Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5-HT1b and 5-HT2a receptors are expressed in growth cones and filopodia. a–j Sensory neuron growth cones immunostained for serotonin receptors, 5-HT1b (green in a, red in g), 5-HT2a (green, d and h) as well as filamentous actin to highlight the extent of the growth cone (Phalloidin, red; b and e). The receptors 5-HT1b and 5HT2a were observed as a widespread punctate distribution throughout the growth cone (g–h). Punctate expression was evident in filopodia (j) with isolated 5-HT1b (green, *), 5-HT2a (red, #) as well as colocalised (arrowheads) puncta on filopodial shafts. There was prominent colocalization of 5-HT2a and 5-HT1b receptors in peripheral areas (i, arrow). Scale bars are 5 μm (a–i) and 1 μm (j). k Turning responses to serotonin were sensitive to chlorpromazine. Attractive turning of growth cones to 5HT-Lo (n = 7, p = <0.0001) and repulsion to 5HT-Hi (n = 8, p = 0.003) were significantly different when cultures were treated with chlorpromazine. l Axon extension was not significantly different in any experimental treatments (ns, p > 0.05). (Mann–Whitney u-test)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Expressing, Isolation, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Lo elicits growth cone attraction through the 5-HT2a receptor. a Growth cone attraction was measured in the presence of 5HT-Lo gradients (p = <0.0001) and TCB-2 gradients (p = 0.0002). Growth cone attraction to 5HT-Lo was abolished by pharmacological application of the selective 5-HT2a receptor antagonist, ritanserin (n = 17, p = 0.0001) and the PLC inhibitor, U-73122 (n = 9, p = 0.0018). b Growth cone attraction to 5HT-Lo was not perturbed when growth cones treated with a specific 5-HT1b antagonist, GR55562 (n = 8, p = <0.0001), or the 5-HT3 antagonist ondansetron (n = 8, p = 0.0082). Depletion of ER calcium with thapsigargin (n = 19, p = 0.0003) abolished attractive turning to 5HT-Lo, while inhibition of VGCCs with nifedipine (n = 15, p = 0.0330) had no significant effect. Turning angles were compared to vehicle and 5HT-Lo and only significant differences shown in (a) and (b). Kruskal–Wallis, Dunn’s multiple comparison test. c, d Axon extension was not significantly different in any experimental treatments (ns, p > 0.05). Kruskal–Wallis, Dunn’s multiple comparison test

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Inhibition, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Hi promotes growth cone repulsion through the 5-HT1b receptor. a Growth cone repulsion was measured in the presence of 5HT-Hi gradients (p = <0.0001) and CP-94253 gradients (p = 0.0018). Growth cone repulsion to 5HT-Hi was altered to levels indistinguishable from random growth (vehicle) with application of the selective antagonist to 5-HT1b, GR55562 (n = 16, p = <0.0001), by activating adenylate cyclase with forskolin (n = 8, p = 0.0003) and restoration of cAMP signals with Sp-cAMPs (n = 8, p = 0.0001). b Repulsion to 5HT-Hi was not perturbed when growth cones were treated with the 5-HT2a receptor antagonist ritanserin (n = 8, p = 0.002) or the 5-HT3 antagonist ondansetron (n = 10, p = 0.0003). Inhibition of ER calcium release with thapsigargin (n = 10, p = <0.0001) or calcium influx with nifedipine (n = 8, p = <0.0001) did not alter growth cone repulsion from 5HT-Hi. Turning angles were compared to vehicle and 5HT-Hi. c, d Axon extension was not perturbed by any pharmacological application (ns, p > 0.05). Turning angles were compared to vehicle and 5HT-Hi. (Kruskal–Wallis, Dunn’s multiple comparison test)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Inhibition, Hi-C, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Lo exposure causes asymmetric distribution of 5-HT receptors to the turning or motile side of growth cones. Schematic a showing growth cone divided into “near” and “far” regions with respect to micropipet position (located on the upper left side). b–d Growth cones immunostained after turning in response to 5HT-Lo and stained for 5-HT2a (green, b), 5-HT1b (red, c) and merged in (d). Dotted line separates the near and far regions of growth cone (near vs far). (e) Representative image of filopodia with 5-HT2a (green), 5-HT1b (red) receptor puncta. (f) There was no significant near/far bias in the number of filopodia following exposure to 5HT-Lo (n = 8, p = 0.9004), 5HT-Hi (n = 13, p = 0.5450) or BDNF (n = 10, p = 0.7907) compared to vehicle. (Data not normally distributed: Kruskal–Wallis, Dunn’s multiple comparison test). g There were significantly more (n = 36, p = 0.0033) 5-HT2a puncta in filopodia on the near side of growth cones exposed to 5HT-Lo compared to all other treatments. h There was no bias in 5-HT1b puncta in filopodia of growth cones. (Mann–Whitney U-test). (i-j) When entire growth cones were analysed, i 5HT-2a (n = 9, p = 0.0006) and j 5HT-1b (n = 9, p = 0.0028) receptor translocation was significantly biased in growth cones exposed to 5HT-Lo. All ratios were compared to vehicle (Data normally distributed: Shapiro–Wilk. One-way ANOVA, Tukey’s multiple comparison test). Scale bars are 5 μm (Fig. 7b–d) and 1 μm (Fig. 7e)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Staining, Comparison, MANN-WHITNEY, Translocation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Lo exposure causes asymmetric distribution of membrane 5-HT2a receptors to the motile side of growth cones. a–h Representative growth cones exposed to vehicle and 5HT-Lo gradients (micropipette on upper left side) and stained for membrane localization of receptors 5-HT1b (green, a, e), 5-HT2a (green, c, g) and merged with actin, (red, b, d, f, h). Dotted line separates the near and far regions of the growth cone. i Increase magnification of representative image h to show the presence of 5-HT2a receptor membrane on most filopodia (*) of the growth cone (yellow *, insets). Scale bars are 5 μm. (j-k)There was no significant near/far bias in the amount of f-actin (/area) (j) and the number of filopodia (k) in growth cones exposed to vehicle and 5HT-Lo. Kruskal–Wallis, Dunn’s multiple comparison post hoc test. (l) There was no significant bias in the total percentage of filopodia containing 5HT2a and 5HT1b puncta (60%). One-way ANOVA followed by Tukey’s multiple comparison post hoc test. (m) There was no significant near/far bias in the amount of 5-HT2a and 5-HT1b puncta per filopodia. (n) Analysis of 5-HT receptor membrane localization showed translocation of 5-HT2a (n = 18, p = <0.0001) to the near side of growth cones exposed to 5HT-Lo while no significant translocation of 5-HT1b (n = 15, p = 0.346) was observed. (Mann–Whitney U-test)

Article Snippet: Immunocytochemistry DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Membrane, Staining, Comparison, Translocation Assay, MANN-WHITNEY

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 1 TRIM32 expression is decreased in failing human hearts and hypertrophic mouse hearts (A and B) Representative Western blots (A) and quantitative results (B) of ANP, β-MHC and TRIM32 protein expression in normal donor hearts and in human hearts with DCM (n = 8 hearts per experimental group; *P < 0.05 compared with donor hearts). (C and D) Representative Western blots (C) and quantitative results (D) of ANP, β-MHC and TRIM32 protein expression in a hypertrophic mouse heart induced by AB for the times indicated (W, weeks) (n = 6 mice per experimental group; *P < 0.05 compared with sham). (E and F) Representative Western blots (E) and quantitative results (F) of ANP, β-MHC and TRIM32 protein expression in cultured NRCMs treated with AngII for 24 or 48 h (n = 4 samples per experimental group; four independent experiments; *P < 0.05 compared with PBS). Results are means +−S.D., and statistical analyses were determined using an unpaired Student’s t test. Molecular masses are indicated in kDa in the Western blot panels.

