Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 1. Oncostatin M (OSM)–induced expression of CCL13. A and B, Normal dermal fibro- blasts (nDF), normal lung fibroblasts (nLF), normal cervical fibroblasts (nCF), and normal synovial fibroblasts (nSF) were treated with 10 ng/ml of OSM for the indicated periods of time. A, Levels of CCL2, CCL13, and CCL11 mRNA were determined by RNase protection assay. Results are representative of 3 independent experiments. B, The amounts of CCL13 and CCL2 in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Values are the mean SD (n 3). P 0.05 versus control. C, Primary human blood neutrophils were stimulated with 10 ng/ml of granulocyte–macrophage colony-stimulating factor (GM-CSF) for 3 hours. The amounts of OSM in culture supernatants were determined by ELISA. Values are the mean SD (n 3). P 0.05 versus control. D, Culture supernatants (SNs) from untreated neutrophils or neutrophils stimulated with GM-CSF for 3 hours were transferred onto normal SFs. The contribution of OSM was evaluated by additional application of blocking antibodies to OSM or control antibodies (ctrl. Ab). The amounts of CCL13 produced in normal SF culture supernatants were then determined by ELISA. Values are the mean SD (n 4). P 0.05 versus control; d P 0.05 versus antibody control.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Expressing, Rnase Protection Assay, Enzyme-linked Immunosorbent Assay, Control, Blocking Assay, Produced

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 2. Significance of OSM and other key cytokines involved in rheumatoid arthritis (RA) pathogenesis for CCL13 expression. A, Normal SFs were treated for 2 hours with 10 ng/ml of OSM, 200 units/ml of interleukin-6 (IL-6)/0.5 g soluble IL-6 receptor (sIL-6R), 10 ng/ml of IL-1, or 10 ng/ml of tumor necrosis factor (TNF). CCL13 mRNA levels were quantified, normalized to L32 levels, and compared with untreated cells. Values are the mean SD (n 4). P 0.05 versus specific control. B, Normal SFs were stimulated as described in A for 2 hours or 12 hours. Values are the mean SD (n 4). P 0.05 versus time-specific control. C, Normal SFs were treated for 3 hours with OSM, alone or in combination with IL-6/sIL-6R, IL-1, or TNF (at the concentrations described in A). Values are the mean SD (n 4). P 0.05 versus control. D, RASFs were stimulated with cytokines as described in A for 3 hours. Values are the mean SD (n 4). P 0.05 versus normal SF control; d P 0.05 versus control. E, OSM and CCL13 protein in the supernatant of unstimulated fibroblasts was determined by ELISA. Values are the mean SD (n 3). P 0.05 versus normal SF. F and G, Both normal SF and RASF supernatants were treated with OSM and either control or OSM-neutralization antibodies. Normal SFs and RASFs were restimulated with OSM-depleted supernatant for 3 hours. Values are the mean SD (n 3). and # P 0.05 versus control; d P 0.05 versus stimulated control. H, Supernatants of untreated RASFs were treated with control antibody, CCL13 antibody, or both CCL13 and OSM-neutralizing antibodies. Depleted supernatants were added to untreated normal SFs. Values are the mean SD (n 3). P 0.05 versus control; d P 0.05 versus control supernatant; # P 0.05 versus CCL13-depleted supernatant. w/o without; OASF osteoarthritis SF (see Figure 1 for other definitions).

