Journal: Journal of Virology

Article Title: Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network

doi: 10.1128/jvi.01194-23

Figure Lengend Snippet: CRISPR/Cas9-mediated knockdown of DCLK1 blocks SARS-CoV-2 replication-transcription processes. (A) LentiCRISPR-v2 vectors expressing anti-DCLK1 synthetic guide RNAs (sgRNAs #1, #2, and #3; lanes 3–5) were used to target DCLK1, while control synthetic guide RNA was used as a control (sgRNA-C, lane 2). Additional controls included untreated cells (Calu-3-wt, lane 1). Puromycin-resistant cells were pooled to determine the relative expression of DCLK1. sgRNA #2-treated cells showed 70% inhibition of DCLK1 (Calu-3-DKO cells) and were selected for further study. (B) Calu-3 cells were infected with SARS-CoV-2 for 48 h and imaged by confocal microscope after immunofluorescence staining for microtubules (green), Spike protein (red), and nuclei (blue). (C) Western blots of total lysates of infected cell lines for Spike protein, N protein, and actin. (D) Reverse transcription-quantitative PCR for average copy number of viral gRNA (left) and N protein mRNA (right) in infected cell lines. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, ns (not significant) P > 0.05.

Article Snippet: Infection of lung adenocarcinoma cells with SARS-CoV-2, treatment with DCLK1 kinase inhibitor, and inactivation of DCLK1 gene Lung adenocarcinoma cells (Calu-3, Cat. No. HTB-55, ATCC) were infected for 4 h at 37°C with SARS-CoV-2 at an MOI = 1.

Techniques: CRISPR, Knockdown, Expressing, Control, Inhibition, Infection, Microscopy, Immunofluorescence, Staining, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Virology

Article Title: Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network

doi: 10.1128/jvi.01194-23

Figure Lengend Snippet: SARS-CoV-2-mediated alteration in proteomic profile of lung epithelial cells are restored following DCLK1 inhibition. Calu-3 cell lysates from uninfected (A1), SARS-CoV-2-infected (A2), infected cells treated with vehicle (DMSO) (A3), or DCLK1-IN-1 (A4) underwent proteomic analysis (experiments performed in triplicate). (A) Principal component analysis of total protein abundance for each sample shows close clustering of total normalized protein abundance (peak area) for A1 and A4 compared to A2 and A3. (B) Heat map clustering for differential protein abundance for each experimental condition. (C) Heat maps show multiple proteins induced by SARS-CoV-2 (A2 and A3, red) and normalization toward levels in uninfected cells (A1, green) following treatment with DCLK1-IN-1 (A4, green). (D) Volcano plots show significantly increased (red circles) and decreased (green circles) protein levels in infected (A2) compared to uninfected (A1) cells. (E) Eight proteins are identified that were induced by infection and normalized by DCLK1-IN-1. (F) Western blots validate proteomic data for a subset of proteins normalized by DCLK1-IN-1 as indicated. (G) Twenty-one genes were downregulated in SARS-CoV-2-infected cells, and expression was restored by DCLK1-IN-1 (see box; the six most downregulated genes are shown in red). (H) Heat map showing six proteins most downregulated by infection (A2) compared to uninfected control (A1). Downregulated protein expression is restored by DCLK1-IN-1 (A4) and not DMSO (A3). (I) Examples of protein levels in infected cells that were restored by DCLK1-IN-1. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns (not significant) P > 0.05.

Article Snippet: Infection of lung adenocarcinoma cells with SARS-CoV-2, treatment with DCLK1 kinase inhibitor, and inactivation of DCLK1 gene Lung adenocarcinoma cells (Calu-3, Cat. No. HTB-55, ATCC) were infected for 4 h at 37°C with SARS-CoV-2 at an MOI = 1.

Techniques: Inhibition, Infection, Quantitative Proteomics, Western Blot, Expressing, Control

Journal: Journal of Virology

Article Title: Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network

doi: 10.1128/jvi.01194-23

Figure Lengend Snippet: SARS-CoV-2 encoded structural and non-structural protein expression is blocked by DCLK1 inhibition. Viral structural (A–C) and regulatory proteins (D–F) of SARS-CoV-2 are significantly downregulated by DCLK1-IN-1 treatment of infected Calu3 cells when compared to untreated infected controls. Analyses were carried out using quantitative proteomics. A1, uninfected; A2, infected; A3, infected and treated with DMSO as vehicle; and A4, infected and treated with DCLK1-IN-1. **P ≤ 0.01, ***P ≤ 0.001, ***P ≤ 0.0001, ns (not significant) P > 0.05.