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Expressing, Western Blot, Cell Culture

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 2 TRIM32 attenuates AngII-induced cardiomyocyte hypertrophy in vitro (A) The protein expression level of TRIM32 in NRCMs after infection with AdshRNA, AdshTRIM32, AdGFP or AdTRIM32 was determined by Western blotting (n = 4 samples per experimental group). Left: representative blots. Right: quantitative results. (B) Representative images of NRCMs infected with AdshRNA, AdshTRIM32, AdGFP or AdTRIM32 and treated with AngII (1 μmol/l) or PBS for 48 h (n = 4 samples per experimental group; blue, nucleus; green, α-actinin; scale bar, 20 μm). (C) Quantitative results of the cell surface area of NRCMs infected with AdshTRIM32 or AdshRNA subjected to AngII (1 μmol/l) or PBS for 48 h (n⩾50 cells per experimental group; *P < 0.05 compared with AdshRNA/PBS; #P < 0.05 compared with AdshRNA/AngII). (D) Quantitative results of the cell surface area of NRCMs infected with AdTRIM32 or AdGFP subjected to AngII (1 μmol/l) or PBS for 48 h (n⩾50 cells per experimental group; *P < 0.05 compared with AdGFP/PBS; #P < 0.05 compared with AdGFP/AngII). (E) Relative mRNA levels of ANP, BNP and β-MHC in AdshTRIM32-infected NRCMs

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: In Vitro, Expressing, Infection, Western Blot

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 3 TRIM32 overexpression attenuates pressure overload-induced cardiac hypertrophy and heart failure (A) Schematic diagram of the construction of TG mice with a full-length murine Trim32 cDNA under the control of the α-MHC promoter. (B and C) Representative Western blots (B) and quantitative results (C) of TRIM32 protein expression in heart tissue from four TG lines and MCT mice (n = 3 mice per experimental group; three independent experiments).

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Over Expression, Control, Western Blot, Expressing

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 4 Generation of global TRIM32-deficient mice (A) One sgRNA targeted a region downstream of the 5′ end of exon 2 in the Trim32 mouse gene. (B) Representative results of the T7E1 assay from pups subjected to microinjection. Sizes are indicated in bp. (C) Representative results of the DNA sequencing from mice with frameshift mutations. (D) Agarose gel photograph illustrating genotyping results of PCR products from WT (+/+), heterozygous (+/−) and TRIM32-KO (−/−) mice. Sizes are indicated in bp. (E) Representative Western blots of TRIM32 expression in heart tissues from global TRIM32-KO mice and their littermate controls (n = 4 mice per experimental group). Molecular masses are indicated in kDa.

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Microinjection, DNA Sequencing, Agarose Gel Electrophoresis, Western Blot, Expressing

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 5 TRIM32 deficiency aggravates pressure overload-induced cardiac hypertrophy and heart failure (A) Statistical results for HW/BW and HW/TL in WT mice and TRIM32-KO mice at 4 weeks after sham or AB (n = 8–10 mice per experimental group). (B) Representative images of gross morphology, H&E staining, WGA staining and PSR staining of hearts from the indicated groups (n = 5 mice per experimental group; scale bar, 20 μm for lower H&E staining,

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Staining

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 6 TRIM32 inhibits activation of Akt-dependent signalling pathways upon hypertrophic stresses

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Activation Assay

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 7 Blockage of Akt-dependent signalling pathways rescues pressure overload-induced cardiac abnormalities in TRIM32-KO mice (A and B) Representative Western blots (A) and quantitative results (B) of total (T-) and phospho- (P-) Akt-dependent signalling pathway protein levels in the hearts from LY294002- or DMSO-treated TRIM32-KO mice 4 weeks after AB surgery (n = 4 mice per experiment group; GAPDH was used as a loading control). Molecular masses are indicated in kDa in (A). (C) Statistical results for HW/BW and HW/TL in the indicated groups (n = 8–10 mice per experimental group). (D) Representative images of gross morphology, H&E staining and PSR staining from hearts in the indicated groups at 4 weeks after AB (n = 5 mice per experimental group; scale bar, 20 μm for lower H&E staining and PSR staining). (E) Statistical results for the cardiomyocyte cross-sectional area in the indicated groups (n⩾100 cells per experimental group). (F) Quantification of left ventricular collagen volume in the indicated groups (n⩾25 fields per experimental group). (G) Measurements of LVESD, LVEDD and FS in the indicated groups (n = 5 mice per experimental group). *P < 0.05 compared with KO/AB/DMSO; n.s. indicates no significant difference. Results are means +−S.D., and statistical analyses were performed using an unpaired Student’s t test or one-way ANOVA.

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Western Blot, Control, Staining

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 is selectively expressed in inner EGL and displays an uneven cytoplasmic distribution during GNP differentiation. a RT-qPCR analysis of Trim32 in P0, P7, P14, and adult mouse cerebella. Data are expressed as means ± SD (n = 3). An asterisk indicates P < 0.05 and triple asterisks indicate P < 0.001. b Immunoblotting analysis of Trim32, Gli1, and MycN in P0, P7, P14, and adult mouse cerebellums. c Immunofluorescence staining of Trim32 (red) in the EGL of P7 Math1-GFP transgenic mouse cerebellum. Nuclei were counterstained with DAPI (blue). EGL external granule layer, ML molecular layer, PCL Purkinje cells layer, and IGL internal granule layer. The scale bars represent 100 µm in the first panel and 25 µm in the second panel. d Confocal analysis (left) and quantification of immunofluorescence intensities (right) of distribution of Trim32 (green) in the dividing GNPs in different phases of the cell cycle. The dashed line highlights the cells that are in the indicated phase of cell cycle. The cell-cycle phases were identified by PH3 staining (red) for DNA. Nuclei were counterstained with DAPI (blue). The scale bars represent 10 µm. Data are expressed as means ± SD (n = 3). ns indicates P > 0.05 and triple asterisks indicate P < 0.001

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Transgenic Assay

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 knockout enhances GNP proliferation in the postnatal developing cerebellum. a Expression of the Math1-GFP protein (GFP, green) in P7 mouse cerebellar sections from the Math1-GFP/Trim32wt mice and Math1-GFP/Trim32KO mice. Nuclei were counterstained with DAPI (blue). Graph in a representing Math1 positive cells normalized to the length of the EGL edge. The scale bar represents 25 µm. Immunofluorescence staining of NeuN (red, b) and Ki67 (red, c) in P7 mouse cerebellar sections from the Math1-GFP/Trim32wt mice and Math1-GFP/Trim32KO mice. Nuclei were counterstained with DAPI (blue). Graph in b and c respectively representing NeuN or Ki67-positive cells normalized to the length of the EGL edge. The scale bar in b represents 50 µm. The scale bar in c represents 25 µm. oEGL outer external granule layer, iEGL inner external granule layer, ML molecular layer, and IGL internal granule layer. d P7 and P18 cerebellar midsagittal sections were stained for DAPI to show the overall morphology of cerebellum in Trim32wt mice and Trim32ko mice. The scale bar represents 500 µm