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 4. Comparison of oncostatin M (OSM)–, interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R)–, IL-1–, and tumor necrosis factor (TNF)–induced signal transduction, showing a potential role of phosphorylated (pY) STAT-5 in CCL13 induction. A, Normal synovial fibroblasts (nSF) were treated with 10 ng/ml of OSM, 200 units/ml of IL-6/0.5 g of sIL-6R, 10 ng/ml of IL-1, or 10 ng/ml of TNF. Lysates were subjected to Western blot analysis, using antibodies specific for the indicated proteins. The blots were stripped and reprobed with antibodies recognizing the proteins irrespective of their activation status or with loading control antibodies. B, Normal SFs were stimulated with OSM as indicated. Western blots of lysates were stained with the antibodies indicated. C, Normal SFs, normal cervical fibroblasts (nCF), normal lung fibroblasts (nLF), and normal dermal fibroblasts (nDF) were stimulated with 10 ng/ml of OSM. Cell lysates were analyzed as described in B. D, Normal SFs were transfected with control or STAT-5 small interfering RNA (siSTAT5). Forty-eight hours later, cells were stimulated with 10 ng/ml of OSM for 3 hours. CCL13 and CCL2 protein in normal SF culture supernatants was measured by enzyme-linked immunosorbent assay. Values are the mean SD (n 4). P 0.05 versus control; d P 0.05 versus stimulated siRNA control; w/o without.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Comparison, Transduction, Western Blot, Activation Assay, Control, Staining, Transfection, Small Interfering RNA, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 5. Role of OSM-induced p38 activation in the stabilization of CCL13 mRNA through inhibition of tristetraprolin (TTP). A and C, Normal SFs and rheumatoid arthritis SFs (RASFs), respectively, were pretreated with 10 M AG490, 10 M SB202190, 10 M U0126, 10 M SP600125, 1 M JAK inhibitor I (JI-1), or the solvent DMSO, and then exposed to 10 ng/ml of OSM for 3 hours. Subsequently, CCL13 protein was quantified in culture supernatants by enzyme-linked immunosorbent assay (ELISA). Values are the mean SD (n 4). and # P 0.05 versus control; d P 0.05 versus OSM-stimulated sample. B, Western blots from treated normal SFs were analyzed using the indicated antibodies. D, Normal SFs were stimulated with 10 ng/ml of OSM for 30 minutes. Then, cells were washed and cultivated for an additional 20 minutes in OSM-free medium containing actinomycin D (4 M). SB202190 (10 M) was added to the medium before cells were restimulated with OSM for the indicated periods of time. CCL13 and GAPDH mRNA levels were analyzed by reverse transcription–polymerase chain reaction. One of 3 representative independent experiments is shown. E, Normal SFs were transfected with TTP or control siRNA. Forty-eight hours later, cells were pretreated with 10 M SB202190 or DMSO for 20 minutes and then stimulated with 10 ng/ml of OSM for 3 hours. CCL13 was measured by ELISA. Values are the mean SD (n 3). P 0.05 versus control; d P 0.05 versus OSM-stimulated sample. See Figure 4 for other definitions.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Activation Assay, Inhibition, Solvent, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Transfection

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 6. Contribution of STAT-5 and ERK-1/2 to CCL13 expres- sion. RASFs were transfected with STAT-5 or control small interfering RNA (siRNA). Forty-eight hours later, cells were preincubated with DMSO or U0126 for 30 minutes, prior to stimulation with 10 ng/ml of OSM for 3 hours. CCL13 protein was measured in culture superna- tants of RASFs, by ELISA. Values are the mean SD (n 4). P 0.05 versus unstimulated control; # P 0.05 versus OSM- stimulated DMSO control; d P 0.05 versus stimulated siRNA control. Inset, Knockdown efficiency was controlled by Western blot analysis of lysates, using a specific antiserum against tyrosine- phosphorylated STAT-5 (pY-STAT5). After stripping, the blots were reprobed with antiserum recognizing STAT-3. See Figure 5 for other definitions.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Transfection, Control, Small Interfering RNA, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Stripping Membranes

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Capsaicin Enhanced the Efficacy of Photodynamic Therapy Against Osteosarcoma via a Pro-Death Strategy by Inducing Ferroptosis and Alleviating Hypoxia.

doi: 10.1002/smll.202306916

Figure Lengend Snippet: Figure 3. Responsive activation of TRPV1-mediated intracellular Ca2+ concentration. A) CLSM images of 143B and HOS cells in various groups after using the Fluo-4 probe to monitor Ca2+ influx. The scale bar is 20 μm. B,C) FC quantitative value of Ca2+ uptake in 143B and HOS cells after different treatments. D) The protein expression level of TRPV1 was determined by western blot analysis. E) Statistical analyses of TRPV1 (the data are presented as the mean ± SD, n = 3, ***p < 0.001).

Article Snippet: Mouse anti-human TRPV1 (cat. 66983-1-Ig), mouse anti-human NQO1 (cat. 67240-1-Ig), rabbit anti-human GCLM (cat. 14241-1-AP), rabbit anti-human PCNA (cat. 10205-2-AP), rabbit anti-human KI67 (cat. 27309-1-AP), and rabbit antihuman HIF-1α (cat. 20960-1-AP) were acquired from Proteintech Corp (USA).