Article Snippet: Infection of lung adenocarcinoma cells with SARS-CoV-2, treatment with DCLK1 kinase inhibitor, and inactivation of DCLK1 gene Lung adenocarcinoma cells (Calu-3, Cat. No. HTB-55, ATCC) were infected for 4 h at 37°C with SARS-CoV-2 at an MOI = 1.

Techniques: Expressing, Inhibition, Infection, Quantitative Proteomics

Journal: Journal of Virology

Article Title: Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network

doi: 10.1128/jvi.01194-23

Figure Lengend Snippet: DCLK1 regulates phosphorylation of nucleocapsid (N) protein. (A) Quantitative phosphoproteomic analysis of SARS-CoV-2-infected Calu-3 cells after treatment with DCLK1-IN-1 (blue bars, A4P) or DMSO (orange bars, A3P). These data are compared with the abundance of phosphopeptides in infected cells (red bars, A2P). Lysates from uninfected/untreated Calu-3 cells are also shown (negative controls, A1P). (B) Amino acid sequence in the SR-rich region of N protein. Phosphorylated serines are shown in red with stars indicating sites that had a reduction in phosphorylation after treatment with DCLK1-IN-1. Reduction in serine phosphorylation by DMSO is indicated by hashtags. (C) DCLK1-IN-1-mediated inhibition of phosphorylation is directed primarily toward conserved serines (red) in the highly conserved SR-rich region of N protein that is found in all omicron variants (BA.1 through BA.5) and the original Wuhan strain (top). (D) DCLK1-IN-1 inhibits Omicron strain viral production in infected Calu-3 cells; TCID50 for DMSO (vehicle) set at 100% and compared with DCLK1-IN-1. (E) Enhanced phosphorylation of eight host proteins due to SARS-CoV-2 infection is repressed by DCLK1-IN-1 (sample designation as in panel A). (F) Examples of host proteins (MAD1L1 and SYMPK) where phosphorylation is restored by DCLK1-IN-1 (blue bars) compared with uninfected Calu-3 cells (green bars). Reduction of protein phosphorylation due to infection (A2P) is not restored by DMSO (A3P). CTD, C-terminal domain; NTD, N-terminal domain.*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns (not significant) P > 0.05.

Article Snippet: Infection of lung adenocarcinoma cells with SARS-CoV-2, treatment with DCLK1 kinase inhibitor, and inactivation of DCLK1 gene Lung adenocarcinoma cells (Calu-3, Cat. No. HTB-55, ATCC) were infected for 4 h at 37°C with SARS-CoV-2 at an MOI = 1.

Techniques: Phospho-proteomics, Infection, Sequencing, Inhibition

Journal: Journal of Virology

Article Title: Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network

doi: 10.1128/jvi.01194-23

Figure Lengend Snippet: SARS-CoV-2-induced alterations in EGFR family members’ kinase activity are restored following DCLK1 inhibition. Proteomic and phospho-proteomic analyses of total cell lysates from uninfected cells (A1), SARS-CoV-2-infected Calu-3 cells (A2), and infected cells treated with DMSO (A3) or DCLK1-IN-1 (A4) identify significant changes in the abundance of cellular kinases. (A) Heat map shows an altered abundance of 21 kinases in infected cells; these changes are partly restored by DCLK1-IN-1. Red and green stars are the known interactors of the M and N proteins, respectively. (B) Phosphorylation signatures for eight kinases are altered by SARS-CoV-2 infection (A2P). The degree of phosphorylation at these sites (A1P) is significantly restored by DCLK1-IN-1 (A4P). Red and green stars represent known interactors based on BiGRID database for SARS-CoV-2 M and N proteins. (C) KEGG pathway analysis of differentially abundant kinases (see Materials and Methods for details). (D) A protein-protein interaction network for 28 proteins shows significant changes in abundance in response to SARS-CoV-2 infection (1.5‐fold, P < 0.05). Non-interacting nodes are not shown. Line colors indicate the strength of data based on experimental results (pink), co‐expression (black), gene‐fusion (red), or co‐occurrence (blue) (http://string-db.org/).