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Knock-Out, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 antagonizes the SHH signaling activity. a RT-qPCR analysis of Trim32, the granule neuronal progenitor marker Maht1, and SHH target genes in P7 cerebellar granule neuronal progenitors (cGNPs) from Trim32wt mice and Trim32KO mice. Data are expressed as means ± SD (n = 3). Double asterisks indicate P < 0.01 and triple asterisks indicate P < 0.001. b Immunoblotting analysis of Trim32, Gli1, Ccnd1, Ccnd2, and MycN in P7 mouse cerebellum from Trim32wt mice and Trim32KO mice. c, d RT-PCR mRNA expression of the SHH target genes Gli1 and MycN in HEK293T cells overexpressing Trim32-GFP or GFP control vector. The cells were cultured with or without SHH for 24 and 48 h. Data are expressed as means ± SD (n = 3). Double asterisks indicate P < 0.01 and triple asterisks indicate P < 0.001. Gli-RE-luciferase activity in human medulloblastoma cell line D283 (e) and HEK293T cells (f), following transfected the indicated vectors. The luciferase activity was evaluated relative to Renilla activity. The means ± SD from three experiments are shown

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Activity Assay, Quantitative RT-PCR, Marker, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation, Cell Culture, Luciferase, Transfection

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 interacts with Gli1 though the NHL domain. a In vivo assay for binding between Trim32 and Gli1. Expression vectors encoding TRIM32-GFP and Flag-tagged Gli1 were transfected into HEK293T cells. Whole cell lysates were immunoprecipitated with anti-Trim32 or anti-FLAG antibody and immunoblotted with anti-Trim32 and anti-Gli1 antibodies. b Co-immunoprecipitation of exogenously expressed Trim32-HA with the endogenous Gli1 in HEK293T cells. c Co-immunoprecipitation of exogenously expressed Trim32-GFP with the Gli1 truncated mutants. d Schematic drawing of full-length Trim32 (Trim32-wt) and deletion mutants. Trim32 contains a RING finger (R), two B-boxes (B), a coiled-coil region (Coil), and an NHL domain (NHL). e Co-immunoprecipitation of exogenously expressed Flag-tagged Gli1 with the Trim32 truncated mutants shown as in d

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: In Vivo, Binding Assay, Expressing, Transfection, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 promotes Gli1 ubiquitination and degradation. a HEK293T cells transfected with Gli1 and increasing doses of Trim32 were collected for immunoblotting with the indicated antibodies. b Quantification of Gli1 and Trim32 protein levels (normalized to actin) from a. The means ± SEM from three experiments are shown. c Cells stably expressing scramble RNA or shRNA targeting Trim32 (#1, #2) were harvested for immunoblotting with the indicated antibodies. d HEK293T cells were transfected with plasmids encoding Flag-tagged Gli1 and control GFP vector or Trim32 or Trim32-ΔR, treated with 20 µg/ml cycloheximide (CHX) and harvested at the indicated times points. The levels of Gli1 and Trim32 in the lysates were investigated by immunoblotting. e Quantification of Gli1 remaining protein levels (normalized to actin) from d. The means ± SEM from three experiments are shown. f Cells were transfected with scramble RNA or shRNA targeting Trim32 (#1), treated with 20 µg/ml cycloheximide (CHX), collected at the indicated time points, and then immunoblotted with the indicated antibodies. g Quantification of Gli1 protein levels (normalized to actin) from f. The means ± SEM from three experiments are shown. h Cells transfected with Gli1 or Trim32 were treated with or without 10 µM MG132 for 8 h, collected, and then immunoblotted with the indicated antibodies. Graph representing quantification of Gli1 protein levels (normalized to actin). i Anti-ubiquitin immunoblotting of immunoprecipitated exogenous Gli1 in HEK293T cells transfected with the indicated plasmids. j The effect of Trim32-wt or Trim32 deletion mutant (Trim32-ΔR) to the activation of Gli-RE-luciferase induced by Gli1 in HEK293T cells. The means ± SD from three experiments are shown. Double asterisks indicate P < 0.01

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Transfection, Western Blot, Stable Transfection, Expressing, shRNA, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Activation Assay, Luciferase

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 knockout increases the incidence of MB in Ptch1+/− mice. Quantitative PCR mRNA (a; mean ± SD; n = 9) and protein levels (b; n = 3) of Trim32 in mouse medulloblastomas (MB) from Ptch1+/− mice and normal cerebella. c RNA expression level of Trim32 negatively correlated with Gli1 (n = 51) in human SHH MB samples. Data are analyzed from Oncomine database. d Kaplan–Meier analysis of MB incidence in 63 Ptch1+/−/Trim32wt mice (gray line) versus 17 Ptch1+/−/Trim32KO mice (black line). Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice, obtained by interbreeding for at least three generations the progeny of Ptch1 heterozygous and Trim32 knock-out mice, were then monitored for the onset of medulloblastoma; (triple asterisks indicate P < 0.001, Logrank test). e Expression of the Math1-GFP protein (GFP, green) in the Math1-GFP mouse medulloblastomas and adjacent cerebellar cortices sections from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice. Nuclei were counterstained with DAPI (blue). The scale bar represents 200 µm. MB medulloblastoma, IGL internal granule layer. RT-qPCR analysis of SHH target genes (f), Trim32 (g), the granule neuronal progenitor marker Maht1 (h), and the differentiated granule cell markers including Tuj1 and NeuN (i) in adult mouse cerebellums and medulloblastomas from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice. Data are expressed as means ± SD (n = 3). An asterisk indicates P < 0.05, double asterisks indicate P < 0.01, and triple asterisks indicate P < 0.001. j Immunoblotting analysis of Trim32, Gli1, Ccnd2, MycN, and NeuN in MB from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, RNA Expression, Expressing, Quantitative RT-PCR, Marker, Western Blot

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: mRNA expression analysis

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Expressing

Journal: Frontiers in Psychiatry

Article Title: Histone deacetylase inhibitors mitigate antipsychotic risperidone-induced motor side effects in aged mice and in a mouse model of Alzheimer’s disease

doi: 10.3389/fpsyt.2022.1020831

Figure Lengend Snippet: Effects of histone deacetylase (HDAC) inhibitors on histone acetylation at the 5htr2a and Drd2 gene promoters in the striatum in young and aged mice. (A) No significant differences of H3K27ac binding at the 5htr2a promoter between young and aged group was found, but a significant increase in H3K27ac binding at the 5ht2a promoter in the striatum was found in aged and young mice co-treated with RIS and VPA or MS-275 compared to those treated with RIS alone. (B) H3K27ac binding at the Drd2 promoter was significantly decreased in aged mice as compared to young mice in Veh treated group. However, H3K27ac binding at the Drd2 promoter in the striatum was observed to be significantly increased in aged mice co-treated with RIS and HDAC inhibitors VPA or MS-275 when compared to those treated with Veh alone. Data are presented as mean ± SEM, ( n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Blots were blocked for 1-hour with 5% non-fat dry milk (Biorad, 1706404) and incubated using 1:1,000 5HT2A (Boster Biological, PA1373), 1:500 D2R (Millipore, AB5084P), or 1:1000 B-actin (Santa Cruz, sc-47778) overnight at 4°C.