Techniques: Activation Assay, Concentration Assay, Expressing, Western Blot

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Capsaicin Enhanced the Efficacy of Photodynamic Therapy Against Osteosarcoma via a Pro-Death Strategy by Inducing Ferroptosis and Alleviating Hypoxia.

doi: 10.1002/smll.202306916

Figure Lengend Snippet: Figure 4. Evaluation of ferroptosis. The production of Lipid-ROS in A) 143B and B) HOS cells after different interventions as measured by CLSM. The scale bars represent 20 μm. C) FC analysis of Lipid-ROS after different interventions in 143B and HOS cells. D,E) Confocal images and FC analyses of ROS generation in 143B and HOS cells treated with different treatments and stained with DCFH-DA (scale bar = 20 μm). F,G) Quantitative value of intracellular C11-BODIPY and DCFH-DA FL intensity (the data are presented as the mean ± SD, n = 3, ***p < 0.001). H) The morphology of mitochondria after various treatments as observed by TEM. The scale bars represent 2 μm. I) The expression level of Gpx4 protein was measured by western blot analysis. J) The expression of TRPV1 and Gpx4 by IF observation after different treatments in 143B cells (scale bar = 100 μm).

Article Snippet: Mouse anti-human TRPV1 (cat. 66983-1-Ig), mouse anti-human NQO1 (cat. 67240-1-Ig), rabbit anti-human GCLM (cat. 14241-1-AP), rabbit anti-human PCNA (cat. 10205-2-AP), rabbit anti-human KI67 (cat. 27309-1-AP), and rabbit antihuman HIF-1α (cat. 20960-1-AP) were acquired from Proteintech Corp (USA).

Techniques: Staining, Expressing, Western Blot

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Capsaicin Enhanced the Efficacy of Photodynamic Therapy Against Osteosarcoma via a Pro-Death Strategy by Inducing Ferroptosis and Alleviating Hypoxia.

doi: 10.1002/smll.202306916

Figure Lengend Snippet: Figure 5. A) In MG63, HOS, and 143B cells, the effects of CAP on the intracellular oxygen level after different treatments were observed and analyzed by confocal microscopy (scale bar = 20 μm) and FC. B) Quantitative value of intracellular Image-iT Green Hypoxia Reagent FL intensity (the data are presented as the mean ± SD, n = 3, ***p < 0.001). C,D) The expression level of HIF-1𝛼protein was analyzed by western blot (the data are presented as the mean ± SD, n = 3, ***p < 0.001). E,F) The expression of TRPV1 and HIF-1𝛼by IF observation after different treatments in 143B and HOS cells (scale bar = 100 μm).

Article Snippet: Mouse anti-human TRPV1 (cat. 66983-1-Ig), mouse anti-human NQO1 (cat. 67240-1-Ig), rabbit anti-human GCLM (cat. 14241-1-AP), rabbit anti-human PCNA (cat. 10205-2-AP), rabbit anti-human KI67 (cat. 27309-1-AP), and rabbit antihuman HIF-1α (cat. 20960-1-AP) were acquired from Proteintech Corp (USA).

Techniques: Confocal Microscopy, Expressing, Western Blot

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Capsaicin Enhanced the Efficacy of Photodynamic Therapy Against Osteosarcoma via a Pro-Death Strategy by Inducing Ferroptosis and Alleviating Hypoxia.

doi: 10.1002/smll.202306916

Figure Lengend Snippet: Figure 8. Evaluation and biosafety of different treatments. A) H&E staining and images of PCNA, Ki67, TRPV1, Gpx4, and HIF-1𝛼. IHC staining in tumor specimens after various treatments (scale bar = 100 μm). B–F) IOD values in various groups were analyzed by processing IHC staining through ImagePro Plus. G) Triple IF staining of TRPV1 (red), Gpx4 (orange), and HIF-1𝛼(green) in tumor specimens after various treatments (scale bar = 50 μm). H) TUNEL staining of tumor sections after various treatments (scale bar = 50 μm). I) H&E staining of major organs of mice after different treatments (scale bar = 100 μm). J) Hematological and blood biochemical tests of mice after various treatments.

Article Snippet: Mouse anti-human TRPV1 (cat. 66983-1-Ig), mouse anti-human NQO1 (cat. 67240-1-Ig), rabbit anti-human GCLM (cat. 14241-1-AP), rabbit anti-human PCNA (cat. 10205-2-AP), rabbit anti-human KI67 (cat. 27309-1-AP), and rabbit antihuman HIF-1α (cat. 20960-1-AP) were acquired from Proteintech Corp (USA).

