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

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, 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 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: 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, 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, Sequencing

Journal: The FASEB Journal

Article Title: Identification of two natural coumarin enantiomers for selective inhibition of TRPV2 channels

doi: 10.1096/fj.201901541rrr

Figure Lengend Snippet: FIGURE 2 Inhibition of 2-APB-induced increase of intracellular calcium in TRPV2-transfected HEK-293 cells by enantiomers B304-1 and B304-2. A, left images, calcium imaging of HEK-293 cells over-expressing TRPV2 channels after treatment with B304-1 alone, 2-APB+B304-1, 2-APB alone and ionomycin. Right panel, an average fluorescence intensity induced by B304-1, 2-APB+B304-1, 2-APB, and ionomycin in TRPV2-expressing HEK-293 cells (red circle) and untransfected cells (blue triangle) (n = 52). B, left images, calcium imaging of over-expressing TRPV2 HEK-293 cells after treatment with B304-2, 2-APB+B304-2, 2-APB, and ionomycin. Right panel, an average fluorescence intensity induced by B304-2, 2-APB+B304-2, 2-APB, and ionomycin in TRPV2-expressing HEK-293 cells (red circle) and untransfected cells (blue triangle) (n = 47). C, left images, calcium imaging of HEK293 cells expressing TRPV2 after treatment with tranilast, 2-APB+tranilast, 2-APB, and ionomycin. Right panel, an average fluorescence intensity induced by tranilast, 2-APB+tranilast, 2-APB, and ionomycin in TRPV2-expressing HEK-293 cells (red circle) and untransfected cells (blue triangle) (n = 43). Fluorescence intensity was normalized with the maximal value of ionomycin (10 μM). The test was repeated three times. Scale bar indicates 100 μm. D,E, Left panels, inhibition of intracellular calcium rise in TRPV2-expressing HEK-293 cells by different concentrations of B304-1 and B304-2 in FlexStation3 calcium fluorescence assay, in comparison with the TRPV2 antagonist 20 μM of tranilast and agonist 2-APB (1 mM). Right panels, summary for inhibition ratio of calcium fluorescence by B304-1, B304-2, and tranilast. Data are presented as the means ± SEM. from three independent tests

Article Snippet: TRPV2 antibodies (ACC-039) were purchased from Alomone (Jerusalem, Israel).

Techniques: Inhibition, Transfection, Imaging, Expressing, Fluorescence, Comparison

Journal: The FASEB Journal

Article Title: Identification of two natural coumarin enantiomers for selective inhibition of TRPV2 channels

doi: 10.1096/fj.201901541rrr

Figure Lengend Snippet: FIGURE 3 Dose-dependent and selective inhibition of TRPV2 by B304-1 and B304-2 in HEK-293 cells expressing TRPV2. A, Top panel, Activation of TRPV2 by 2-APB (2 mM), and inhibition of TRPV2 current by B304-1 (30 μM) or B304-2 (30 μM) or tranilast (20 μM). Bottom panel, representative current traces of TRPV2 in response to voltage from −80 to +80 mV. B, Summary for inhibition ratio of TRPV2 current by B304-1 (30 μM), B304-2 (30 μM), and tranilast (20 μM). Data are presented as the means ± SEM. from 5 to 7 independent tests. C, Curve fitting analysis of dose-dependent activation of TRPV2 by 2-APB with an EC50 of 1223.2 ± 32.0 μM (n = 8), each data point is expressed as the mean ± SEM. D, Curve fitting in blue represents dose-dependent inhibition of TRPV2 by B304-1 with an IC50 of 22.2 ± 7.8 μM. Curve fitting in red represents dose-dependent inhibition of TRPV2 by B304-2 with IC50 at 3.7 ± 0.7 μM; Data are expressed as the means ± SEM from 5 to 9 independent tests. E, left panel, whole-cell current of TRPV1 was induced by 1 μM of capsaicin, and co-application of 200 μM of B304-1 or B304- 2 had no inhibition on TRPV1. Right panel, current-voltage curves of TRPV1 in response to voltage ramps from −100 to +100 mV under control condition (0) after addition of 1.0 μM of capsaicin (1), co-addition of 200 μM of B304-1 and 1 μM of capsaicin (2), co-addition of 200 μM of B304-2 and 1 μM of capsaicin (3) and wash out to baseline (4). (F) left panel, TRPV3 current was activated by 200 μM 2-APB, and co-application of B304-1 or B304-2. Right panel, current-voltage curves of TRPV3 in response to voltage ramps from −100 to +100 mV under control condition (0) after addition of 200 μM 2-APB (1), co-addition of 200 μM B304-1 and 200 μM 2-APB (2), co-application of 200 μM B304-2 and 200 μM 2-APB (3) and wash out (4). (G) left panel, activation of TRPV4 was induced by 100 nM of GSK or inhibited by 130 mM BaCl2, in comparison with co-application of 200 μM B304-1 or B304-2 that had no inhibitory effect. Right panel, current-voltage curves of TRPV4 in response to voltage ramps from −100 to +100 mV under control condition (0) after addition of 100 nM of GSK (1), co-addition of 200 μM B304-1 and 100 nM of GSK (2), co-addition of 200 μM B304-2 and 100 nM of GSK (3) and 130 mM BaCl2 (4)