Techniques: Histone Deacetylase Assay, Binding Assay

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A)/(B) Coronal sections including the caudate putamen region from two Htr2a Cre BAC mouse lines (KM207 and KM208) with Cre-dependent GFP reporter distribution: Image S1A is from http://www.gensat.org/imagenavigator.jsp?imageID=69385 . Image S1B is from http://www.gensat.org/imagenavigator.jsp?imageID=83872 . Comparison of Htr2a mRNA , autoradiography and HTR2A-EGFP-CT protein : arrows shown in these 3 figures indicated the corresponding brain regions with these three different methods. (C) Expression of Htr2a mRNA in the adult C57 mouse brain: Images are from Allen Mouse Brain Atlas ( http://mouse.brainmap.org/experiment/siv?id=81671344 ). (D) [ 3 H] MDL100,907 (0.4 nM) autoradiography labeling of HTR2A binding sites in the Wistar rat brain section (V: layer V cortex, CPu: caudate putamen, Pn: pon, 7: facial nucleus and M05: motor trigeminal nucleus.) (E) Sagittal view of distribution of HTR2A-EGFP-CT fusion protein in the whole brain cleared tissue from Htr2a EGFP-CreERT2/+ mouse line. Comparison of HTR2A-EGFP-CT protein and Htr2a transcripts in the CPu: The red star with dashed line with arrows in and denoted the patch-like pattern (striosome) in the CPu. (F) The distribution of the HT2A-EGFP-CT fusion protein in coronal section at the level of the CPu from Htr2a EGFP-CreERT2/+ mouse line. (G) Distribution of Htr2a mRNA in the brain from C57 mice. Data resource from Allen Mouse Brain Atlas ( http://mouse.brainmap.org/experiment/siv?id=81671344 .

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Comparison, Autoradiography, Expressing, Labeling, Binding Assay

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) Schema of the transgenic modifications at the murine Htr2a gene in exon 3. For Htr2a -EGFP-CT-IRES-CreERT2 ( Htr2a EGFP-CreERT2 ) mouse line, EGFP was inserted into the C terminus (CT) of the receptor, after residue 452. An IRES-CreERT2 cassette was inserted between the Htr2a stop codon and the 3’UTR. Blue boxes represent exons of murine Htr2a ; the green box is EGFP; the red box shows the STOP codon. The gray box is the IRES followed by a pink box for CreERT2. The schematic was created with https://www.biorender.com/ . (B) Validation of Egfp and Htr2a mRNA expressions in the mouse brain: RNAscope experiments were performed to probe Egfp and Htr2a mRNA in C57 ( Htr2a +/+ ) and Htr2a EGFP-CreERT2/EGFP-CreERT2 mice. The Htr2a (red) probe can be detected in C57 mice; Egfp (green) and Htr2a (red) probes showed co-localization (yellow) in Htr2a EGFP-CreERT2/EGFP-CreERT2 mice. Images were taken under an Olympus slide scanner with 10X objective and an Olympus confocal microscope with a 60X objective. (C) The expression pattern of HTR2A in cortical pyramidal neurons (soma, apical dendrites, and apical tufts). Images were taken with a 20X objective under an Olympus confocal microscope. (D) Paravalbumin is the GABAergic interneuron marker. The brain sections from Htr2a EGFP-CreERT2/+ mice were stained with anti-parvalbumin and anti-GFP antibodies. The yellow arrowhead denotes parvalbumin-positive and GFP-positive interneurons. Images were captured under a 60X objective with an Olympus confocal microscope. (E) Cortical L5a marker NECAB1 was used to locate the cortical layer 5a distribution. HTR2A-EGFP-CT fusion receptors with anti-GFP staining showed its distribution in the NECAB1-positive layer. (F) With CTIP2 as a cortical L5b/L6 marker, HTR2A-EGFP-CT did not show overlap with the Ctip2-positive layers. (G) An anti-HTR2A antibody showed the same pattern as the anti-GFP distribution associated with HTR2A-EGFP-CT. Note, all brain sections in - from Htr2a EGFP-CreERT2/+ mice were stained with an anti-GFP antibody for HTR2A-EGFP-CT and different cortical markers (NECAB1, CTIP2), as well as with an anti-HTR2A antibody. (H) The distribution pattern of HTR2A-EGFP-CT labeling in the dorsal striatum. The Mu opioid receptor (MOR) is rich in the striosome and subcallosal stria of the Caudate-Putamen (CPu). HTR2A-EGFP-CT signals showed the same expression pattern with MOR distribution in the patch-like (striosome) area and stria. Experiments in this figure were conducted with 3-4 mice with similar results. Images were taken using an Olympus VS120 slide scanner or an Olympus confocal microscope under 60X objective.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Transgenic Assay, Residue, Microscopy, Expressing, Marker, Staining, Labeling

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: Brain sections from Htr2a EGFP-CreERT2/+ mice were stained with a GFP antibody to amplify the HTR2A-EGFP-CT signal and then were acquisitioned with an Olympus slide scanner under 10X objective. The abbreviations for the brain areas are located in the Supplementary Figure. The raw images were uploaded to open-source website, A Mouse Imaging Server (AMIS, https://amis2.docking.org/ .

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Staining, Imaging

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A/B) Schematic representation of the cytoarchitectonic areas of the frontal lobe of the brain. (A) The sagittal view of medial and lateral brain region, as well as horizontal view of the ventral regions on the frontal lobe show mPFC (medial prefrontal cortex), OFC (orbitofrontal cortex, as ventral PFC), and insular cortex (dorsal and ventral agranular insular cortex, AId and AIv; posterior agranular insular cortex, AIp; dysgranular insular cortex, DI). The schema was adapted from (B) Coronal sections from rostral to caudal view for mPFC, OFC, and insular cortex. The schema was adapted from (C) AchE staining: sequential brain sections through the frontal lobe area were used to visualize the distribution of HTR2A-EGFP-CT in the mPFC. The cytoarchitecture features of AchE staining showed strongest staining in the dorsal prelimbic cortex that was absent in the ventral prelimbic cortex. The blue arrow indicates the boundary between the dorsal and ventral prelimbic cortex. HTR2A-EGFP-CT showed its distributions in the dorsal prelimic cortex but was not present in the ventral prelimbic cortex and infralimbic cortex. Images scale bar: 500 µm. Similar results were obtained with 3 animals. MOs: secondary motor cortex, MOp: primary motor cortex, AAC: anterior cingulate area, PL: prelimbic cortex, IL: infralimbic cortex, and fr: anterior forceps.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Staining