Techniques: Staining, Immunohistochemistry, TUNEL Assay

Journal: Nature Communications

Article Title: Dual recruitment of two CCM2 molecules to KRIT1 suppresses KLF4 expression

doi: 10.1038/s41467-026-69595-7

Figure Lengend Snippet: a Schematic of the domain organization of KRIT1 and CCM2. ARD, ankyrin repeat domain; FERM, band 4.1, ezrin, radixin, moesin domain; PTB, Phosphotyrosine binding domain; HHD, harmonin homology domain. Locations of NPxY1, NPxF2, and NPxF3 motifs are indicated. b EA.hy926 cells, or EA.hy926 cells stably expressing CCM2-Myc or CCM2-Myc mutants deficient in KRIT1 binding (II/AA or C/D) were infected with lentivirus encoding shRNA targeting CCM2 or control shRNA and levels of KLF4 and CCM2 were assessed by immunoblotting. TLK2 was assessed as a loading control. We note that while mutants were less well expressed than the wild type CCM2-Myc, they reach levels comparable to endogenous CCM2 in control cells, showing the importance of KRIT1 binding for KLF4 regulation. c KLF4 expression levels in experiments described in ( b ) were assessed. Data are normalized to levels seen in CCM2 knockdown cells without CCM2-Myc expression. Bars represent mean ± SD, n = 6 independent experiments. Data were analysed for differences from the control knockdown condition by one-way ANOVA with Dunnett’s multiple comparisons test. Corrected p values are indicated when significant ( p < 0.05). d Myc-Trap pulldowns were performed on EA.hy926 cells stably expressing CCM2-Myc or CCM2-Myc mutants, and co-precipitating endogenous KRIT1 was assessed by immunoblotting. Image is representative of at least 3 independent experiments. The following antibodies were used in Fig. 1: anti-KLF4 (Cell Signaling Technologies), anti-CCM2 (Protein Tech), anti-TLK2 (Santa Cruz Biotech), anti-KRIT1 (AbCam), and anti-Myc (GenScript) (See Supplementary Table ).

Article Snippet: The following antibodies were used in Fig. 1: anti-KLF4 (Cell Signaling Technologies), anti-CCM2 (Protein Tech), anti-TLK2 (Santa Cruz Biotech), anti-KRIT1 (AbCam), and anti-Myc (GenScript) (See Supplementary Table ).

Techniques: Binding Assay, Stable Transfection, Expressing, Infection, shRNA, Control, Western Blot, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation

doi: 10.3390/ijms23105367

Figure Lengend Snippet: TRPV2 levels were decreased in both AD patients and APP/PS1 mice. ( A ) The TRPV2 levels were measured in the brain whole-protein extracts from mice at the indicated age (E is embryos, P is days and M is months). The data are represented as the mean ± SEM ( n = 4 mice/group). * p < 0.05, and ** p < 0.01 are compared to the E15.5 by the Tukey’s test. ( B ) Cortex and hippocampus protein extracts were obtained at 6 months of age from wild-type (WT) and two transgenic AD mice models (APP/PS1) who were subjected to the TRPV2 antibody. The data are represented as the mean ± SEM for four different mice per genotype. * p < 0.05, and ** p < 0.01 compared to controls as obtained by the Student’s t -test. ( C ) Transcriptional expression levels of TRPV2 in entorhinal, temporal, and frontal cortex and hippocampus tissues of the patients with AD in the cross-platform database (entorhinal cortex: GSE26927, GSE26972, GSE48350, GSE5281; hippocampus: GSE28146, GSE29378, GSE36980, GSE48350, GSE5281; temporal cortex: GSE29652, GSE36980, GSE37263, GSE5281; frontal cortex: GSE12685, GSE36980, GSE48350, GSE5281, GSE53890, GSE66333; control, n = 19; AD patient, n = 19). ( D ) Transcriptional downregulation of TRPV2 in the entorhinal cortex of the tissue samples from patients with AD in the GSE26972 database (control, n = 19; AD patient, n = 19). ( E ) Transcriptional downregulation of TRPV2 in the hippocampus of the tissue samples from patients with AD in the GSE28146 database (control, n = 19; AD patient, n = 19).