Article Snippet: Infection of lung adenocarcinoma cells with SARS-CoV-2, treatment with DCLK1 kinase inhibitor, and inactivation of DCLK1 gene Lung adenocarcinoma cells (Calu-3, Cat. No. HTB-55, ATCC) were infected for 4 h at 37°C with SARS-CoV-2 at an MOI = 1.

Techniques: Activity Assay, Inhibition, Infection, Phospho-proteomics, Expressing

Journal: Journal of Virology

Article Title: Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network

doi: 10.1128/jvi.01194-23

Figure Lengend Snippet: Expression of viral proteins and RNAs are blocked by DCLK1-IN-1 in the K18-hACE2 model of SARS-CoV-2 infection. K18-hACE2 transgenic mice are a model for SARS-CoV-2 infection. (A) Western blots of total cell lysates from necropsied lungs of uninfected mice (U1–U6) or mice infected with SARS-CoV-2 (M1–M6); lysates were prepared 5 days post-infection, and each group consisted of three male and three female mice. (B and C) Quantitative evaluation of band intensities for host proteins shown in panel A; band intensities for Spike and N proteins were not quantified since these proteins were undetectable in uninfected mice. (D) Representative H&E stained lung for each treatment group (n = 6). Histopathological outcomes for DMSO-treated or DCLK1-IN-1-treated mice were compared with infected (group 1) and uninfected (group 4) mice; the experiment was replicated once with similar results (data not shown). (E) Total RNAs from mouse lung by qRT-PCR with copy numbers of viral genomic RNA (left panel) and N protein mRNA (right panel) calculated from C t values. As controls, group 4 mice were negative for both RNAs; for group 2 mice, RNA levels were similar to those of group 1 (data not shown). (F) Western blots for Spike and N proteins from total lung cell lysates for two representative mice in each group. (G) Quantified band intensities of Spike and N proteins (repeated twice, n = 6) were quantitated using Image J software. (H) Quantitative qRT-PCR for IL-6, TNF-α, and IL-1β mRNAs in DCLK1-IN-1-treated and control mice using total RNAs isolated from mouse lung (n = 3 per group). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns (not significant) P > 0.05.

Article Snippet: Infection of lung adenocarcinoma cells with SARS-CoV-2, treatment with DCLK1 kinase inhibitor, and inactivation of DCLK1 gene Lung adenocarcinoma cells (Calu-3, Cat. No. HTB-55, ATCC) were infected for 4 h at 37°C with SARS-CoV-2 at an MOI = 1.

Techniques: Expressing, Infection, Transgenic Assay, Western Blot, Staining, Quantitative RT-PCR, Software, Control, Isolation

Journal: Journal of Virology

Article Title: Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network

doi: 10.1128/jvi.01194-23

Figure Lengend Snippet: DCLK1-IN-1 shows efficacy in the treatment of SARS-CoV-2-infected K18-hACE2 mice. Lungs from K18-hACE2 transgenic mice were obtained at necropsy 5 days post-infection with SARS-CoV-2. (A) Confocal microscopy shows immunofluorescence staining for SP-C (cyan), Spike (red), Dclk1 (magenta), and CD206 (green) from infected mice and infected mice treated with DCLK1-IN-1 (blue, nucleus; ×20 magnification). (B) Spike-negative and CD206+ M2-like macrophages stain for SP-C and DCLK1 and are highlighted in close contact with infected cells (×40 magnification). (C) Quantitation of staining intensities for Spike and Dclk1 for all groups as indicated. (D) Lungs stained for Spike (red), CD206 (green), Dclk1 (magenta), SP-C (cyan), and nuclei (blue) were scanned by AxioScan (magnification ×10) and images shown for DMSO treated (control, upper panel) and DCLK1-IN-1 treated (lower panel). (E) Representative images of lungs from groups of mice stained for hACE2 (green), Spike (red), Dclk1 (cyan), S100A9 (magenta), and nuclei (blue). (F) Infected lung cells co-expressing Dclk1 and S100A9 are highlighted (magnification ×40). *P ≤ 0.05, **P ≤ 0.01, ns (not significant) P > 0.05.