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A-I) Motor activities in the open field Baseline activities (0-30 min; pre-administration) and post-injection activities following administration (31-120 min) of the vehicle (Veh), LSD (0.3 mg/kg), DOI (1 mg/kg) or psilocin (Psil, 1 mg/kg); n=9-10 mice/genotype/treatment. Note, the C57BL/6J and Htr2a EGFP-CreERT2/EGFP-CreERT2 mice are termed C57 and Htr2a EGFP-CreERT2 mice, respectively, below and in the figure panels. All data represented as means ±SEMs; N=9-10 mice/genotype/treatment. (A-C) RMANOVA for baseline: time [F(23,1587)=23.922, p <0.001], time by genotype [F(23,1587)=8.900, p <0.001], time by treatment [F(69,1587)=14.677, p <0.001], time by genotype by treatment [F(69,1587)=7.859, p <0.001], genotype [1,69)=30.277, p <0.001], treatment [F(3,69)=24.114, p <0.001], and genotype by treatment interaction [F(3,69)=11.598, p <0.001]. RMANCOVA for post-injection: time [F(17,1071)=2.600, p <0.001], time by genotype [F(17,1071)=12.309, p <0.001], time by treatment [F(51,1071)=10.977, p <0.001], time by genotype by treatment [F(51,1071)=8.914, p <0.001], genotype [1,63)=16.278, p <0.001], treatment [F(3,63)=26.809, p <0.001], and genotype by treatment interaction [F(3,63)=8.972, p <0.001]. (D-F) Rearing activities in the same mice. RMANOVA for baseline: time [F(23,1587)=23.548, p <0.001], time by genotype [F(23,1587)=9.221, p <0.001], time by treatment [F(69,1587)=7.544, p <0.001], time by genotype by treatment interaction [F(69,1587)=1.840, p =0.020], genotype [1,69)=5.228, p =0.025], treatment [F(3,69)=12.397, p <0.001]. RMANCOVA for post-injection: time by genotype [F(17,1071)=2.670, p <0.001], time by treatment [F(51,1071)=6.033, p <0.001], time by genotype by treatment [F(51,1071)=1.699, p =0.002], genotype [1,63)=15.125, p <0.001], treatment [F(3,63)=11.016, p <0.001], and genotype by treatment interaction [F(3,63)=4.016, p =0.011]. (G-I) Stereotypical activities in these same mice. RMANOVA for baseline: time [F(23,1587)=139.510, p <0.001], time by genotype [F(23,1587)=7.485, p <0.001], time by treatment [F(69,1587)=5.973, p <0.001], time by genotype by treatment interaction [F(69,1587)=2.485, p <0.001], genotype [1,69)=7.491, p =0.008], and treatment [F(3,69)=7.632, p <0.001]. RMANCOVA for post-injection: time by genotype [F(17,1071)=3.353, p <0.001], time by treatment [F(51,1071)=7.105, p <0.001], time by genotype by treatment interaction [F(51,1071)=3.910, p <0.001], and treatment [F(3,63)=7.226, p <0.001].

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Injection

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A-C) Cumulative motor activities Cumulative baseline activities (0-30 min; pre-administration) and cumulative post-injection activities following administration (31-120 min) of the vehicle (Veh), LSD (0.3 mg/kg), DOI (1 mg/kg) or psilocin (Psil, 1 mg/kg); n=9-10 mice/genotype/treatment. Note, the C57BL/6J and Htr2a EGFP-CreERT2/EGFP-CreERT2 mice are termed C57 and Htr2a EGFP-CreERT2 mice, respectively, below and in the figure panels. The symbol “*” is for genotype difference (black); “+” is for within C57 effect (blue); “^” for within Htr2a EGFP-CreET2 effect (green), and “#” for overall treatment effects (purple). All data are represented as means ± SEMs. (A) Locomotor activities in C57 and Htr2a EGFP-CreERT2 mice. Two-way ANOVA for baseline: genotype [F(1,69)=8.968, p =0.004]; for C57 vs . Htr2a EGFP-CreERT2 mice (black): ** p <0.01, overall genotype effects. Two-way ANCOVA for post-injection: genotype [F(1,68)=19.433, p <0.001], treatment [F(3,68)=29.894, p <0.001], and genotype by treatment interaction [F(3,68)=11.143, p <0.001]; Bonferroni post-hoc tests; for C57 vs . Htr2a EGFP-CreERT2 mice (black): *** p <0.001, DOI; for C57 mice (blue): + p <0.05, Veh vs. DOI; for Htr2a EGFP-CreERT2 mice (green): ^^^ p<0.001, Veh, LSD, Psil vs. DOI. (B) Rearing activities in the same mice. Two-way ANOVA for baseline: genotype [F(1,69)=9.307, p =0.003]; for C57 vs . Htr2a EGFP-CreERT2 mice (black): ** p <0.01, overall genotype effects. Two-way ANCOVA for post-injection: genotype [F(1,68)=17.900, p <0.001] and treatment [F(3,68)=13.765, p <0.001]; Bonferroni post-hoc tests; for C57 vs . Htr2a EGFP-CreERT2 mice (black): ** p <0.01, overall genotype effects; overall treatment effects (purple): ## p <0.01, Psil vs. LSD and DOI; ### p <0.001, Veh vs. LSD and DOI. (C) Stereotypical activities in these same mice. Two-way ANOVA for baseline: genotype [F(1,69)=44.086, p <0.001]; for C57 vs . Htr2a EGFP-CreERT2 mice (black): *** p <0.001, overall genotype effects. Two-way ANCOVA for post-injection: treatment [F(3,68)=8.338, p <0.001]; Bonferroni post-hoc tests; overall treatment effects (purple): # p<0.05, Veh vs. DOI; ## p<0.01, Veh vs. LSD or Psil vs. DOI; ### p<0.001, Psil vs. LSD. (D-F) Head twitch responses, grooming, and retrograde walking These responses were scored beginning immediately after vehicle or psychedelic administration and followed over the next 30 min; n=9-10 mice/genotype/treatment. (D) Head twitch responses in C57 and Htr2a EGFP-CreERT2 animals following administration (31-60 min) of the Veh, 0.3 mg/kg LSD, 1 mg/kg DOI, or 1 mg/kg Psil. Two-way ANOVA: genotype [F(1,69)=9.236, p =0.003], treatment [F(3,69)=51.113, p <0.001], and genotype by treatment interaction [F(3,69)=2.878, p =0.042]; Bonferroni post-hoc tests: for C57 vs. Htr2a EGFP-CreERT2 mice (black): *** p <0.001, DOI; for C57 mice (blue): + p <0.05, LSD vs. DOI; +++ p <0.001, Veh vs. all psychedelics, or Psil vs. DOI; for Htr2a EGFP-CreERT2 mice (green): ^ p <0.05, Psil vs. LSD; ^^ p <0.01, Veh vs. Psil; ^^^ p <0.001, Veh vs. LSD and DOI. (E) Groom time in the same mice. Two-way ANOVA: genotype [F(1,69)=11.465, p <0.001], treatment [F(3,69)=15.125, p <0.001], and genotype by treatment interaction [F(3,69)=6.855, p <0.001]; Bonferroni post-hoc tests: for C57 vs Htr2a EGFP-CreERT2 mice (black): *** p <0.001, LSD and DOI; for C57 mice (blue): +++ p <0.001; Veh vs. LSD and DOI or or Psil vs. LSD and DOI; for Htr2a EGFP-CreERT2 mice (green): ^^ p <0.01, Psil vs. Veh and LSD. (F) Retrograde walking in these same mice. Two-way ANOVA for treatment [F(3,69)=21.713, p <0.001]; Bonferroni post-hoc tests: overall treatment effects (purple): ### p <0.001, Veh vs. LSD and DOI, or Psil vs. LSD and DOI. (G) Prepulse inhibition C57 and Htr2a EGFP-CreERT2 mice were injected with the Veh, 0.3 mg/kg LSD, 1 mg/kg DOI, or 1 mg/kg Psil and tested 10 min later; n=10-15 mice/genotype/treatment. RMANOVA: PPI [F(2,210)=329.097, p <0.001], PPI by genotype interaction [F(2,210)=23.856, p <0.001], PPI by treatment interaction [F(6,210)=3.342, p =0.004], genotype [F(1,105)=45.808, p <0.001], and treatment [F(3,105)=19.562, p <0.001]; Bonferroni post-hoc tests: for C57 vs Htr2a EGFP-CreERT2 mice (black): *** p <0.001, overall genotype effects; for treatment effects (purple): # p <0.05, Veh vs. Psil; ## p <0.01, Veh vs. LSD; ### p <0.001, Veh vs. DOI. Some schematics were created with https://www.biorender.com/ .

