Journal: International Journal of Molecular Sciences

Article Title: Negative Cellular Effects of Urban Particulate Matter on Human Keratinocytes Are Mediated by P38 MAPK and NF-κB-dependent Expression of TRPV 1

doi: 10.3390/ijms19092660

Figure Lengend Snippet: Urban particulate matter upregulates transient receptor potential vanilloid 1 (TRPV 1) gene expression in human keratinocytes. ( A ) HaCaT cells were transfected with the TRPV 1-Luc reporter along with a Renilla-luciferase expression vector that was driven by a thymidine-kinase promoter using DharmaFECT ® Duo transfection reagent according to the manufacturer’s protocols. After 24 h, the cells were incubated in the presence of the indicated concentrations of UPM for 14 h. The cells were subjected to luciferase reporter assay. p < 0.05 vs. control. ( B – D ) HaCaT cells were incubated with the indicated concentrations of UPM for two days. Western blot analysis for TRPV 1 was performed on the cell lysates ( B ), and the mRNA levels of the indicated genes were measured using quantitative real-time PCR (qRT-PCR) analysis ( C ). The expressed results are relative to the untreated cells after normalization against the GAPDH level. In addition, viability of cells exposed to urban particulate matter (UPM) was measured using WST-1 assay ( D ). * p < 0.05 vs. control. The results were confirmed using four independent experiments. Each experiment was conducted in duplicate. The data are expressed as the mean ± SD.

Article Snippet: The membranes were incubated overnight at 4 °C with an anti-TRPV 1 antibody (Invitrogen, Carlsbad, CA, USA) and an anti-β-actin antibody (Sigma–Aldrich).

Techniques: Expressing, Transfection, Luciferase, Plasmid Preparation, Incubation, Reporter Assay, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, WST-1 Assay

Journal: International Journal of Molecular Sciences

Article Title: Negative Cellular Effects of Urban Particulate Matter on Human Keratinocytes Are Mediated by P38 MAPK and NF-κB-dependent Expression of TRPV 1

doi: 10.3390/ijms19092660

Figure Lengend Snippet: UPM-induced expression of TRPV 1 is mediated through activation of p38 mitogen-activated protein kinase (MAPK) and NF-κB. HaCaT cells were treated with UPM (200 ppm) and then incubated for two days in the presence of the indicated concentration of MAPK and NF-κB inhibitors. ( A ) Total RNA was isolated from the cells, and the mRNA levels of the indicated genes were measured using real-time quantitative RT-PCR. The expressed results are relative to the untreated cells after normalization against the GAPDH level. * p <0.05 vs. the untreated control, o p <0.05 vs. the UPM-treated control. The results were confirmed using four independent experiments. Each experiment was conducted in duplicate. The data are expressed as the mean ± SD. ( B ) The cell lysates were subjected to Western blot analysis for TRPV 1. UPM, urban particulate matter; SB, SB203580; SP, SP600125; PD, PD98059; PDTC, ammonium pyrrolidinedithiocarbamate.

Article Snippet: The membranes were incubated overnight at 4 °C with an anti-TRPV 1 antibody (Invitrogen, Carlsbad, CA, USA) and an anti-β-actin antibody (Sigma–Aldrich).

Techniques: Expressing, Activation Assay, Incubation, Concentration Assay, Isolation, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Negative Cellular Effects of Urban Particulate Matter on Human Keratinocytes Are Mediated by P38 MAPK and NF-κB-dependent Expression of TRPV 1

doi: 10.3390/ijms19092660

Figure Lengend Snippet: Mechanisms involved in the effect of UPM on TRPV 1-induced signaling. UPM induces the activation of p38 MAPK and NF-κB, which sequentially upregulate expression of TRPV1 gene. TRPV1 contributes to the increase of calcium influx, production of cytokines, as well as the decrease of cell proliferation. However, it is not involved in the production of ROS.

Article Snippet: The membranes were incubated overnight at 4 °C with an anti-TRPV 1 antibody (Invitrogen, Carlsbad, CA, USA) and an anti-β-actin antibody (Sigma–Aldrich).

Techniques: Activation Assay, Expressing

Journal:

Article Title: The effects of Sympathetic Outflow on Upregulation of Vanilloid Receptors TRPV 1 in Primary Afferent Neurons Evoked by Intradermal Capsaicin

doi: 10.1016/j.expneurol.2009.12.011

Figure Lengend Snippet: Effects of administration of a P2X purinergic receptor agonist or PKC activator on the baseline staining of TRPV 1 , p-PKC, and CGRP in DRG neurons in the absence of capsaicin injection in naïve, sham-sympathectomized and sympathectomized rats

Article Snippet: Briefly, primary antibodies used included 1) anti-TRPV 1 (1:1,000, guinea-pig polyclonal; Chemicon, Temicula, CA); 2) anti-CGRP (1:1,000, mouse monoclonal; Chemicon, Temicula, CA); and 3) anti-p-PKCα or p-PKCε (1:500, Ser 657 or Ser 729, rabbit; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Staining, Injection

Journal: International Journal of Molecular Sciences

Article Title: Nogo-A Induced Polymerization of Microtubule Is Involved in the Inflammatory Heat Hyperalgesia in Rat Dorsal Root Ganglion Neurons

doi: 10.3390/ijms221910360

Figure Lengend Snippet: KO of Nogo-A decreased the co-localization of TRPV1 with ace-α-tubulin in the CFA- induced inflammatory pain. ( A ) The co-location of ace-α-tubulin and TRPV1 was decreased in the DRG neurons of Nogo-A KO with CFA injection. ( B ) The total TRPV1 (the monomer, glycosylation, and polymerization of TRPV-1) were included for the quantification and it was significantly decreased in DRG of Nogo-A KO rat with CFA injection, compared with that of the WT rat (* p < 0.05, unpaired t -test, n = 5). ( C ) The total TRPV1 was significantly higher in Nogo-A KO rat with CFA injection than that of WT after intrathecal injection of 10 μg paclitaxel, indicating that increase of the polymerization microtubule with paclitaxel could compensate the TRPV1 decrease resulted from the disassembly of microtubule due to KO of Nogo-A (** p < 0.01, unpaired t -test, n = 4). The scale bar in ( A ) is 100 μm.

Article Snippet: The following commercially available antibodies were used as primary antibodies: ace-α-tubulin (1:10,000, sc23950, Santa Cruz Biotechnology, Dallas, Texas, USA), pCRMP2 T514 polyclonal antibody (1:1000, ab62478; Abcam, Waltham, MA, USA) guinea pig anti-TRPV-1 polyclonal antibody (1:1000, AB5566; MilliporeSigma, Burlington, MA, USA), GADPH (1:2000, TA-08, Zhongshan Golden Bridge Biotechnology, Guangdong, China), and β-actin (1:2000, TA-09, Zhongshan Golden Bridge Biotechnology, Guangdong, China).

Techniques: Injection