Article Snippet: TRPV2 antibodies (ACC-039) were purchased from Alomone (Jerusalem, Israel).

Techniques: Inhibition, Expressing, Activation Assay, Control, Comparison

Journal: The FASEB Journal

Article Title: Identification of two natural coumarin enantiomers for selective inhibition of TRPV2 channels

doi: 10.1096/fj.201901541rrr

Figure Lengend Snippet: FIGURE 5 Reversal of TRPV2 agonist-induced inhibition of mouse brown adipocyte differentiation by B304-1 and B304-2. A, Oil red O staining and the number of 6-day-differentiated mouse brown adipocytes after pharmacological treatment by 2-APB alone, 2-APB+tranilast, 2-APB+B304-1, and 2-APB+B304-2. The control group indicates differentiation medium with solvent (0.2% of DMSO). Scale bar indicates 100 μm. B,C, Ucp1 (B), Pparγ (C) mRNA levels after various treatment in 6-day-differentiated mouse brown adipocytes. D, Total cDNAs per well after indicated pharmacological treatments in 6-day-differentiated brown adipocytes. Data are the means ± SEM, n = 3. *P < .05, ***P < .001, and ****P < .0001 vs control group; #P < .05, ###P < .001, and ####P < .0001 vs 2-APB group. One-way ANOVA followed by two-tailed t test with Bonferroni correction

Article Snippet: TRPV2 antibodies (ACC-039) were purchased from Alomone (Jerusalem, Israel).

Techniques: Inhibition, Staining, Control, Solvent, Two Tailed Test

Journal: The FASEB Journal

Article Title: Identification of two natural coumarin enantiomers for selective inhibition of TRPV2 channels

doi: 10.1096/fj.201901541rrr

Figure Lengend Snippet: FIGURE 4 TRPV2 expressions in brown pre-adipocytes and adipocytes, and inhibition of TRPV2-mediated current by B304-1 and B304- 2 in differentiated brown adipocytes. A, Real-time PCR analyses of TRPV1, TRPV2, TRPV3, and TRPV4 expressions in differentiated brown adipocytes after 35 thermal cycles. Control (C) lanes indicate the bands from template plasmid cDNAs. Lanes with (+) indicate reverse transcripts of indicated genes, and lanes labeled with (−) indicate reactions without reverse transcription. B, Western blot analysis of TRPV2, TRPV1, and TRPV3 expression in 6-day-differentiated brown adipocytes. C, Real-time PCR analysis of TRPV2 expression in pre-adipocytes (red) and 6-day- differentiated brown adipocytes (blue). mRNA levels are expressed as the means ± SEM. from six independent tests, **P < .01 vs pre-adipocytes. One-way ANOVA followed by two-tailed t test with Bonferroni correction. D, E, Western blot analysis (D) and densitometry quantification (E) of TRPV2 expression in pre-adipocytes, and 6-day-differentiated brown adipocytes. F, Left panels, whole-cell currents of TRPV2 activated by agonist 2 mM 2-APB in pre-adipocytes (top) or 6-day-differentiated brown adipocytes (bottom). Right panels, current-voltage curves from left panels for pre-adipocytes (top) and 6-day differentiated adipocytes (bottom) in response to 2-APB (2 mM) or tranilast (50 µM) at time points indicated by 0, 1, 2, and 3. G,H, Left panels, TRPV2-like currents activated by 2 mM 2-APB and inhibition of the current by co-application of 100 μM B304-1 (G) or co-addition 100 μM B304-2 (H). Right panels, current-voltage curves from left panels in response to voltage ramps from −100 to +100 mV. (G) under control condition (0) after addition of 2 mM 2-APB (1), co-addition of 100 μM B304-1 and 2 mM 2-APB (2), and washout (3). (H) under control condition (0) after addition of 2 mM 2-APB (1), co-addition of 100 μM B304-2 and 2 mM 2-APB (2), and back to baseline (3)

Article Snippet: TRPV2 antibodies (ACC-039) were purchased from Alomone (Jerusalem, Israel).

