Journal: World Journal of Gastroenterology

Article Title: Anti-inflammatory effect of cannabinoid agonist WIN55, 212 on mouse experimental colitis is related to inhibition of p38MAPK

doi: 10.3748/wjg.v22.i43.9515

Figure Lengend Snippet: Representative figures of the expression of cannabinoid receptor type 1 and type 2 receptors in mouse colon by immunohistochemistry staining, which are from 4 separate experiments with similar results. A: Expression of cannabinoid (CB)1 receptors; B: Expression of CB2 receptors; (magnification × 400, respectively). The expressions of CB1 receptor in the colonic epithelial cells and CB2 receptor in the submucosal immune cells are marked by arrows and presented as positive results with brown-yellow staining.

Article Snippet: The first antibody was rabbit anti-CB1 receptor or rabbit anti-CB2 receptor polyclonal antibody (1:20 dilution for both; Enzo, Plymouth Meeting, PA, United States).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: World Journal of Gastroenterology

Article Title: Anti-inflammatory effect of cannabinoid agonist WIN55, 212 on mouse experimental colitis is related to inhibition of p38MAPK

doi: 10.3748/wjg.v22.i43.9515

Figure Lengend Snippet: Expression of cannabinoid receptor type 1 by western blot analysis. A: Collection of colonic tissues specimens from C57BL/6 mice in different experimental groups and the western blotting operation were performed as described in the Materials and Methods; B: Representative strips from 4 separate experiments with similar results are presented. Semi-quantitative blot analysis of cannabinoid (CB)1 receptors was carried out by ImageJ software. Data are presented as mean ± SD ( n = 4). a P < 0.05 vs control; c P < 0.05 vs dextran sulfate sodium (DSS) + vehicle (Veh).

Article Snippet: The first antibody was rabbit anti-CB1 receptor or rabbit anti-CB2 receptor polyclonal antibody (1:20 dilution for both; Enzo, Plymouth Meeting, PA, United States).

Techniques: Expressing, Western Blot, Software

Journal: Molecular Cancer

Article Title: Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition

doi: 10.1186/1476-4598-9-196

Figure Lengend Snippet: ErbB2-positive human breast tumors express cannabinoid receptors . (A) Representative images of human breast tumors negative (upper row) or positive (lower row) for CB 1 , CB 2 and ErbB2 receptors (brown). Scale bars: 200 μm; insets: 100 μm. (B) Percentage of tumors scored as positive or negative for cannabinoid receptor expression amongst the ErbB2-negative (n = 64) and ErbB2-positive (n = 23) populations.

Article Snippet: Tissue-tek embedded tumor sections were fixed in PFA and incubated with anti-CB 1 receptor, anti-CB 2 receptor, anti-CD31 (Pharmingen/BD Biosciences, San Jose, California), anti-CD45 (Pharmingen/BD Biosciences), anti-Ki67 (Neomarkers/Lab Vision, Fremont, California) or anti-cleaved-caspase 3 (Cell Signaling Technology, Danvers, Massachusetts) antibodies.

Techniques: Expressing

Journal: Molecular Cancer

Article Title: Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition

doi: 10.1186/1476-4598-9-196

Figure Lengend Snippet: Cannabinoids inhibit breast tumor growth in vivo and the number of tumors generated per animal . (A) Volume time-course (scale bar: 1 cm) of the first tumor appeared in each animal. (B) Representative images (H&E staining) of the histopathology of the MMTV-neu-derived mammary tumors. Scale bars (from left to right): 200 μm, 100 μm and 50 μm. (C) Percentage of animals with 1, 2, 3, 4 or more tumors at the end of the treatment (90 days) in each experimental group. (D) Total tumor burden (total tumor volume per animal) determined 90 days after cannabinoid or vehicle treatment. (E) Volume of the tumors appeared in second, third or subsequent positions, 40 days after their appearance. The small size of the cannabinoid-treated groups is due to the very few second or third tumors appeared early enough to last 40 days in the animals before the end of the treatment (90 days after the appearance of the first tumor).