Article Snippet: Infection of lung adenocarcinoma cells with SARS-CoV-2, treatment with DCLK1 kinase inhibitor, and inactivation of DCLK1 gene Lung adenocarcinoma cells (Calu-3, Cat. No. HTB-55, ATCC) were infected for 4 h at 37°C with SARS-CoV-2 at an MOI = 1.

Techniques: Infection, Transgenic Assay, Confocal Microscopy, Immunofluorescence, Staining, Quantitation Assay, Control, Expressing

Journal: Journal of Virology

Article Title: Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network

doi: 10.1128/jvi.01194-23

Figure Lengend Snippet: Proposed mechanism of action for efficacy of DCLK1 kinase inhibitor in SARS-CoV-2 infection. SARS-CoV-2 enters target cells through interactions of Spike protein with cellular surface protein ACE2. Subsequently, Spike protein is cleaved by transmembrane serine protease 2. After entry into the cytosol, gRNA containing 5′ cap (red circles) and a poly(A) tail is released from nucleocapsid (N) protein core particles and translated to produce polyproteins (pp1a and pp1b). These polyproteins are cleaved into 16 non-structural proteins that assemble into RTCs. RTCs synthesize new gRNAs via negative-strand RNA intermediates and also produce subgenomic mRNAs that encode structural and accessory proteins. The exact composition and involvement of cellular proteins in the regulation of RTCs are poorly defined. Viral particles are assembled by coating gRNAs with heavily phosphorylated N proteins to produce structures that bud into the endoplasmic reticulum-golgi intermediate compartment. During this process, viral particles acquire a lipid bilayer containing Spike, membrane (M), and envelope (E) proteins. Doublecortin motifs of DCLK1 can help with the movement of RTCs and transport viral particles by regulating microtubule dynamics. Thus, DCLK1-IN-1 acts on multiple steps of the viral replication cycle. gRNA, genomic RNA; RTC, replication-transcription complex.

Article Snippet: Infection of lung adenocarcinoma cells with SARS-CoV-2, treatment with DCLK1 kinase inhibitor, and inactivation of DCLK1 gene Lung adenocarcinoma cells (Calu-3, Cat. No. HTB-55, ATCC) were infected for 4 h at 37°C with SARS-CoV-2 at an MOI = 1.

Techniques: Infection, Membrane

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Mouse qPCR primer/probe sets

Article Snippet: Gpr55 , Mm02621622_s1 , 102.

Techniques: Amplification

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Expression of GPR55 in human NSCs. RNA from hNSCs was extracted and cDNA and PCR amplification were performed. GPR55 was amplified and yielded a product of 311 bp. GAPDH was used as control and yielded a product of 260 bp (A). HEK293 cells overexpressing human GPR55 were used as a positive control while HEK293 cells were used as a negative control. RNA for qPCR analysis (B–E) was isolated from whole cells of cultures either in undifferentiation conditions or after 10 days of differentiation conditions (removal of growth factors from culture medium). Shown are individual ΔCt values (n = 5, each individual point refers to RNA extracted from a separate culture) for NSC markers (B), extracellular signal receptors (mGluR1 is mGlu1 receptor) (C) and CB1 (CNR1) and CB2 (CNR2) receptors (D). Expression of GPR55 in undifferentiated and differentiation conditions (10 days) was compared to expression of GPR55 by HEK293 hGPR55 overexpressing cells and HEK293 cells (E).

Article Snippet: Gpr55 , Mm02621622_s1 , 102.