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Injection, Inhibition

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A-B) Null and startle activities in prepulse inhibition Mice were injected with the Veh, 0.3 mg/kg LSD, 1 mg/kg DOI, or 1 mg/kg Psil and tested 10 min later; n=10-15 mice/genotype/treatment. Note, the C57BL/6J and Htr2a EGFP-CreERT2/EGFP-CreERT2 mice are termed C57 and Htr2a EGFP-CreERT2 mice, respectively, below and in the figure panels. The results are presented as means ± SEMs; n=10-15 mice/genotype/treatment. (A) Null activities in C57 and Htr2a EGFP-CreERT2 mice. Two-way ANOVA: genotype [F(1,105)=33.886, p <0.001] and treatment [F(3,105)=32.101, p <0.001]; Bonferroni post-hoc tests: for C57 vs . Htr2a EGFP-CreERT2 mice (black): *** p <0.001, overall genotype effects; overall treatment effects (purple): ### p <0.001, Veh vs. LSD and DOI or LSD and Psil vs. DOI. (B) Startle activities in C57 and Htr2a EGFP-CreERT2 mice. Two-way ANOVA: genotype [F(1,105)=51.786, p <0.001], treatment [F(3,105)=12.810, p <0.001], and genotype by treatment interaction [F(3,105)=3.282, p =0.024]; Bonferroni post-hoc tests: for C57 vs . Htr2a EGFP-CreERT2 mice (black): *** p <0.001, Veh, LSD, and Psil; for C57 (blue): + p <0.05, Veh vs. Psil; +++ p <0.001, LSD vs. Psil; for Htr2a EGFP-CreERT2 mice (green): ^ p <0.05, Veh vs. DOI; ^^^ p <0.001, LSD vs. DOI.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Inhibition, Injection

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) [ 3 H] ketanserin saturation binding assay was conducted to examine the affinity (Kd, nM) and receptor levels (Bmax, pmole/mg) of HTR2A in the whole cortex of C57 and Htr2a EGFP-CreERT2 mice. Non-specific binding was determined with 10 µM clozapine. Data are means ± SEMs (n=4 samples/genotype) and analyzed by unpaired t-test. Kd values: C57(1.09 ± 0.324 nM) vs. Htr2a EGFP-CreERT2 (0.75 ± 0.13nM), [t(6)=1.005, p =0.354]; Bmax values: C57 (0.25 ±0.018 pmole/mg) vs. Htr2a EGFP-CreERT2 (0.46 ± 0.025 pmole/mg), [t(6)=6.745, p =0.0005] (*** p <0.001, C57 vs. Htr2a EGFP-CreERT2 ). (B) Real-time qPCR was used to determine Htr2a mRNA levels from the whole cortex of C57 mice ( Htr2a +/+) , Htr2a EGFP-CreERT2/+ , and Htr2a EGFP-CreERT2/EGFP-CreERT2 mice. Data are means ± SEMs (n=4 samples/genotype). One-way ANOVA [(F(2,9)=129.4, p <0.0001] followed by Tukey’s post-hoc test showed *** p <0.001, C57 vs. Htr2a EGFP-CreERT2/+ ; ****p <0.0001, C57 vs. Htr2a EGFP-CreERT2/EGFP-CreERT2 . (C) LSD-mediated HTR2A downregulation: Mice (C57 and Htr2a EGFP-CreERT2 ) were injected with vehicle or LSD (0.5 mg/kg, i.p.) for 5 consecutive days and then were euthanized 24 hr later. The whole cortices were dissected followed by receptor purification using Wheat-Germ beads pull-down. Western blot against anti-HTR2A antibody detected the receptor expression levels. Data represent the means ± SEMs (n=5-7). Two-way ANOVA: treatment [F(1,21)=27.11, p <0.0001], genotype [F(1,21)=24.09, p <0.0001], and treatment by genotype interaction [F(1,21)=0.1832, p =0.673] (overall genotype effects (black): ****p<0.0001, C57 vs. Htr2a EGFP-CreERT2/EGFP-CreERT2 ; overall treatment effect (purple): #### p <0.0001, vehicle vs. LSD). The schematic was created with https://www.biorender.com/ .

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Saturation Assay, Binding Assay, Injection, Purification, Western Blot, Expressing

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) Htr2a EGFP-CreERT2 mice crossed with the Cre-dependent reporter line (Ai9 mice here) were used to visualize HTR2A expressing cells with tdTomato expression. The bicistronic design of Htr2a-EGFP-IRES-CreERT2 produced a HTR2A-EGFP-CT fusion protein and CreERT2 recombinase. Tamoxifen activates CreERT2 recombinase, leading to Cre-loxP recombination and then turning on tdTomato expression. The schema was created with https://www.biorender.com/ . (B) The expression pattern of tdTomato (red) and HTR2A-EGFP-CT fusion proteins (green) in the mPFC subregion were identified using immunohistochemistry staining for HTR2A expression patterns and AchE staining for the cytoarchitecture. MOs: secondary motor cortex; AAC: anterior cingulate area; PL: prelimbic cortex; IL: infralimbic cortex; and fr: anterior forceps (C) L5a pyramidal neurons were used for recording neuronal firing activity during focal 5-HT (10 µM) application. Addition of 5-HT resulted in an initial decrease in firing followed by increased firing shown 3 min after application. Data are presented as means ± SEMs (n=7) and were analyzed using paired t-test to compare basal activity and firing after 5-HT application, [t(6)=3.177, p=0.0191] (*p<0.05) and one sample t-test was used for normalization of basal activity for the 5-HT response, p=0.0469 (*p<0.05). (D) Representative recording of HTR2A + /L5a pyramidal neuronal firing after M100907 (200 nM) and 5-HT (10 µM). M100907 effects on firing were analyzed using paired t-test, [t(10)=2.667, p=0.0236] (*p<0.05 vs. baseline) and normalized M100907 effects on firing were analyzed with one-sample t-test, [t(11)=4.421, p =0.001] (***p=0.001). Data are represented as means ± SEMs (n=11). The effects of M100907 + 5-HT on firing were analyzed with paired t-test, [t(11)=2.036, p=0.067] and normalized effects were analyzed using one-sample t-test. Data are presented as means ± SEMs (n=12). (E) Representative recording of HTR2A + /L5a neuronal firing after administration of WAY100635 (100 nM) and 5-HT (10 μM). WAY100635 effects on firing were analyzed with paired t-test, [t(6)=3.115, p=0.021] (*p<0.05, vs baseline) and normalized effects on firing were analyzed with one-sample t-test, [t(6)=7.228, p=0.0004] (***p<0.001). Data are presented as means ± SEMs (n=7). WAY100635 + 5-HT effects on firing were analyzed with paired t-test, [t(8)=2.159, p=0.0629] and normalized WAY100635 + 5-HT effects on firing were analyzed with one sample t-test p =0.0039 (**p=0.01). Data are represented as means ± SEMs (n=9). (F) A representative biocytin-labeled L5 pyramidal neuron showed its location in the HTR2A-EGFP-CT positive cortical band layer in the dorsal prelimbic cortex. After recording, the patched neuron was injected with biocytin. The brain sections were stained with an anti-GFP antibody to locate the L5a cortical band. Images were taken under 20X objective with an Olympus confocal microscope.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Expressing, Produced, Immunohistochemistry, Staining, Activity Assay, Labeling, Injection, Microscopy