Article Snippet: Tissue-tek embedded tumor sections were fixed in PFA and incubated with anti-CB 1 receptor, anti-CB 2 receptor, anti-CD31 (Pharmingen/BD Biosciences, San Jose, California), anti-CD45 (Pharmingen/BD Biosciences), anti-Ki67 (Neomarkers/Lab Vision, Fremont, California) or anti-cleaved-caspase 3 (Cell Signaling Technology, Danvers, Massachusetts) antibodies.

Techniques: In Vivo, Generated, Staining, Histopathology, Derivative Assay

Journal: Molecular Cancer

Article Title: Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition

doi: 10.1186/1476-4598-9-196

Figure Lengend Snippet: Cannabinoids inhibit breast cancer metastasis to the lungs in vivo . (A) Metastatic lung nodules (pointed by arrows). (B) Percentage of animals with lung metastases. Macroscopic metastases were visible to the naked eye and microscopic lesions were detectable only by H&E staining. These latter lesions were found only in JWH-133-treated animals. (C) Gelatin zymographies of vehicle- and cannabinoid-treated tumors. Four representative tumors are shown per experimental group. PC: positive control (conditioned medium of H71080 cells stimulated with the phorbol ester PMA). Arrows point to the latent (pro-MMP) and active MMP bands according to the positive control and the expected MMP molecular weights. Non-contiguous parts of the same gel are shown. Graphs show the densitometric analysis of MMP2 and MMP9 activities. Data are expressed in arbitrary units. *, p < 0.05 vs vehicle-treated tumors; §, p = 0.068 vs vehicle-treated tumors.

Article Snippet: Tissue-tek embedded tumor sections were fixed in PFA and incubated with anti-CB 1 receptor, anti-CB 2 receptor, anti-CD31 (Pharmingen/BD Biosciences, San Jose, California), anti-CD45 (Pharmingen/BD Biosciences), anti-Ki67 (Neomarkers/Lab Vision, Fremont, California) or anti-cleaved-caspase 3 (Cell Signaling Technology, Danvers, Massachusetts) antibodies.

Techniques: In Vivo, Staining, Positive Control

Journal: Molecular Cancer

Article Title: Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition

doi: 10.1186/1476-4598-9-196

Figure Lengend Snippet: THC inhibits Akt in vivo . (A) Mouse ErbB2 and (B) rat ErbB2 (neu transgene) mRNA expression in vehicle- and cannabinoid-treated tumors as determined by real-time quatitative PCR. (B and C) Western blot and densitometric analysis of phospho-Akt (B) and phospho-S6 ribosomal protein (C) levels in MMTV-neu-derived tumors treated with vehicle or THC. Eight representative tumors are shown. Total Akt (B) and α-tubulin (C) were used for normalization. Non-contiguous parts of the same gel are shown. Optical densities are expressed in arbitrary units.

Article Snippet: Tissue-tek embedded tumor sections were fixed in PFA and incubated with anti-CB 1 receptor, anti-CB 2 receptor, anti-CD31 (Pharmingen/BD Biosciences, San Jose, California), anti-CD45 (Pharmingen/BD Biosciences), anti-Ki67 (Neomarkers/Lab Vision, Fremont, California) or anti-cleaved-caspase 3 (Cell Signaling Technology, Danvers, Massachusetts) antibodies.