Article Snippet: The TRPV2 (1:1000, polyclonal antibody, # ALO-ACC-032-50) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Transgenic Assay, Expressing, Control, Tissue, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation

doi: 10.3390/ijms23105367

Figure Lengend Snippet: CBD enhanced microglial Aβ phagocytosis via the TRPV2 channels. ( A ) Primary microglia cells were plated at a density of 5 × 10 4 in poly-D-lysine-coated wells of 24-well plates containing 10% FBS DMEM medium. After 24 h of treatment with CBD (5 μM), FITC-Aβ42 (1 μg/mL) was added to the medium. Immunofluorescence analysis of the microglial phagocytosis of FITC-Aβ42 was performed after allowing uptake for 4 h in microglia cells. ( B ) Quantification of the internalized FITC-Aβ42 using ImageJ software ( n = 36 to 42 per group). a.u., arbitrary units from 3/4 individual mice. ( C ) FITC-Aβ42 uptake index in the presence or absence of CBD after uptake for the indicated time in microglia cells, calculated based on the phagocytosis assay. ( D ) Microglial Aβ42 uptake was analyzed using Western blotting after 4 h of incubation with Aβ42 oligomer in the presence or absence of CBD. ( E ) Quantification of the protein levels using Image J software ( n = 4 per group). * p < 0.05 and ** p < 0.01 are compared with the Aβ by the Student’s t -test; ### p < 0.001 between Aβ and CBD + Aβ by Student’s t -test. The scale bars = 20 μm.

Article Snippet: The TRPV2 (1:1000, polyclonal antibody, # ALO-ACC-032-50) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Immunofluorescence, Analysis, Software, Phagocytosis Assay, Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation

doi: 10.3390/ijms23105367

Figure Lengend Snippet: The TRPV2-mediated phagocytosis in microglia cells was attenuated by inhibiting PDK, Akt, or PERK. FITC-Aβ42 uptake index in BV2 microglia cells was analyzed using the phagocytosis assay. ( A ) The presence of CBD (5 μM), 2APB (250 μM), and CAP (capsaicin: 2 μM) after 24 h ( n = 6). ** p < 0.01 and *** p < 0.001 are compared with the control by Tukey’s test, and # p < 0.05 and ## p < 0.01 are compared with Aβ by Tukey’s test. ( B ) BV2 microglia with knocked-down TRPV2 treated with or without CBD after 24 h ( n = 4). * p < 0.05 is between Aβ and CBD + Aβ by Student’s t -test; # p < 0.05 is between Aβ and CBD + Aβ upon siRNA by Student’s t -test. ( C – F ) BV2 microglia preadded with Tra (tranilast, 75 μM), akti (Akt inhibitor, 10 μM), PDK1 inhibitor (GSK2334470, 1 μM), and PERK inhibitor (GSK2656157, 1 μM) and treated with or without CBD after 24 h ( n = 5). ** p < 0.01 and *** p < 0.001 are compared with the control by Tukey’s test; ## p < 0.01 is between Aβ and CBD + Aβ by Student’s t -test; $ p < 0.05, $$ p < 0.01, and $$$ p < 0.001 are between CBD + Aβ and CBD + Aβ upon inhibitors by Student’s t -test.

Article Snippet: The TRPV2 (1:1000, polyclonal antibody, # ALO-ACC-032-50) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Phagocytosis Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation

doi: 10.3390/ijms23105367

Figure Lengend Snippet: Relative mRNA levels of microglial phagocytic receptors and inflammation factors in the microglia cells treated with or without CBD (5 μM). Primary microglial cells were treated with or without CBD after 12 h, and the mRNA levels were determined through qPCR. The data are represented as the mean ± SEM ( n = 6). * p < 0.05, ** p < 0.01, and *** p < 0.001 are compared with the control by Tukey’s test; # p < 0.05 and ## p < 0.01 are between Aβ and CBD + Aβ by Student’s t -test.

Article Snippet: The TRPV2 (1:1000, polyclonal antibody, # ALO-ACC-032-50) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Control, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation

doi: 10.3390/ijms23105367

Figure Lengend Snippet: Relative mRNA levels of microglial phagocytic receptors and inflammation factors in microglia cells after knockdown of TRPV2. BV2 microglia were treated with or without CBD (5 μM) after 12 h, and mRNA levels were determined by qPCR. The data are represented as the mean ± SEM ( n = 4). * p < 0.05, ** p < 0.01, and *** p < 0.001 are compared with wild type treated with Aβ by Tukey’s test; # p < 0.05 and ### p < 0.001 mean CBD + Aβ with or without siRNA by Student’s t -test. $ p < 0.05 and $$ p < 0.01 mean CBD knockdown of TRPV2 with or without CBD by Student’s t -test.