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: Htr2a EGFP-CreERT2/+ mice were crossed with Ai9 mice. Mice were treated with tamoxifen (100 mg/kg, i.p.) at p39-p42 for 4 days and then brains were perfused at 14 days post-injection (14 dpi) at p56. The brain sections were stained with GFP antibody. Images were captured in two channels for tdTomato (red) and GFP (green) signals under an Olympus slide scanner with 10X objective. The GFP signal as a HTR2A-EGFP-CT fusion protein and the tdTomato signal as HTR2A-positive cells were shown here. Experiments were conducted on 4 brains with similar results. The raw images have been uploaded to open-source website, A Mouse Imaging Server (AMIS, https://amis2.docking.org/ ).

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Injection, Staining, Imaging

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: Htr2a EGFP-CreERT2/+ x Ai9 or C57 x Ai9 ( Htr2a +/+ x Ai9) mice were treated with vehicle (corn oil) or tamoxifen (100 mg/kg) at p39 to p42 with 4 injections and then brains were collected at p56. A whole brain mapping was conducted to determine whether CreERT2 recombinase turned on tdTomato protein expression after TAM treatment. The brain sections were stained with DAPI and then images were rendered by an Olympus VS120 slide scanner. Experiments were conducted with 4 animals with similar results.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: Htr2a EGFP-CreERT2/+ x Ai9 mice were used for this experiment. Individual recording neurons (HTR2A-tdTomato-positive) from different brain sections of each treated group (purple: 5-HT; blue: M100907+5-HT; and Green: WAY100365+5-HT) are depicted in the ACC or dPL. (A) HTR2A signals with anti-HTR2A staining from Htr2a Cre/+ mouse (B) HTR2A - A242S-EGFP-CT signals with anti-GFP staining from Htr2a A242S-EGFP-Cre/+ (C-D) Comparison of different Cre-dependent reporters (Ai9 mice vs PHP.eB.flex.tdT AAV virus) among Htr2a mouse lines

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Staining, Comparison, Virus

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) The schema depicts the transgenic designs of each mouse line. As described previously, the Htr2a -EGFP-CT-IRES-CreERT2 ( Htr2a EGFP-CreERT2 ) was inserted with EGFP in the C-terminus of the Htr2a followed by adding the IRES-CreERT2 sequence after exon 3 as an inducible Htr2a mouse line. Htr2a -IRES-Cre mouse line ( Htr2a Cre ) was engineered IRES-Cre sequence at the end of exon 3. A humanized mouse line, Htr2a -A242S-EGFP-CT-IRES-Cre ( Htr2a A242S-EGFP-Cre ), was created with one point mutation at the alanine 242 residue of the murine Htr2a to the serine residue of the human HTR2A and this was followed at the murine 452 residue by an EGFP insertion; the IRES-Cre was inserted after the exon 3 coding sequence. In the diagrams, the blue boxes represent exons of the murine Htr2a gene; the green box is Egfp ; the red box is the stop codon. The gray box is the IRES (internal ribosome entry site) followed by a pink box for Cre or CreERT2 recombinase. The white box is the A242S point mutation. The schematic was created with https://www.biorender.com/ . (B) HTR2A distribution in the brain among four mouse lines ( Htr2a +/+ , Htr2a Cre/+ , Htr2a EGFP-CreERT2/+ , and Htr2a A242S-EGFP-Cre/+ ). For this study, mice were perfused, and the brain sections were immunostained with an anti-HTR2A antibody to visualize the HTR2A distribution. (C) The HTR2A receptor from four mouse lines ( Htr2a +/+ , Htr2a Cre/Cre , Htr2a EGFP- CreERT2/EGFP-CreERT2 , and Htr2a A242S- EGFP-Cre/A242S-EGFP-Cre ). Here, mice were euthanized and then cortices were dissected followed by receptor purification using Wheat-Germ beads pull-down. Western blot against anti-HTR2A antibody to detect receptor expression level. In Htr2a +/+ and Htr2a Cre/Cre mice, the HTR2A was detected between 50-75 KD; for EGFP insertion lines, the HTR2A-EGFP-CT fusion protein was detected around 100 KD. (D) Representative image of Htr2a Cre/+ mice expressing DIO-eYFP in the mPFC was stained with anti-GFP for eYFP signal amplification at 20x (left) and 63x magnification (left inset). Example electrophysiological trace of a HRT2A + neuron expressing DIO-eYFP during bath application of DCZ. Representative image of Htr2a Cre/+ mice expressing DIO-HA-hM3Dq-IRES-mCitrine in the mPFC was stained with anti-GFP for mCitrine at 20x (right) and 63x magnification (right inset). Example electrophysiological trace of a HRT2A + neuron expressing DIO-HA-hM3Dq-IRES-mCitrine during bath application of DCZ.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Transgenic Assay, Sequencing, Mutagenesis, Residue, Purification, Western Blot, Expressing, Staining, Amplification

