to the profibrotic stiff microenvironment and myofibroblast population in lung fibrosis

doi: 10.1091/mbc.E17-01-0026

Figure Lengend Snippet: Expression of fibrillar collagens in EMT-competent lung epithelial cells and primary lung fibroblasts on stimulation with TGF-β1 for 3 d. (A, B) mRNA levels of COL1A1 (A) and COL3A1 (B) assessed by qRT-PCR using three technical replicates. POLR2A was used as endogenous gene. (C) Total secreted collagens of primary myofibroblasts assessed with the Sircol assay. Statistical analysis as in . Note the vertical log scale in A and B.

Article Snippet: Real-time PCRs were performed on 40 ng of each cDNA sample using TaqMan Gene Expression Master Mix and TaqMan gene-specific primer pairs and probes for human genes encoding collagen type I, alpha 1 ( COL1A1 ) (Hs00164004_m1), collagen type III, alpha 1 ( COL3A1 ) (Hs00164103_m1), and the RNA polymerase II polypeptide A ( POLR2A , used as a reference gene) (Hs00172187_m1), and for mouse genes encoding Col1a1 (Mm00801666_g1), Col3a1 (Mm01254476_m1), and Polr2a (Mm00839502_m1, used as a reference gene) (Applied Biosystems).

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Molecular Neuroscience

Article Title: Oppositional Effects of Serotonin Receptors 5-HT1a, 2, and 2c in the Regulation of Adult Hippocampal Neurogenesis

doi: 10.3389/fnmol.2010.00014

Figure Lengend Snippet: Qualitative results . (A–G) 5-HT1a, 2a and 2c receptor expression pattern in vivo , and in vitro . (A) Anti-5-HT1aR staining (DAB reaction) shows receptor expression in neurons both in the hilus and granule cell layer including the subgranular zone, Scale bar 80 μm. (B) Proliferating precursor cells [Nestin, green (B1) ] in cell culture show an intense 5-HT1aR expression [blue, (B2) ], Scale bar 10 μm. (C) 5-HT1a receptor expression in the dentate gyrus [Doublecortin, green (C1) ; 5-HT1aR, red (C2) ]. (D) The receptor is detectable in horizontal type-2 cells revealed as an overlap with DCX-EGFP [green (D1) , 5-HT1aR in red (D2) . (E) Single focal plane of the hippocampus after in situ hybridization with riboprobe for the 5-HT1a receptor, immunofluorescence for BrdU, and DAPI; the confocal image reveals strong receptor expression in CA1, fainter in the dentate gyrus (DG), and no staining in the CA3 region. (F) 5-HT2a receptor expression pattern (red) in the dentate gyrus reveals a dense staining in the hilus whereas anti-5-HT2c receptor staining [ (G) , red] mostly marks the granule cell layer (Nestin in green). (H) Expression analysis of 5-HT receptors: PCR analysis was done from reverse transcribed RNA obtained from neural precursor cells under proliferative conditions. There is abundant expression of 5-HT1aR (Lane 1) and 5-HT2aR (Lane 2). Also low levels of 5-HT2c receptors expressed in the precursor cells can be seen (Lane 3). (I,J) Anti-BrdU DAB reaction. A single i.p., injection of the 5-HT1aR agonist 8-OH DPAT 2h pre-BrdU increases the number of BrdU-positive cells in the subgranular zone (SGZ) 24 h later compare to control (CTR in J ; GCL, granule cell layer; Scale bar 120 μm).

Article Snippet: DIG-labeled riboprobe (nucleotide region 1251–1688 of the transcript for mouse Htr1a; NCBI reference NM_008308.4) was synthesized in vitro with Promega Riboprobe System – SP6/T7, DIG RNA labeling mix (Roche), and purified with RNAeasy mini kit (Qiagen).

Techniques: Expressing, In Vivo, In Vitro, Staining, Cell Culture, In Situ Hybridization, Immunofluorescence, Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Oppositional Effects of Serotonin Receptors 5-HT1a, 2, and 2c in the Regulation of Adult Hippocampal Neurogenesis

doi: 10.3389/fnmol.2010.00014

Figure Lengend Snippet: Effects of acute and chronic 5-HT1a, 2, and 2c receptor agonists and antagonists treatment . (A,B) 5-HT1aR effects. (A) Acute treatment with the 5-HT1a receptor agonist 8-OH DPAT produces a significant increase in cell proliferation 1 day after BrdU injection, whereas chronic stimulation for 1 week has no effect. In contrast, acute 5-HT1a receptor blocking with WAY100135 causes no change in cell proliferation but decreases the number of BrdU-positive cells 1 week later. (B) Confocal analysis of the differentiation profile reveals a net increase in the proportion of BrdU/DCX-positive cells after acute stimulation, and a decrease of newborn neurons (BrdU+/NeuN+) upon chronic treatment with the antagonist. (C–H) Effects of acute and chronic 5-HT2 receptor agonist and antagonist treatment, and 5-HT2c receptor stimulation on precursor cell proliferation and differentiation in the adult dentate gyrus. (C) As compared to control, acute treatment with the 5-HT2 receptor antagonist Cinanserin leads to a large increase in the number of proliferating cells 1 day after BrdU injection, but shows no differences 1 week later. In contrast, acute stimulation with the agonist α-methyl-5-HT-maleate as well as chronic treatment over 7 days significantly decreases the number of BrdU-positive cells. Acute 5-HT2c receptor agonist treatment also decreases cell proliferation, but has no effect on survival. Phenotypic analysis reveals a significant decrease in BrdU+/Dcx+ cells after acute 5-HT2R antagonist treatment (D) that result in a largely net increase in the number of BrdU+ cells of undetermined phenotype (E) . Phenotypic analysis of acute and chronic effects of the 5-HT2R agonist reveal a net decrease in type-1/2a and type-2b cells after acute treatment (F) , and a significantly reduced number of newborn neurons after 7 days (G) . Acute 5-HT2c receptor agonist treatment exerts a shift from type-1/2a (which decreases) to type-3 and newly postmitotic cells (which increases), with the type-2b stage being unaffected (H) . Data present the absolute number of BrdU-positive cells per dentate gyrus as well as their phenotype by percentage and absolute number, mean ± SD. (I) In vitro , self-renewal potential of neural precursor cells indicated by the capacity to form spheres when plated at clonal densities. Upon 5-HT1aR antagonist (NAN-190) addition as well as upon 5-HT2R stimulation with α-methyl-5-HT the self-renewal potential was significantly decreased, whereas the 5-HT1aR agonist 8-OH DPAT had no effect when added to the culture. (J) Neuronal differentiation of precursor cells was detected by β-III-tubulin antibody staining, and the number of neurons was counted. 5-HT2R antagonist Cinanserin (Cin) potently decreased neuronal differentiation from adult neural precursor cells suggesting an essential role for 5-HT2R in neuronal differentiation. 5-HT2R agonist addition but not 5-HT2cR agonist (WAY161503) also inhibited neuronal differentiation.

Article Snippet: DIG-labeled riboprobe (nucleotide region 1251–1688 of the transcript for mouse Htr1a; NCBI reference NM_008308.4) was synthesized in vitro with Promega Riboprobe System – SP6/T7, DIG RNA labeling mix (Roche), and purified with RNAeasy mini kit (Qiagen).

Techniques: Injection, Blocking Assay, In Vitro, Staining