Techniques: Expressing, Amplification, Control, Positive Control, Negative Control, Isolation

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Expression of GPR55 in primary murine hippocampal NSCs. RNA from primary hippocampal NSC cultures from C57BL/6 and GPR55−/− was extracted and cDNA and PCR amplification were performed. GPR55 was amplified and yielded a product of 420 bp. GAPDH was used as a control and yielded a product of 292 bp (A). DNA was also extracted from tail clips of C57BL/6 and GPR55−/− mice to determine genotype (B). Two sets of primers were used to detect amplification of the WT GPR55 allele (441 bp) or GPR55−/− mutant allele (301 bp). RNA for qPCR analysis was isolated from whole cells of primary hippocampal NSC cultures derived from C57BL/6 and GPR55−/− animals. Individual data points represent separate NSC harvests; n = 5. Shown are individual ΔCt values of NSC markers (C), extracellular signal receptors (mGluR5, mGlu5 receptor) (D) and CB1 (CNR1) and CB2 (CNR2) receptors (E) for samples from C57BL/6 and GPR55−/− cultures.

Article Snippet: Gpr55 , Mm02621622_s1 , 102.

Techniques: Expressing, Amplification, Control, Mutagenesis, Isolation, Derivative Assay

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Effects of GPR55 agonists on hNSC proliferation. hNSCs were plated on T25 flasks coated with laminin. Cells were allowed to adhere overnight prior to vehicle, agonist or antagonist treatment. Vehicle refers to medium with 0.01% DMSO. Cells were then treated for 48 h with GPR55 agonists (O‐1602 1 μM, LPI 1 μM, ML184 1 μM), GPR55 antagonist ML193 (5 μM) or both. ML193 was added to cells 30 min prior to addition of agonists. BrdU (10 μM) was added for the final 1 h of treatment. Cells were then collected and stained for BrdU incorporation and total DNA (7‐AAD) for flow cytometric analysis. (A) Representative dot plots of vehicle, agonist and antagonist treatment. Treatment of hNSCs with GPR55 agonists ML184 (B), O‐1602 (C) and LPI (D) increased proliferation rates while pretreatment with ML193 attenuated these effects. ML193 treatment alone did not alter proliferation rates. Data are displayed as percentage of cells in S phase for the last 1 h of treatment as compared to vehicle control. Results are expressed as mean ± SEM; n = 7 experiments. *P<0.05, one‐way ANOVA with Dunnet's post hoc test was used to detect statistical significance.

Article Snippet: Gpr55 , Mm02621622_s1 , 102.

Techniques: Staining, BrdU Incorporation Assay, Control

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Effects of GPR55 agonists on hNSC differentiation. hNSCs were cultured under differentiating conditions (maintenance medium without bFGF and EGF) and treated with either vehicle (0.01% DMSO), the selective GPR55 agonist ML184 (1 μM), selective antagonist ML193 (5 μM) or a combination of ML184 and ML193 for 10 days. Representative images of vehicle‐treated (A), ML184 (B), ML193 (C), ML184 + ML193 (D) and undifferentiated (E) cells stained for βIII‐tubulin (red) and DAPI (blue). Scale bars are 100 μm. (F, H) Representative histograms of flow cytometric analysis of βIII‐tubulin and S100β respectively. For flow cytometric analysis, cells were incubated with antibodies against human βIII‐tubulin (APC) and S100β (Alexa‐fluor 488). Cytometric acquisition was performed using a BD FACS Canto II flow cytometer and analysed with FlowJo software. Treatment with ML184 increased the expression of βIII‐tubulin after 10 days, while ML193 attenuated these effects. ML193 treatment decreased the formation of neurons as compared to differentiated vehicle control. Quantitative analysis of βIII‐tubulin+ cells (G) and S100β + cells (I) as compared to differentiated control from flow cytometric analysis of five experiments. mRNA was derived from whole cells collected from each experiment and analysed via qPCR for neuronal markers (βIII‐tubulin, J; Map2, K) and astrocytic markers (S100β, L; GFAP, M). Data are presented as fold change of vehicle‐treated cells under differentiating conditions for 10 days. Individual data points represent separate, individual experiments. *P<0.05, one‐way ANOVA and Dunnet's post hoc test.

Article Snippet: Gpr55 , Mm02621622_s1 , 102.