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Control, Plasmid Preparation, Labeling, Reverse Transcription, Western Blot, Expressing, Two Tailed Test

Journal: The FASEB Journal

Article Title: Identification of two natural coumarin enantiomers for selective inhibition of TRPV2 channels

doi: 10.1096/fj.201901541rrr

Figure Lengend Snippet: FIGURE 6 SAR of murraxocin derivatives indicates 2′-OH is required for TRPV2 inhibition. A, Murraxocin derivatives used in this work. B,C, TRPV2 current was activated by 2 mM 2-APB before application of murraxocin derivative (300 μM) or B304-2 (300 μM)

Article Snippet: TRPV2 antibodies (ACC-039) were purchased from Alomone (Jerusalem, Israel).

Techniques: Inhibition

Journal: The FASEB Journal

Article Title: Identification of two natural coumarin enantiomers for selective inhibition of TRPV2 channels

doi: 10.1096/fj.201901541rrr

Figure Lengend Snippet: FIGURE 7 Putative binding sites for B304-1 and B304-2 in rat TRPV2 channel. A, Side view (up) and top-down (down) view B304-1 with rat TRPV2 subunits. B, A side view (up) and top-down view (down) for B304-2 with rat TRPV2 subunits. C, Residue sequence alignment and comparison of the S5-P-S6 regions between rat TRPV2 and mouse TRPV2 channels. D,E, Current traces of mouse TRPV2 T599A (D) and I600A (E) mutants expressed in HEK-293 cells in responses to 2-APB alone and 2-APB with B304-1 or B304-2 or tranilast

Article Snippet: TRPV2 antibodies (ACC-039) were purchased from Alomone (Jerusalem, Israel).

Techniques: Binding Assay, Residue, Sequencing, Comparison

Journal: Neoplasia (New York, N.Y.)

Article Title: β-Catenin mediated TAM phenotype promotes pancreatic cancer metastasis via the OSM/STAT3/LOXL2 axis

doi: 10.1016/j.neo.2024.101096

Figure Lengend Snippet: β-catenin signaling in TAMs promotes PDAC EMT through OSM production. (A) Heatmap showing differential expression of EMT and stemness-associated genes between control and MCM-treated PDAC cells (n=3). (B) GO and KEGG analysis revealed enriched biological processes in MCM-treated PANC-1 cells, with significant enrichment in wound healing, stem cell maintenance, and JAK/STAT signaling pathways. (C) GSEA demonstrated significant enrichment of OSM signaling, JAK/STAT signaling, proinflammatory and profibrotic pathways. (D) Immunoblotting analysis showing the effects of CTNNB1 or OSM knockdown on EMT markers in MCM-treated PDAC cells (n=3). (E) Immunoblotting analysis and quantification showing the impact of IL6ST knockdown on EMT markers following MCM treatment (n=3). (F) IHC staining showing α-SMA, E-cadherin, and LOXL2 expression in control and MCM-treated Panc02 xenograft tumors. Scale bar, 50 μm. (G) IHC staining showing OSM expression in adjacent normal tissue and tumor regions from PDAC patients (n=3). Scale bar, 50 μm. Results are presented as mean ± SEM. Data were compared using one-way ANOVA (D, E). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes were incubated with the following primary antibodies: ARG1 (ProteinTech Group, Inc.; cat. no 16001-1-AP; 1:4,000), CD206 (ProteinTech Group, Inc.; cat. no 18704-1-AP; 1:500), β-catenin (ProteinTech Group, Inc.; cat. no 51067-2-AP; 1:1,000; Cell Signaling Technology, Inc.; cat. no 8480; 1:1,000), Cyclin D1 (ProteinTech Group, Inc.; cat. no 60186-1-Ig; 1:5,000), c-Myc (ProteinTech Group, Inc.; cat. no 10828-1-AP; 1:2,000), OSM (ProteinTech Group, Inc.; cat. no 27792-1-AP; 1:1,000), E-Cadherin (ProteinTech Group, Inc.; cat. no 20874-1-AP; 1:5,000 or 1:20,000), N-Cadherin (ProteinTech Group, Inc.; cat. no 22018-1-AP; 1:2,000), Vimentin (ProteinTech Group, Inc.; cat. no 10366-1-AP; 1:5,000), Snail (ProteinTech Group, Inc.; cat. no 1:500), LOXL2 (ProteinTech Group, Inc.; cat. no 67139-1-Ig; 1:1,000), p-STAT3 Tyr705 (BIOSS; cat. no bsm-33301M; 1:1,000), STAT3 (ProteinTech Group, Inc.; cat. no 10253-2-AP; 1:2,000), ZEB1 (ProteinTech Group, Inc.; cat. no 10253-2-AP; 1:2,000), ZO-1 (Cell Signaling Technology, Inc.; cat. no 8193; 1:1,000), CD163 (ABclonal; cat. no A26411PM; 1:1,000), CD204 (ABclonal; cat. no A2401; 1:1,000), NLRP3 (ABclonal; cat. no A24833; 1:1,000), and β-actin (ProteinTech Group, Inc.; cat. no 81115-1-RR; 1:5,000).