Article Snippet: The TRPV2 (1:1000, polyclonal antibody, # ALO-ACC-032-50) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation

doi: 10.3390/ijms23105367

Figure Lengend Snippet: Relative mRNA levels of microglial phagocytic receptors and inflammation factors in microglia cells after knockdown of Trem2. BV2 microglia were treated with or without CBD (5 μM) after 12 h, and mRNA levels were determined by qPCR. The data are represented as the mean ± SEM ( n = 4). * p < 0.05, ** p < 0.01, and *** p < 0.001 are compared with wild type treated with Aβ (1 μg/mL) by Tukey’s test; # p < 0.05, ## p < 0.01, and ### p < 0.001 mean CBD + Aβ with or without siRNA by Student’s t -test. $ p < 0.05, $$ p < 0.01, and $$$ p < 0.001 mean CBD knockdown of Trem2 with or without CBD by Student’s t -test.

Article Snippet: The TRPV2 (1:1000, polyclonal antibody, # ALO-ACC-032-50) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Knockdown, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation

doi: 10.3390/ijms23105367

Figure Lengend Snippet: CBD induced autophagy in microglial cells via TRPV2 by promoting the upregulation of Akt. ( A ) BV2 microglia cells incubated with Aβ42 were analyzed using Western blot in the presence of CBD (5 μM) for the indicated time. ( B ) BV2 microglial cells were pretreated with Tra (tranilast 75 μM) for 1 h and then treated with CBD for 24 h. Band densitometry quantification of TRPV2. The autophagy flux expression was normalized to GAPDH. The phosphorylation of Akt was normalized to the total Akt level ( n = 4 per group). * p < 0.05, ** p < 0.01, and *** p < 0.001 are compared with the control by Tukey’s test. # p < 0.05 mean CBD + Aβ with or without siRNA by Student’s t -test.

Article Snippet: The TRPV2 (1:1000, polyclonal antibody, # ALO-ACC-032-50) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Incubation, Western Blot, Expressing, Phospho-proteomics, Control

Journal: International Journal of Molecular Sciences

Article Title: Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation

doi: 10.3390/ijms23105367

Figure Lengend Snippet: CBD improved energy metabolism via mitochondrial functions in microglia cells. ( A ) Total ATP production in BV2 microglia under different conditions as indicated. Aβ (1 μg/mL) and/or CBD (5 μM) were added to the cells for 24 h prior to measurement ( n = 6 from three independent experiments). The data are represented as the mean ± SEM. * p < 0.05 and *** p < 0.001 are compared with the controls by Tukey’s test; ### p < 0.001 means CBD + Aβ and CBD by Student’s t -test; $$ p < 0.01 and $$$ p < 0.001 is CBD with or without Aβ by Student’s t -test. ( B ) Total ATP production in WT and TRPV2 knockdown microglia cells treated with or without CBD for 24 h ( n = 6 from three independent experiments). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the controls by Tukey’s test; ## p < 0.01 is between Aβ and CBD + Aβ by Student’s t -test; $$ p < 0.01 is between Aβ and CBD + Aβ upon siRNA by Student’s t -test. ( C ) BV2 microglial cells were analyzed for Tom 20 expression in mit (mitochondria) and cyt (cytoplasm) by performing immunoblot analysis under different conditions as indicated using the mitochondrial extraction kit. ( D ) Band densitometry quantification of TRPV2 was normalized to GAPDH ( n = 4 per group). ** p < 0.01 and *** p < 0.001 are compared with control by Tukey’s test; # p < 0.05 and ## p < 0.01 are compared with Aβ by Tukey’s test; $ p < 0.05 is between Aβ and CBD + Aβ by Student’s t -test. ( E ) ROS production and ( F ) MMP of BV2 microglial cells under different conditions as indicated. After 1 h of treatment with Rapa (rapamycin, 10 μM), Aβ and/or CBD were added to the cells for 24 h prior to measurement ( n = 12 from three independent experiments). The fluorescence intensities of DCF and TMRE were quantified and expressed as a percentage relative to the controls. The data are represented as the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 are compared with the controls by Tukey’s test; ### p < 0.001 are compared with Aβ by Tukey’s test; $$ p < 0.01 is CBD with or without Aβ and Rapa by Student’s t -test; %%% p < 0.001 is Aβ with or without CBD and Rapa by Student’s t -test.

Article Snippet: The TRPV2 (1:1000, polyclonal antibody, # ALO-ACC-032-50) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Knockdown, Expressing, Western Blot, Analysis, Extraction, Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation

doi: 10.3390/ijms23105367

Figure Lengend Snippet: Primers and their sequences for fluorescence quantitative PCR.

Article Snippet: The TRPV2 (1:1000, polyclonal antibody, # ALO-ACC-032-50) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Fluorescence, Real-time Polymerase Chain Reaction