Techniques: Cell Culture, Staining, Incubation, Flow Cytometry, Software, Expressing, Control, Derivative Assay

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Effects of O‐1602, a GPR55 agonist, on murine NSC proliferation in vivo. C57BL/6(WT) and GPR55−/− mice were implanted with subdermal osmotic pumps set for a dispersion time of 14 days. Pumps were connected to brain infusion kits for direct infusion into the left hippocampus via continuous cannulation. Mice were treated with ACSF +0.05% EtOH (vehicle) or O‐1602 (4 μg·kg−1·day−1). BrdU (100 mg·kg−1) was injected i.p. twice a day for the first 2 days and once a day for days 3 and 4. Brain tissue was harvested at day 14. Schematic representation of experimental time points is displayed in (A). (B) Quantitative analysis of total Ki67 positive cells per dentate gyrus per mouse. Statistical analysis showed significant increases in Ki67+ cells within the SGZ of the dentate gyrus in O‐1602‐treated WT animals as compared to vehicle controls; n = 8 per group. Vehicle‐treated GPR55−/− animals (n = 6) displayed significantly lower Ki67+ cells in the SGZ as compared to WT vehicle‐treated animals. O‐1602 treatment had no significant effect in GPR55−/− animals; n = 7; *P<0.05, one‐way ANOVA and Tukey's multiple comparisons test. (C) Representative confocal microscopy images depicting localization of Ki67 (green) within the dentate gyrus from 30 μm brain sections of WT and GPR55−/− mice. Scale bar = 200 μm.

Article Snippet: Gpr55 , Mm02621622_s1 , 102.

Techniques: In Vivo, Dispersion, Injection, Confocal Microscopy

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Effects of O‐1602 on adult murine hippocampal neurogenesis in vivo. C57BL/6(WT) and GPR55−/− mice were implanted with subdermal osmotic pumps set for a dispersion time of 14 days. Experimental methods were the same as described for Figure 5. (A) Representative confocal microscopy images depicting localization of BrdU (red) and DCX (green) within the dentate gyrus of the hippocampus from 30 μm brain sections of WT and GPR55−/− mice. Scale bar = 100 μm. Arrows indicate BrdU+/DCX+ double labelled cells. Quantitative analysis of DCX (B), BrdU (C), DCX/BrdU (D) total positive cells per dentate gyrus per mouse. Analysis shows significant increases in DCX+, BrdU+ and DCX+/BrdU+ cells within the SGZ of the dentate gyrus after treatment with O‐1602 as compared to vehicle control; n = 8 per group. Vehicle‐treated GPR55−/− animals (n = 6) exhibit a significant reduction in the number of DCX+, BrdU+ and DCX+/BrdU+ cells in the SGZ as compared to WT vehicle‐treated control animals. Treatment with O‐1602 in GPR55−/− animals had no significant effect on the total number of DCX+, BrdU+ or DCX+/BrdU+ cells in the SGZ. (E) Analysis also determined that the percentage of total BrdU+ cells that were also DCX+ was increased in WT animals treated with O‐1602, yet results were not significant; P > 0.05. GPR55−/− animals demonstrated a significantly reduced rate of BrdU+ cells becoming DCX+ as compared to WT vehicle‐treated control. O‐1602 treatment had no effect on this percentage as compared to vehicle‐treated GPR55−/− mice. Bars represent mean ± SEM; *P<0.05, one‐way ANOVA and Tukey's multiple comparisons test.

Article Snippet: Gpr55 , Mm02621622_s1 , 102.

Techniques: In Vivo, Dispersion, Confocal Microscopy, Control

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Human qPCR primer/probe sets

Article Snippet: Gpr55 , Mm02621622_s1 , 102.

Techniques: Amplification

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Mouse qPCR primer/probe sets

Article Snippet: GPR55 , Hs00271662_s1 , 80.

Techniques: Amplification

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Expression of GPR55 in human NSCs. RNA from hNSCs was extracted and cDNA and PCR amplification were performed. GPR55 was amplified and yielded a product of 311 bp. GAPDH was used as control and yielded a product of 260 bp (A). HEK293 cells overexpressing human GPR55 were used as a positive control while HEK293 cells were used as a negative control. RNA for qPCR analysis (B–E) was isolated from whole cells of cultures either in undifferentiation conditions or after 10 days of differentiation conditions (removal of growth factors from culture medium). Shown are individual ΔCt values (n = 5, each individual point refers to RNA extracted from a separate culture) for NSC markers (B), extracellular signal receptors (mGluR1 is mGlu1 receptor) (C) and CB1 (CNR1) and CB2 (CNR2) receptors (D). Expression of GPR55 in undifferentiated and differentiation conditions (10 days) was compared to expression of GPR55 by HEK293 hGPR55 overexpressing cells and HEK293 cells (E).