Techniques: Quantitative Proteomics, Control, Protein-Protein interactions, Western Blot, Knockdown, Immunohistochemistry, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Oncostatin M induced by STAT5-activating oncogenes promotes disease progression in hematologic malignancies

doi: 10.1038/s41392-025-02491-6

Figure Lengend Snippet: Treatment of JAK2 p.V617F + animals with an anti-OSM receptor fusion protein or monoclonal anti-OSM antibody ameliorates polycythemia and osteosclerosis. a Structure of the anti-OSM receptor fusion protein (“anti-OSMR-FP”): the extracellular domains (D) 1–4 of the murine OSMR receptor were fused to D2–3 of murine gp130 and C-terminally Fc- and HA-tagged. , , Figure generated with BioRender. b Outline of the in vivo anti-OSMR-FP treatment: Intraperitoneal injections (6 µg/g body weight) were administered every two days from day 7 posttransplantation and maintained for 30 days in Jak2 p.V617F + BM recipients. N = 5 for each treatment group. c , d Hemoglobin (HGB) in PBS-, control peptide-, and anti-OSMR-FP-treated animals one month after treatment ( c ) and over time ( d ). N = 5 for each treatment group. * p < 0.05. The data are presented as the means ± SEMs. e Assessment of GM-CSF levels via bead-based flow cytometry in mice 10 weeks after BM transplantation. * p < 0.05. f Hematoxylin/eosin staining of bone specimens from JAK2 p.V617F + mice treated with control peptide (left) or anti-OSMR-FP (right). g Osteosclerosis score. N ≥ 4. * p < 0.05. Statistical analysis was conducted via the Mann‒Whitney test. The data are presented as the means ± SEMs. h Outline of the in vivo treatment of mice transplanted with Jak2 p.V617F + BM with a rat-derived monoclonal anti-mOSM antibody (αOSM). Intraperitoneal injections started on day 7 posttransplantation and were administered weekly for a total of four injections. N = 4 for each treatment group. i , j Hemoglobin (HGB) ( i ) and white blood cell count (WBC) ( j ) in isotype- vs. αOSM-treated animals, as assessed one month after BM transplantation. N = 4 vs. 4. * p < 0.05. The data are presented as the means ± SEMs. k Evaluation of GM-CSF levels on day 80 posttransplantation via bead-based flow cytometry. * p < 0.05. The data are presented as the means ± SEMs

Article Snippet: Another cohort of mice received treatment with a rat-derived monoclonal anti-mOSM antibody (MAB4951; R&D Systems, Minneapolis, MN, USA) at a dose of 1 μg/g body weight or a corresponding rat IgG 2B (clone 823157, R&D) starting one week after transplantation by weekly i.p. injection.