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) The HTR2A proteins in the whole brain mapping from Htr2a Cre/+ mice were shown using an anti-HTR2A antibody. (B) The distribution of HTR2A-A242S-EGFP-CT fusion proteins from Htr2a A242S-EGFP-Cre/+ mice were mapped in the whole brain using an anti-GFP antibody. The raw images have been uploaded to open-source website, A Mouse Imaging Server (AMIS, https://amis2.docking.org/ ). (C) Cre reporter mice (Ai9) crossed with three Htr2a mouse lines. The distribution patterns of HTR2A and HTR2A + tdTomato from an inducible Htr2a EGFP-CreERT2/+ x Ai9 mouse and from constitutive Cre lines ( Htr2a Cre/+ xAi9 and Htr2a A242S-EGFP-Cre/+ x Ai9) are shown. All mice were perfused at p56. The brain sections were stained with anti-HTR2A or anti-GFP antibodies. Images were captured in two channels for tdTomato (red) and anti-HTR2A or anti-GFP (green) signals under and Olympus slide-scanner with 10X objective. Experiments were conducted with 3-4 brains with similar results. (D) Cre reporter virus (PHP.eB.flex.tdT) with inducible CreERT2 and constitutive Cre-mouse Htr2a lines. The Htr2a mouse lines were retro-orbitally injected with PHP.eB.flex.tdTomato . The tdTomato patterns of expression among the 3 mouse lines from virally-treated groups are compared with Htr2a EGFP-CreERT2/+ x Ai9 mice in the area of the somatosensory cortex . Mice were perfused and then stained with anti-HTR2A for Htr2a Cre/+ mice and with anti-GFP for Htr2a EGFP-CreERT2/+ and Htr2a A242S-EGFP-Cre/+ mice. Images were captured under an Olympus slide-scanner with 10X objective. (CPu-s: striosome, TUO: olfactory tubercle, IG: induseum griseum, SH: septohippocampal nucleus).

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Imaging, Staining, Virus, Injection, Expressing

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A-D) Head twitch responses and prepulse inhibition (PPI) Head twitch responses were scored over 30 min beginning after injection of the vehicle (Veh), LSD (0.3 mg/kg), DOI (1 mg/kg), or Psil (1 mg/kg). For PPI, mice received the same regimen of treatment and were tested in PPI 10 min later as described in the Methods section. Note, the C57BL/6J and Htr2a A242S-EGFP-Cre/A242S-EGFP-Cre mice are termed C57 and Htr2a A242S-EGFP-Cre mice, respectively, below and in the figure panels. All data are represented as means ± SEMs. (A) Head twitch responses in C57 and Htr2a A242S-EGFP-Cre animals immediately following administration of the Veh, LSD, DOI, or Psil over the 30 min following administration; n=9-10 mice/genotype/treatment. Two-way ANOVA: treatment [F(3,70)=43.033, p <0.001], and genotype by treatment interaction [F(3,70)=6.784, p <0.001]; Bonferroni post-hoc tests: for C57 vs. Htr2a A242S-EGFP-Cre mice (black): * p <0.05, Psil; *** p <0.001, DOI; for C57 mice (blue): +++ p <0.05, Veh vs. all psychedelics or Psil vs. DOI; for Htr2a A242S-EGFP-Cre mice (green): ^^^ p <0.05, Veh vs . all psychedelics. (B) Prepulse inhibition: C57 and Htr2a A242S-EGFP-Cre mice were injected with the Veh, LSD, DOI, or Psil and tested 30 min later; n=9-10 mice/genotype/treatment. RMANOVA: PPI [F(2,140)=113.340, p <0.001], genotype [F(1,70)=4.989, p =0.029], and treatment [F(3,70)=23.203, p <0.001]; Bonferroni post-hoc tests: for C57 vs Htr2a A242S-EGFP-Cre mice (black): ** p <0.01, Veh; for C57 mice (blue): + p <0.05, Psil vs. Veh; ++ p ≤0.001, LSD vs. Veh; +++ p ≤0.001, DOI vs. Veh; for Htr2a A242S-EGFP-Cre mice (green): ^^^ p <0.001, all psychedelics vs. Veh. (C) Null activities in C57 and Htr2a A242S-EGFP-Cre mice. In the two-way ANOVA Levene’s test of homogeneity was violated and it was still violated when null activity was standardized to genotype or vehicle. Neither the Mann-Whitney U test nor the Wilcoxin W test for genotype (two-tailed) were significant for any treatment. Kruskal-Wallis one-way ANOVA for treatment (two-tailed): [h(3)=27.582, p <0.001]; Bonferroni post-hoc tests for C57 mice (blue); + p <0.05; Veh vs. Psil; ++ p <0.01, Psil vs. DOI; +++ p <0.001, Veh vs. LSD and DOI; for Htr2a A242S-EGFP-Cre mice (green): ^ p <0.05; Veh vs. Psil or Psil vs. DOI; ^^ p <0.01, Veh vs. LSD; ^^^ p <0.001, Veh vs. DOI. (D) Startle activities in C57 and Htr2a A242S-EGFP-Cre mice. Two-way ANOVA: genotype [F(1,70)=4.295, p =0.042], treatment [F(3,70)=7.547, p <0.001], and genotype by treatment interaction [F(3,70)=3.740, p =0.015]; ANOVA followed by Bonferroni post-hoc tests: for C57 vs . Htr2a A242S-EGFP-Cre mice (black): * p <0.05, Psil; ** p <0.01, Veh; for C57 (blue): ++ p <0.05, LSD vs. Psil; for Htr2a A242S-EGFP mice (green): ^^^ p <0.001, Veh vs. DOI.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Inhibition, Injection, Activity Assay, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: Htr2a EGFP-CreERT2/+ x Ai9 x Thy1 GFP/+ mice were used and were treated with tamoxifen (100 mg/kg for 4 days) to induce tdTomato expression. Since HTR2A-EGFP-CT is very dim without antibody amplification, it was difficult to directly detect. Thus, in this experiment we used tdTomato to label HTR2A-containing neurons and Thy1-GFP to observe their distribution in the cortex. Images were captured under an Olympus slide-scanner with 10X objective. Experiments were conducted on 3 brains with similar results.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Expressing, Amplification

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) RNAscope was used to detect Htr2a and Egfp transcripts in the Htr2a EGFP-CreERT2/EGFP-CreERT2 mice. Brain sections were also stained with anti-GFP antibody to detect protein signals of the HTR2A-EGFP-CT fusion protein. Experiments were conducted with 4 animals containing similar results. Images were taken under a 10X objective with an Olympus VS120 slide-scanner (scale bar 1mm). (B) In the motor cortex area, Egfp transcripts were co-localized with Lamp5 in Layer2/3 and Layer5b, and with parvalbumin positive interneurons. Cplx3 as a layer6b marker showed its co-localization with Egfp transcripts. Images were taken under 10X objective with an Olympus VS120 slide-scanner and under a 20X objective with an Olympus FV3000RS confocal microscope. Experiments were conducted with 3 animals achieving similar results.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Staining, Marker, Microscopy

Journal: Journal of Microbiology and Biotechnology

Article Title: The Effect of Lactobacillus gasseri BNR17 on Postmenopausal Symptoms in Ovariectomized Rats

doi: 10.4014/jmb.2105.05032

Figure Lengend Snippet: Serum estradiol (E2) ( A ), Calcitonin ( B ), Osteocalcin ( C ), and 5-HT-2A ( D ) were measured after 14 weeks of initial administration ( n = 6~8 rats per group). Sophora japonica fruit extract (SJFE) treatment was used to create the positive control.

Article Snippet: The concentrations of estradiol (E2, CSB-E05110r), osteocalcin (CSB-E05129r), calcitonin (CSB-E05132r), estradiol (CSB-E05110r), and Serotonin 2A (5-HT-2A, CSB-E14956r) were assessed using an ELISA kit (CUSABIO, China).

Techniques: Positive Control