Article Snippet: GPR55 , Hs00271662_s1 , 80.

Techniques: Expressing, Amplification, Control, Positive Control, Negative Control, Isolation

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Expression of GPR55 in primary murine hippocampal NSCs. RNA from primary hippocampal NSC cultures from C57BL/6 and GPR55−/− was extracted and cDNA and PCR amplification were performed. GPR55 was amplified and yielded a product of 420 bp. GAPDH was used as a control and yielded a product of 292 bp (A). DNA was also extracted from tail clips of C57BL/6 and GPR55−/− mice to determine genotype (B). Two sets of primers were used to detect amplification of the WT GPR55 allele (441 bp) or GPR55−/− mutant allele (301 bp). RNA for qPCR analysis was isolated from whole cells of primary hippocampal NSC cultures derived from C57BL/6 and GPR55−/− animals. Individual data points represent separate NSC harvests; n = 5. Shown are individual ΔCt values of NSC markers (C), extracellular signal receptors (mGluR5, mGlu5 receptor) (D) and CB1 (CNR1) and CB2 (CNR2) receptors (E) for samples from C57BL/6 and GPR55−/− cultures.

Article Snippet: GPR55 , Hs00271662_s1 , 80.

Techniques: Expressing, Amplification, Control, Mutagenesis, Isolation, Derivative Assay

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Effects of GPR55 agonists on hNSC proliferation. hNSCs were plated on T25 flasks coated with laminin. Cells were allowed to adhere overnight prior to vehicle, agonist or antagonist treatment. Vehicle refers to medium with 0.01% DMSO. Cells were then treated for 48 h with GPR55 agonists (O‐1602 1 μM, LPI 1 μM, ML184 1 μM), GPR55 antagonist ML193 (5 μM) or both. ML193 was added to cells 30 min prior to addition of agonists. BrdU (10 μM) was added for the final 1 h of treatment. Cells were then collected and stained for BrdU incorporation and total DNA (7‐AAD) for flow cytometric analysis. (A) Representative dot plots of vehicle, agonist and antagonist treatment. Treatment of hNSCs with GPR55 agonists ML184 (B), O‐1602 (C) and LPI (D) increased proliferation rates while pretreatment with ML193 attenuated these effects. ML193 treatment alone did not alter proliferation rates. Data are displayed as percentage of cells in S phase for the last 1 h of treatment as compared to vehicle control. Results are expressed as mean ± SEM; n = 7 experiments. *P<0.05, one‐way ANOVA with Dunnet's post hoc test was used to detect statistical significance.

Article Snippet: GPR55 , Hs00271662_s1 , 80.

Techniques: Staining, BrdU Incorporation Assay, Control

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Effects of GPR55 agonists on hNSC differentiation. hNSCs were cultured under differentiating conditions (maintenance medium without bFGF and EGF) and treated with either vehicle (0.01% DMSO), the selective GPR55 agonist ML184 (1 μM), selective antagonist ML193 (5 μM) or a combination of ML184 and ML193 for 10 days. Representative images of vehicle‐treated (A), ML184 (B), ML193 (C), ML184 + ML193 (D) and undifferentiated (E) cells stained for βIII‐tubulin (red) and DAPI (blue). Scale bars are 100 μm. (F, H) Representative histograms of flow cytometric analysis of βIII‐tubulin and S100β respectively. For flow cytometric analysis, cells were incubated with antibodies against human βIII‐tubulin (APC) and S100β (Alexa‐fluor 488). Cytometric acquisition was performed using a BD FACS Canto II flow cytometer and analysed with FlowJo software. Treatment with ML184 increased the expression of βIII‐tubulin after 10 days, while ML193 attenuated these effects. ML193 treatment decreased the formation of neurons as compared to differentiated vehicle control. Quantitative analysis of βIII‐tubulin+ cells (G) and S100β + cells (I) as compared to differentiated control from flow cytometric analysis of five experiments. mRNA was derived from whole cells collected from each experiment and analysed via qPCR for neuronal markers (βIII‐tubulin, J; Map2, K) and astrocytic markers (S100β, L; GFAP, M). Data are presented as fold change of vehicle‐treated cells under differentiating conditions for 10 days. Individual data points represent separate, individual experiments. *P<0.05, one‐way ANOVA and Dunnet's post hoc test.

Article Snippet: GPR55 , Hs00271662_s1 , 80.

Techniques: Cell Culture, Staining, Incubation, Flow Cytometry, Software, Expressing, Control, Derivative Assay

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Effects of O‐1602, a GPR55 agonist, on murine NSC proliferation in vivo. C57BL/6(WT) and GPR55−/− mice were implanted with subdermal osmotic pumps set for a dispersion time of 14 days. Pumps were connected to brain infusion kits for direct infusion into the left hippocampus via continuous cannulation. Mice were treated with ACSF +0.05% EtOH (vehicle) or O‐1602 (4 μg·kg−1·day−1). BrdU (100 mg·kg−1) was injected i.p. twice a day for the first 2 days and once a day for days 3 and 4. Brain tissue was harvested at day 14. Schematic representation of experimental time points is displayed in (A). (B) Quantitative analysis of total Ki67 positive cells per dentate gyrus per mouse. Statistical analysis showed significant increases in Ki67+ cells within the SGZ of the dentate gyrus in O‐1602‐treated WT animals as compared to vehicle controls; n = 8 per group. Vehicle‐treated GPR55−/− animals (n = 6) displayed significantly lower Ki67+ cells in the SGZ as compared to WT vehicle‐treated animals. O‐1602 treatment had no significant effect in GPR55−/− animals; n = 7; *P<0.05, one‐way ANOVA and Tukey's multiple comparisons test. (C) Representative confocal microscopy images depicting localization of Ki67 (green) within the dentate gyrus from 30 μm brain sections of WT and GPR55−/− mice. Scale bar = 200 μm.

Article Snippet: GPR55 , Hs00271662_s1 , 80.

Techniques: In Vivo, Dispersion, Injection, Confocal Microscopy

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Effects of O‐1602 on adult murine hippocampal neurogenesis in vivo. C57BL/6(WT) and GPR55−/− mice were implanted with subdermal osmotic pumps set for a dispersion time of 14 days. Experimental methods were the same as described for Figure 5. (A) Representative confocal microscopy images depicting localization of BrdU (red) and DCX (green) within the dentate gyrus of the hippocampus from 30 μm brain sections of WT and GPR55−/− mice. Scale bar = 100 μm. Arrows indicate BrdU+/DCX+ double labelled cells. Quantitative analysis of DCX (B), BrdU (C), DCX/BrdU (D) total positive cells per dentate gyrus per mouse. Analysis shows significant increases in DCX+, BrdU+ and DCX+/BrdU+ cells within the SGZ of the dentate gyrus after treatment with O‐1602 as compared to vehicle control; n = 8 per group. Vehicle‐treated GPR55−/− animals (n = 6) exhibit a significant reduction in the number of DCX+, BrdU+ and DCX+/BrdU+ cells in the SGZ as compared to WT vehicle‐treated control animals. Treatment with O‐1602 in GPR55−/− animals had no significant effect on the total number of DCX+, BrdU+ or DCX+/BrdU+ cells in the SGZ. (E) Analysis also determined that the percentage of total BrdU+ cells that were also DCX+ was increased in WT animals treated with O‐1602, yet results were not significant; P > 0.05. GPR55−/− animals demonstrated a significantly reduced rate of BrdU+ cells becoming DCX+ as compared to WT vehicle‐treated control. O‐1602 treatment had no effect on this percentage as compared to vehicle‐treated GPR55−/− mice. Bars represent mean ± SEM; *P<0.05, one‐way ANOVA and Tukey's multiple comparisons test.

Article Snippet: GPR55 , Hs00271662_s1 , 80.

Techniques: In Vivo, Dispersion, Confocal Microscopy, Control

Journal: British Journal of Pharmacology

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

doi: 10.1111/bph.14387

Figure Lengend Snippet: Human qPCR primer/probe sets

Article Snippet: GPR55 , Hs00271662_s1 , 80.

Techniques: Amplification