Techniques: Generated, In Vivo, Control, Flow Cytometry, Transplantation Assay, Staining, Derivative Assay, Cell Counting

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 3. Normal human epidermal keratinocytes (NKs) secrete oncostatin M (OSM) upon keratinocyte growth factor (KGF) stimulation inducing fibroblast activation. (a) The OSM messenger RNA (mRNA) expression analyzed in NKs after 24hours treatment with recombinant KGF compared with untreated NKs and kFCM and sFCM treatment compared with incubation with nFCM. The graph represents mean values of three independent experiments, and error bars represent the mean±SD of these experiments with n ¼ 3 for each sample analyzed with analysis of variance (ANOVA) þ Kruskal–Wallis test. (b) OSM protein measured by ELISA secreted by NKs upon direct treatment with recombinant KGF (n ¼ 6) compared with untreated NKs (n ¼ 7) and upon incubation with kFCM and sFCM compared with nFCM (each n ¼ 5). The graph represents mean values of two independent experiments analyzed with ANOVAþ Tukey test. (c) Collagen type I-a1 (ColIa1) and (d) fibroblast activation protein (FAP) mRNA expression in normal fibroblasts (NFs), KFs, and scleroderma fibroblasts (SFs) with and without OSM treatment. (e) Fibroblast migration after treatment with recombinant OSM (10ng ml 1). (f) OSM mRNA expression upon KGF-treatment in NKs transfected with control small interfering RNA (CTRL siRNA) and siRNA against KGFR (each n ¼ 3). The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (g) The OSM mRNA expression in keloid tissue (n ¼ 4) and scleroderma tissue (n ¼ 4) compared with normal skin (n ¼ 5). The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with ANOVA þ Kruskal–Wallis test.

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Activation Assay, Expressing, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Migration, Transfection, Control, Small Interfering RNA, MANN-WHITNEY

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 4. STAT3 is activated in fibroblasts upon keratinocyte growth factor (KGF)–induced oncostatin M (OSM) secretion. Western blot analyis was performed for STAT3 and phosphorylated STAT3 (pSTAT3) using b-actin as a loading control. (a) STAT3 is constitutively expressed in normal fibroblasts (NFs) but phosphorylation only occurs upon direct OSM treatment or treatment with OSM-containing keratinocyte-conditioned media (KCMs) as confirmed by the OSM ELISA in Figure 3c. (b) Direct KGF treatment had no unspecific effect on STAT3 phosphorylation. NFs showed decreased STAT3 phosphorylation when treated with KCM:kFCM:siKGF compared with KCM:kFCM:siCTRL, but increased phosphorylation after treatment KCM:nFCM:KGF compared with KCM:nFCM:mock. (c) pSTAT3 and STAT3 expression status in NFs, keloid fibroblasts (KFs), and scleroderma fibroblasts (SFs) upon treatment with recombinant OSM. (d) pSTAT3 and STAT3 expression in NFs treated with rOSM and KCM in the presence or absence of OSM-neutralizing antibodies ( þ antiOSM).

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Western Blot, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Recombinant

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 5. STAT3 mediates fibroblast activation. (a) Collagen type I-a1 (ColIa1) expression and (b) fibroblast migration cannot be induced upon oncostatin M (OSM) stimulation when pretreated with the STAT3 inhibitor S3I-201. The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (c) The expression of the STAT3 target gene urokinase-type plasminogen activator (uPA) is induced upon direct OSM treatment and upon incubation with KCM (keratinocyte-conditioned medium):kFCM, KCM:sFCM, and KCM:rKGF. The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with analysis of variance (ANOVA)þ Kruskal–Wallis test and Mann–Whitney test, respectively. (d) uPA mRNA expression in normal fibroblasts (NFs), keloid fibroblasts (KFs), and scleroderma fibroblasts (SFs) upon treatment with recombinant OSM. (e) The induction of fibroblast migration measured upon OSM treatment and incubation with KCM:kFCM or KCM:sFCM is completely suppressed when the uPA expression is decreased by means of small interfering RNA transfection. The graphs in d and e represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (f) The uPA expression is significantly higher in keloid and scleroderma tissue compared with normal skin. The graphs represent mean values of four independent experiments, and error bars represent the mean±SD of these experiments analyzed with ANOVAþ Kruskal–Wallis test. (g) OSM-induced STAT3-mediated uPA expression can be suppressed by pretreatment with S3I-201. The graph represents mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test.

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Activation Assay, Expressing, Migration, MANN-WHITNEY, Incubation, Recombinant, Small Interfering RNA, Transfection