Techniques: In Vivo, Expressing, Western Blot, Derivative Assay

Journal: Molecular Cancer

Article Title: Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition

doi: 10.1186/1476-4598-9-196

Figure Lengend Snippet: Akt downregulation is involved in cannabinoid antitumoral action . (A and B) Viability of N202.1A cells in response to (A) increasing concentrations of THC or JWH-133 or (B) 6 μM THC or 10 μM JWH-133 with or without 2 μM SR141716 (SR1) and/or SR144528 (SR2) for 48 h. Data are expressed as % of vehicle-treated cells, set at 100%. (C) Growth of N202.1A-derived xenografts treated with THC (left panel) or JWH-133 (right panel) with or without SR2. (D) Phospho-Akt and phospho-S6 ribosomal protein (p-S6) levels in N202.1A cells challenged with THC, as determined by Western blot. Total Akt and α-tubulin levels were used for normalization. (E) Phospho-Akt and total Akt levels in N202.1A cells retrovirally transduced with myristoylated Akt (pBABE-Myr-Akt) or the corresponding empty vector (pBABE). (F) Cell viability of pBABE- or myr-Akt-transduced N202.1A cells in response to THC exposure for 72 h. (G and H) Time course of the volume of pBABE-N202.1A-derived (left panels) and myr-Akt-N202.1A-derived (right panels) tumors treated with THC (G), JWH-133 (H) or the corresponding vehicle. *, p < 0.05; **, p < 0.01 vs vehicle-treated cells or tumors; #, p < 0.05; ##, p < 0.01 vs cannabinoid alone-treated cells (B) or vs THC-treated pBABE-transduced cells (F).

Article Snippet: Tissue-tek embedded tumor sections were fixed in PFA and incubated with anti-CB 1 receptor, anti-CB 2 receptor, anti-CD31 (Pharmingen/BD Biosciences, San Jose, California), anti-CD45 (Pharmingen/BD Biosciences), anti-Ki67 (Neomarkers/Lab Vision, Fremont, California) or anti-cleaved-caspase 3 (Cell Signaling Technology, Danvers, Massachusetts) antibodies.

Techniques: Derivative Assay, Western Blot, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: Astroglial calcium transfer from endoplasmic reticulum to mitochondria determines synaptic integration

doi: 10.1101/2020.12.08.415620

Figure Lengend Snippet: A Scheme showing ER (in green) -mitochondria (in red) calcium transfer and the different drugs used in to study the role of this pathway. B Representative electron microscopy image from CB1-KO astrocytes. M: mitochondria. ER: endoplasmic reticulum. Scale bar: 0,5 μm. C,D WIN effect on cytosolic (D) and mitochondrial (D) calcium (Area under the curve, AUC) in negative control (CB 1 −), Vehicle control (Veh), thapsigargin (TG), 2APB and MCU inhibitor 11 (MCU-i11) conditions. Baseline: 1 min before WIN treatment. ΔF/F0 traces are shown in Figure S4A,B respectively. E Representative images from CB1 transfected astrocytes in Vehicle, WIN-1 min and WIN-30 min conditions. In cyan, CB1; in green, p-AKT (T296). Scale bar: 10 μm. F Normalized total intensity from cells show in (E) in control CB 1 − (Ctrl) and CB 1 + transfected conditions. G,H WIN effect on cytosolic (G) and mitochondrial (H) calcium (AUC) in negative control CB 1 −, Vehicle control (Veh) and AKT-inhibitor (MK) conditions. Baseline: 1 min before WIN treatment. ΔF/F0 traces are shown in Figure S4C,D respectively. I WIN effect on mitochondrial calcium (AUC) in CB 1 + trasfected cells co-trasnfected with dnMCU, MICU1-S124A or MICU1-WT. Baseline: 1 min before WIN treatment. ΔF/F0 traces are shown in Figure S4E. *p-value<0.05; **p-value<0.01; ***p-value<0.001; ****p-value<0.0001. #p-value<0.05; ##p-value<0.01; ###p-value<0.001 in comparison to Vehicle (CB 1 +) condition.

Article Snippet: Cells were pre-incubated in a blocking solution (10% normal goat serum, 0.1% triton X-100 and 0.2 M glycine in PBS) for 1 h and then incubated with primary antibody in the same blocking solution for 2 h. Primary antibodies were rabbit anti-TOM20 (1:500; Santa Cruz, sc-11415), mouse anti-myc (1:500; Roche, 11667149001), rabbit anti p-AKT (T296) (1:100; Cell Signalling Technology, 9614) or guinea pig anti-CB 1 (1:300; Abyntek, CB 1 -gp-AF530).

Techniques: Electron Microscopy, Negative Control, Transfection, Comparison

Journal: bioRxiv

Article Title: Astroglial calcium transfer from endoplasmic reticulum to mitochondria determines synaptic integration

doi: 10.1101/2020.12.08.415620

Figure Lengend Snippet: A Immunohistochemistry representative images from mouse hippocampi injected with AAV-GFAP-Mito-GCaMP6s (in green). Astrocyte marker S100β (in red). Scale bar: 20 μm. B Percentage of cell colocalization of Mito-GCaMP6s and S100β (astrocytic marker) or NeuN (neuronal marker) in Hippocampi (Hp) and Cortex (Cx). C Experimental two-photon imaging approach for in vivo recording of mitochondrial calcium dynamics in mouse somatosensory cortex. D Representative two-photon image. Squared image shows mitochondrial particles (yellow arrow heads). Scale bar: 100 μm. E Representative time-point two-photon images of the somatosensory cortex from CB 1 WT mouse injected with AAV-GFAP-Mito-GCaMP6s. THC (10 mg/kg) treatment was applied at time 0. On the right magnified images from time 0 and time 30 min (white squares). Scale bar: 30 μm. F Normalized calcium peak traces from CB 1 WT and CB 1 KO mice injected with Vehicle or THC. Blue and gray rectangles show the time window analyzed in G and H. G,H Average percentage of calcium events (normalized to day 1) between 0 to 15 min (G) or 15 to 30 min (H) in CB 1 WT and CB 1 KO mice treated with Vehicle or THC. I Scheme showing experimental fiber photometry imaging approach. J Representative traces from the cortex of a CB 1 WT mouse injected with AAV-GFAP-Mito-GCaMP6s. Vehicle-injected (left panel) or THC-injected (10 mg/kg, right panel) mouse traces. Red dots correspond to detected transients above threshold (median+2*MAD). Blue and gray rectangles show the time window of the analyzed “period 1” and “period 2” respectively. Note that the first minute before and after injection was removed to exclude mouse-handling/injection effects. K,M Cortical or L,N hippocampal amplitude of transients in CB 1 WT and CB 1 KO mice treated with Vehicle (VEH) or THC during the 2 time periods highlighted in (J). *p-value<0.05; **p-value<0.01; ****p-value<0.0001.

Article Snippet: Cells were pre-incubated in a blocking solution (10% normal goat serum, 0.1% triton X-100 and 0.2 M glycine in PBS) for 1 h and then incubated with primary antibody in the same blocking solution for 2 h. Primary antibodies were rabbit anti-TOM20 (1:500; Santa Cruz, sc-11415), mouse anti-myc (1:500; Roche, 11667149001), rabbit anti p-AKT (T296) (1:100; Cell Signalling Technology, 9614) or guinea pig anti-CB 1 (1:300; Abyntek, CB 1 -gp-AF530).

Techniques: Immunohistochemistry, Injection, Marker, Imaging, In Vivo

Journal: bioRxiv

Article Title: Astroglial calcium transfer from endoplasmic reticulum to mitochondria determines synaptic integration

doi: 10.1101/2020.12.08.415620

Figure Lengend Snippet: a Scheme depicting the experimental approach. eCBs: endocannabinoids. Glu: glutamate. b Representative traces obtained before (Basal) and after neuronal depolarization (ND). c Percentage of potentiated synapses recorded in WT and DN22-CB 1 -KI hippocampal slices. d Time course of normalized success rate in WT and DN22-CB 1 -KI conditions. Arrow indicates neuronal depolarization (ND). e Normalized success rate before (Basal) and after neuronal depolarization (ND) time in WT and DN22-CB 1 -KI conditions. Red and white circles represent potentiated and non-potentiated synapses, respectively. *p-value<0.05.

Article Snippet: Cells were pre-incubated in a blocking solution (10% normal goat serum, 0.1% triton X-100 and 0.2 M glycine in PBS) for 1 h and then incubated with primary antibody in the same blocking solution for 2 h. Primary antibodies were rabbit anti-TOM20 (1:500; Santa Cruz, sc-11415), mouse anti-myc (1:500; Roche, 11667149001), rabbit anti p-AKT (T296) (1:100; Cell Signalling Technology, 9614) or guinea pig anti-CB 1 (1:300; Abyntek, CB 1 -gp-AF530).

Techniques: