Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: Cytosolic and mitochondrial calcium concentration of TRPA1- or TRPV1-positive DRG neurons during exposure to 10 µM cisplatin. ( A ) Immunostaining of transient receptor potential ankyrin 1 (TRPA1)- or transient receptor potential vanilloid 1 (TRPV1)-positive DRG neurons and live imaging of Fluo-4 (green) and Rhod-2 (red). ( B ) The cytosolic calcium of TRPV1-positive DRG neurons concentration instantly increased to 1.067 ± 0.018 (* p < 0.05) after 5 min and continuously increased to 1.394 ± 0.068 (** p < 0.01) after 70 min. The mitochondrial calcium concentration declined to 1.095 ± 0.009 (* p < 0.05) after 5 min for TRPV1-positive DRG neurons and steadily declined to 0.577 ± 0.044 after 70 min (** p < 0.01). ( C ) During the exposure of 10 µM cisplatin, the cytosolic calcium concentration of TRPA1-positive neurons increased after 5 min to 1.024 ± 0.011 (** p < 0.01) until 70 min at 1.240 ± 0.074 (*** p < 0.001). The mitochondrial calcium concentration was lower after 10 min at 0.928 ± 0.029 (*** p < 0.001) until it reached 0.790 ± 0.050 at 70 min (*** p < 0.001). ( D ) The cytosolic calcium concentration of TRPV1-positive DRG neurons was increased after 15 min to 65 min compared to TRPA1-positive DRG neurons (* p < 0.05, ** p < 0.01). ( E ) The mitochondrial calcium concentration of TRPA1- or TRPV1-positive DRG neurons was different after 5 min until the end of the experiment (* p < 0.05, ** p < 0.01). TRPV1-positive sensory neurons had a lower mitochondrial calcium concentration during exposure to 10 µM cisplatin after 15 min (* p < 0.05, ** p < 0.01). n = 6 cells per condition. * = significant effect between TRPA1+ and TRPV1+ DRG neurons. Scale = 50 µm.

Article Snippet: The cells were first stained with extracellular TRPA1 or TRPV1 antibodies (Alomone Labs, Jerusalem, Israel).

Techniques: Concentration Assay, Immunostaining, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: Cytosolic and mitochondrial calcium concentration of TRPA1- or TRPV1-positive DRG neurons during exposure to 10 µM oxaliplatin. ( A ) Immunostaining of TRPA1- or TRPV1-positive DRG neurons and live imaging of Fluo-4 (green) and Rhod-2 (red) before and after application of 10 µM cisplatin or oxaliplatin. ( B ) TRPA1-positive DRG neurons showed an immediate increase of the cytosolic calcium concentration to 1.037 ± 0.006 (** p < 0.01) after 5 min and continuously increased to 1.372 ± 0.062 after 70 min (*** p < 0.001). The mitochondrial calcium concentration decreased after 5 min to 0.967 ± 0.008 (*** p < 0.001) and declined to 0.712 ± 0.010 after 70 min (*** p < 0.001). ( C ) The TRPV1-positive DRG neurons showed an immediate increase of the cytosolic calcium concentration to 1.049 ± 0.005 (*** p < 0.001) after 5 min and increased to 1.480 ± 0.034 after 70 min (*** p < 0.001). The mitochondrial calcium concentration decreased to 0.959 ± 0.020 (* p < 0.05) after 5 min and to 0.725 ± 0.077 (** p < 0.01) after 70 min (* p < 0.05). ( D ) No difference could be determined in the cytosolic calcium concentration of TRPA1- or TRPV1-positive DRG neurons ( p > 0.05). ( E ) No difference could be determined in the mitochondrial calcium concentration of TRPA1- or TRPV1-positive DRG neurons ( p > 0.05). n = 6 cells per condition. * = significant effect between TRPA1+ and TRPV1+ DRG neurons. Scale = 50 µm.

Article Snippet: The cells were first stained with extracellular TRPA1 or TRPV1 antibodies (Alomone Labs, Jerusalem, Israel).

Techniques: Concentration Assay, Immunostaining, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: Relative cytosolic and mitochondrial calcium concentration during exposure to 10 µM cis- or oxaliplatin of TRPA1- or TRPV1-positive DRG neurons. ( A ) No differences in the cytosolic calcium concentration of TRPA1-positive DRG neurons after exposure to 10 µM cis- or oxaliplatin could be determined ( p > 0.05). ( B ) The cytosolic calcium of TRPV1-positive DRG neurons concentration during exposure to cis- or oxaliplatin showed no difference ( p > 0.05). ( C ) During exposure to 10 µM cis- or oxaliplatin, no difference in the mitochondrial calcium concentration of TRPA1-positive DRG neurons could be determined ( p > 0.05). ( D ) While exposing TRPV1-positive DRG neurons, differences between the two chemotherapeutics on the mitochondrial calcium concentration could be determined after 20 min. The mitochondrial calcium concentration of TRPV1-positive DRG neurons exposed to oxaliplatin was higher at 0.856 ± 0.033 compared to 0.727 ± 0.040 (* p < 0.05). The mitochondrial calcium concentration was higher for oxaliplatin-exposed TRPV1-positive DRG neurons until 0.786 ± 0.058 after 40 min (* p < 0.05). n = 6 cells per condition. * = significant effect between cis- and oxaliplatin, ** p < 0.01

Article Snippet: The cells were first stained with extracellular TRPA1 or TRPV1 antibodies (Alomone Labs, Jerusalem, Israel).

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: ROS production of TRPA1- or TRPV1-positive DRG neurons during exposure to 10 µM cisplatin. ( A ) Fluorescence of CellRox (green) in TRPA1- or TRPV1-positive DRG neurons during exposure to 10 µM cisplatin at different time points. ( B ) TRPA1-positive sensory neurons showed an instant increase of the ROS production to 1.431 ± 0.042 (*** p < 0.001) after 5 min and reached its peak at 2.100 ± 0.197 after 30 min (*** p < 0.001). After 50 min, the ROS production dropped below the control level at 50 min to 0.607 ± 0.036 (*** p < 0.001) and further declined to 0.370 ± 0.035 (*** p < 0.001) after 70 min. ( C ) After exposure to 10 µM cisplatin of the TRPV1-positive DRG neurons, the ROS production increased to 1.238 ± 0.042 (*** p < 0.001) after 5 min and dropped below control level at 50 min at 0.873 ± 0.068 (* p < 0.05) and steadily declined to 0.756 ± 0.072 (** p < 0.01) after 70 min. n = 6 cells per condition. * = significant effect compared to control level. Scale = 50 µm.

Article Snippet: The cells were first stained with extracellular TRPA1 or TRPV1 antibodies (Alomone Labs, Jerusalem, Israel).

Techniques: Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: ROS production of TRPA1- or TRPV1-positive DRG neurons during exposure to 10 µM oxaliplatin. ( A ) Fluorescence of CellRox (green) of TRPA1- or TRPV1-positive DRG neurons at different time points. ( B ) The ROS production of TRPA1-positive DRG neurons increased after 5 min to 1.421 ± 0.103 (** p < 0.01) during exposure to 10 µM oxaliplatin and dropped to control level after 40 min at 1.31 ± 0.194 ( p > 0.05). ( C ) The TRPV1-positive sensory neurons had an increased ROS production after 5 min at 1.080 ± 0.020 (** p < 0.01) and dropped to the control level after 10 min at 1.033 ± 0.036 ( p > 0.05). After 30 min, the ROS production declined below the control level at 0.810 ± 0.079 (* p < 0.05) and steadily declined to 0.429 ± 0.050 after 70 min (*** p < 0.001). n = 6 cells per condition. * = significant effect compared to control level. Scale = 20 µm.

Article Snippet: The cells were first stained with extracellular TRPA1 or TRPV1 antibodies (Alomone Labs, Jerusalem, Israel).

Techniques: Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: The ROS production of TRPA1- or TRPV1-positive DRG neurons during exposure to cis- or oxaliplatin. ( A ) A difference could be determined for TRPA1-positive DRG neurons at 30 min with a higher ROS production for cisplatin-exposed DRG neurons at 2.100 ± 0.197 (* p < 0.05). After 45 min (1.194 ± 0.223; * p < 0.05) until 70 min (1.022 ± 0.233; * p < 0.05), the ROS production was higher for oxaliplatin-exposed neurons. ( B ) The ROS production of TRPV1-positive DRG neurons was higher for cisplatin-exposed neurons over the entire experimental period (* p < 0.05, ** p < 0.01). n = 6 cells per condition. * = significant effect compared to control level.

Article Snippet: The cells were first stained with extracellular TRPA1 or TRPV1 antibodies (Alomone Labs, Jerusalem, Israel).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: Antibodies and dilution used for the TRPA1- and TRPV1-positive staining.

Article Snippet: The cells were first stained with extracellular TRPA1 or TRPV1 antibodies (Alomone Labs, Jerusalem, Israel).

Techniques: Staining

Journal: Pain Research & Management

Article Title: Exploring the Analgesic Initiation Mechanism of Tuina in the Dorsal Root Ganglion of Minor CCI Rats via the TRPV1/TRPA1-cGMP Pathway

doi: 10.1155/2024/2437396

Figure Lengend Snippet: Primary and secondary antibodies used for Western blotting.

Article Snippet: Primary , Rabbit anti-TRPA1 polyclonal IgG , 1 : 1000 , Proteintech Group, Chicago, USA.

Techniques: Western Blot, Concentration Assay

Journal: PLoS ONE

Article Title: Pharmacogenetic inhibition of lumbosacral sensory neurons alleviates visceral hypersensitivity in a mouse model of chronic pelvic pain

doi: 10.1371/journal.pone.0262769

Figure Lengend Snippet: Primary and secondary antibodies used.

Article Snippet: Rabbit polyclonal anti-TRPA1 , St John’s Laboratory , STJ193121 , 1:100.

Techniques:

Journal: Molecular Neurodegeneration

Article Title: TRPA1 channels promote astrocytic Ca 2+ hyperactivity and synaptic dysfunction mediated by oligomeric forms of amyloid-β peptide

doi: 10.1186/s13024-017-0194-8

Figure Lengend Snippet: TRPA1 channels mediate astrocytic hyperactivity. a Immunohistochemistry of mouse stratum radiatum astrocytes showing that TRPA1 channels expression ( green ) was located in GFAP-positive processes ( magenta ) but also went over in distal processes excluding GFAP staining (e.g. white arrowheads ). b , c . Within the astrocytic population, proportion of astrocytes displaying calcium activity and frequency of astrocyte calcium activity in physiological condition ( light grey ; n = 43), under 100 nM Aβo application ( orange ; n = 12), 40 μM HC 030031 application ( dark grey ; n = 7) and 100 nM Aβo + 40 μM HC 030031 co-application ( cyan ; n = 10). d Frequency histogram revealing that Aβo-induced hyperactivity is reduced when HC 030031 is co-applied ( cyan ). Related theoretical Poisson distributions were shown accordingly. e , f . Within single astrocyte arbor, proportion of subregions displaying calcium activity and frequency of astrocyte calcium activity in physiological condition ( light grey ; n = 7), under 100 nM Aβo application ( orange ; n = 13), 40 μM HC 030031 application ( dark grey ; n = 7) and 100 nM Aβo + 40 μM HC 030031 co-application ( cyan ; n = 5). Results are compared with the physiological condition with *, p < 0.05; **, p < 0.01 and ***, p < 0.001 or the Aβo condition with #, p < 0.05; ##, p < 0.01 and ###, p < 0.001

Article Snippet: Tissue sections were then incubated overnight at 4 °C with either an anti-NeuN antibody (AbCys, France, mouse monoclonal; 1:500), anti-GFAP antibody (Molecular Probes, USA, mouse monoclonal; 1:1000), anti-TRPA1 antibody (Novus, USA, rabbit polyclonal; 1:100) or anti-Iba-1 antibody (Wako, USA, rabbit polyclonal; 1:500).

Techniques: Immunohistochemistry, Expressing, Staining, Activity Assay

Journal: Molecular Neurodegeneration

Article Title: TRPA1 channels promote astrocytic Ca 2+ hyperactivity and synaptic dysfunction mediated by oligomeric forms of amyloid-β peptide

doi: 10.1186/s13024-017-0194-8

Figure Lengend Snippet: Young APP/PS1–21 mice are devoid of amyloid deposit and reactive astrocyte whereas gradually overexpress TRPA1. a Thioflavin S staining for β-amyloid deposits ( green ) and NeuN immunostaining ( magenta ) in the hippocampus of 1-month-old ( left ) and 6-month-old ( right ) APP/PS1–21 mice showing the progression of the number of amyloid deposits. b GFAP ( cyan ) and NeuN ( magenta ) immunostainings in the stratum radiatum of a P30 WT ( left ) and APP/PS1–21 ( right ) mice. c Western-blot analysis of protein levels of TRPA1 channels in hippocampus extracts from P19 and P30 WT and APP/PS1–21 mice (3 different extracts of P30 WT and APP/PS1–21 mice are shown). Histogram showing quantification of TRPA1 channels expression normalized to protein loading levels ( n = 6 hippocampus in each group at P19 and 8 hippocampus in each group at P30). d Western-blot analysis of protein levels of GFAP in hippocampus extracts from P19 and P30 WT and APP/PS1–21 mice (extracts of same P30 lysates depicted in c are shown). Histogram showing quantification of GFAP expression normalized to protein loading levels ( n = 7 hippocampus in each group at P19 and P30). Results are compared with the WT mice with *, p < 0.05; **, p < 0.01 and ***, p < 0.001

Article Snippet: Tissue sections were then incubated overnight at 4 °C with either an anti-NeuN antibody (AbCys, France, mouse monoclonal; 1:500), anti-GFAP antibody (Molecular Probes, USA, mouse monoclonal; 1:1000), anti-TRPA1 antibody (Novus, USA, rabbit polyclonal; 1:100) or anti-Iba-1 antibody (Wako, USA, rabbit polyclonal; 1:500).

Techniques: Staining, Immunostaining, Western Blot, Expressing

Journal: Molecular Neurodegeneration

Article Title: TRPA1 channels promote astrocytic Ca 2+ hyperactivity and synaptic dysfunction mediated by oligomeric forms of amyloid-β peptide

doi: 10.1186/s13024-017-0194-8

Figure Lengend Snippet: Aβo increases the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) from CA1 pyramidal neurons in a TRPA1 channels dependent manner. a Representative traces of voltage-clamp recordings from CA1 pyramidal cells held at −65 mV in physiological condition ( grey ) and under Aβo 100 nM application ( orange ). Examples of single EPSC with higher time resolution are shown in the corresponding insets. b Time course of the frequency of sEPSCs in physiological condition ( light grey ; n = 5), under application of 100 nM Aβo ( orange ; n = 7), 40 μM HC 030031 ( dark grey ; n = 5) or co-application of 100 nM Aβo + 40 μM HC 030031 ( cyan ; n = 4). c Histogram showing the sEPSCs frequency at a time matching the astrocyte calcium activity measurements (i.e. 5 to 10 min after drugs application). Results are compared with the physiological condition with *, p < 0.05; **, p < 0.01 and ***, p < 0.001 or the Aβo condition with #, p < 0.05; ##, p < 0.01 and ###, p < 0.001. d Representative traces of voltage-clamp recordings from CA1 pyramidal cells held at −65 mV in APP/PS1–21 mice ( orange ) and their WT littermates ( grey ). Examples of single EPSC with higher time resolution are shown in the corresponding insets. e Time course of the normalized frequency of sEPSCs in WT ( dark grey ; n = 7) and APP/PS1–21 mice ( cyan ; n = 8) under application of 40 μM HC 030031. f Histogram showing the sEPSCs frequency in basal condition in WT ( light grey ; n = 7), APP/PS1–21 mice ( orange ; n = 8) and 5 to 10 min after HC 030031 application ( dark grey and cyan respectively). Results are compared to the WT basal activity with *, p < 0.05; **, p < 0.01 and ***, p < 0.001 or the APP/PS1–21 basal activity with #, p < 0.05; ##, p < 0.01 and ###, p < 0.001

Article Snippet: Tissue sections were then incubated overnight at 4 °C with either an anti-NeuN antibody (AbCys, France, mouse monoclonal; 1:500), anti-GFAP antibody (Molecular Probes, USA, mouse monoclonal; 1:1000), anti-TRPA1 antibody (Novus, USA, rabbit polyclonal; 1:100) or anti-Iba-1 antibody (Wako, USA, rabbit polyclonal; 1:500).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Transient Receptor Potential Ankyrin 1 Channel Localized to Non-Neuronal Airway Cells Promotes Non-Neurogenic Inflammation

doi: 10.1371/journal.pone.0042454

Figure Lengend Snippet: ( A ) Total RNAs were extracted from primary small airways epithelial cells (SAEC), human type II alveolar epithelial cells (A549), human primary smooth muscle cells (HBSMC), human embryonic lung fibroblasts (IMR90) and primary normal human lung fibroblasts (NHLF) and relative TRPA1 mRNA amounts were measured by Taqman Real-Time PCR assay. are normalized to the reference gene, β-actin. Each column represents mean ± SEM of n>2 independent experiments. Immunohistochemical analysis of TRPA1 expression in samples taken from human ( B ) or Trpa1 +/+ and Trpa1 −/− mouse airways and lung ( C ). Representative images of TRPA1 immunostaining show intense staining in epithelial and smooth muscle cells in human tissue. No staining is detected in human samples incubated with the normal serum peptide (Negative control). ( C ) Incubation with TRPA1 antibody shows a strong staining in epithelial and smooth muscle cells in tissues taken from Trpa1 +/+ mice, but not in those from Trpa1 −/− mice. Preadsorption of the TRPA1 antibody with the peptide used for immunization abolished staining (Peptide). Staining for cytokeratin and α-smooth muscle actin (α-SMA) overlaps with the TRPA1 staining in the bronchial epithelium and smooth muscle layer in serial section of human and mice airways/lung tissues ( B and C ). Scale bar 100 µm.

Article Snippet: In another set of experiments, Trpa1 +/+ or Trpa1 −/− mice were perfused via the right ventricle with 20 ml of PBS, and lung and trigeminal ganglia were removed in 10% formalin, and after embedding in paraffin, 4 μm sections were immunohistochemically stained with rabbit polyclonal TRPA1 antibody (1∶500, 2 hours 37°C, AVIVA System Biology) which recognizes the middle region of the protein, (residues 540–589) or with rabbit polyclonal TRPV1 antibody (1∶200, overnight 4°C, Alomone Labs).

Techniques: Real-time Polymerase Chain Reaction, Immunohistochemical staining, Expressing, Immunostaining, Staining, Incubation, Negative Control

Journal: PLoS ONE

Article Title: Transient Receptor Potential Ankyrin 1 Channel Localized to Non-Neuronal Airway Cells Promotes Non-Neurogenic Inflammation

doi: 10.1371/journal.pone.0042454

Figure Lengend Snippet: Potency (EC 50 and CI) of the various TRPA1 agonists in different non-neuronal cell types of the human respiratory tract.

Article Snippet: In another set of experiments, Trpa1 +/+ or Trpa1 −/− mice were perfused via the right ventricle with 20 ml of PBS, and lung and trigeminal ganglia were removed in 10% formalin, and after embedding in paraffin, 4 μm sections were immunohistochemically stained with rabbit polyclonal TRPA1 antibody (1∶500, 2 hours 37°C, AVIVA System Biology) which recognizes the middle region of the protein, (residues 540–589) or with rabbit polyclonal TRPV1 antibody (1∶200, overnight 4°C, Alomone Labs).

Techniques:

Journal: PLoS ONE

Article Title: Transient Receptor Potential Ankyrin 1 Channel Localized to Non-Neuronal Airway Cells Promotes Non-Neurogenic Inflammation

doi: 10.1371/journal.pone.0042454

Figure Lengend Snippet: Intracellular calcium response was used to assess agonist-induced TRPA1 activation in small airways epithelial cells (SAEC) ( A ), normal human lung fibroblasts (NHLF) ( B ) and bronchial smooth muscle cells (HBSMC) ( C ). Typical traces and pooled data of the concentration-dependent calcium response evoked by the selective TRPA1 agonists, cinnamaldehyde (CNM, typical traces and black circles) and acrolein (ACR, grey circles), in all different cell types in primary culture. Similarly to CNM and ACR, cigarette smoke extract (CSE, black triangles) produces in all the different types of cells a concentration-dependent calcium response. Responses to CNM, ACR and CSE are prevented by selective TRPA1 antagonists, HC-030031 (HC, 10 µM) and AP18 (10 µM). The activating peptide (SLIGKV-NH 2 ) of the PAR-2 receptor (PAR-2 AP, 100 µM) elicits a calcium response that is not modified by TRPA1 antagonists. Veh is a combination of vehicles of HC and AP18. Values represent mean ± SEM of n>25 cells. * P <0.05 vs. Veh.

Article Snippet: In another set of experiments, Trpa1 +/+ or Trpa1 −/− mice were perfused via the right ventricle with 20 ml of PBS, and lung and trigeminal ganglia were removed in 10% formalin, and after embedding in paraffin, 4 μm sections were immunohistochemically stained with rabbit polyclonal TRPA1 antibody (1∶500, 2 hours 37°C, AVIVA System Biology) which recognizes the middle region of the protein, (residues 540–589) or with rabbit polyclonal TRPV1 antibody (1∶200, overnight 4°C, Alomone Labs).

Techniques: Activation Assay, Concentration Assay, Modification

Journal: PLoS ONE

Article Title: Transient Receptor Potential Ankyrin 1 Channel Localized to Non-Neuronal Airway Cells Promotes Non-Neurogenic Inflammation

doi: 10.1371/journal.pone.0042454

Figure Lengend Snippet: ( A ) Intratracheal instillation (i.t., 30 µl) of substance P (SP, 25 nM), capsaicin (CPS, 100 µM) in C57BL/6 mice increases Evans blue dye extravasation (PPE) in trachea and bronchi an effect prevented by L-733,060 (NK1 receptor antagonist, NK1-A; 2 µmol/kg, i.v.) and capsazepine (CPZ; 30 mg/kg, i.p.) but not by HC-030031 (HC; 300 mg/kg, i.g.). ( B ) L-733,060 and HC, but not CPZ prevent acrolein (ACR, 5 mM, i.t.)- and ( C ) cigarette smoke extract (CSE, 1 OD, i.t.)-evoked increase in PPE in C57BL/6 mouse trachea and bronchi. Each column represents mean ± SEM of at least 5 mice. Veh is the vehicle of the various agonists. § P <0.05 vs. Veh; * P <0.05 vs. treated group. ( D ) Cigarette smoke (CS)-evoked increase in KC in BAL from Trpa1 +/+ is reduced in Trpa1 −/− mice. Each column represents mean ± SEM. § P <0.05 vs. air-exposed Trpa1 +/+ mice . # P <0.05 vs. Trpa1 +/+ CS group. ( E ) ACR (5 mM, i.t.), but not CPS (100 µM, i.t.) or SP (25 nM, i.t.) increases KC release in BAL. HC, but not L-733,060, reduces the increase in KC release by ACR ( E ) or CSE instillation (1 OD, i.t.) ( F ). ( G ) ACR (5 mM, i.t.)-evoked increase in KC in BAL is practically absent in BAL of Trpa1 −/− mice. Resiniferatoxin-desensitization (RTX-DES) abolished PPE-induced by ACR (5 mM, i.t.) ( H ), while did not modify the ACR-evoked KC release ( I ). Veh-DES is the vehicle of RTX. Each column represents mean ± SEM of at least 5 mice. § P <0.05 vs. Veh or Veh- Trpa1 +/+ ; * P <0.05 vs. treated group; # P <0.05 vs. ACR- Trpa1 +/+ .

Article Snippet: In another set of experiments, Trpa1 +/+ or Trpa1 −/− mice were perfused via the right ventricle with 20 ml of PBS, and lung and trigeminal ganglia were removed in 10% formalin, and after embedding in paraffin, 4 μm sections were immunohistochemically stained with rabbit polyclonal TRPA1 antibody (1∶500, 2 hours 37°C, AVIVA System Biology) which recognizes the middle region of the protein, (residues 540–589) or with rabbit polyclonal TRPV1 antibody (1∶200, overnight 4°C, Alomone Labs).

Techniques:

Journal: Cell Death & Disease

Article Title: TRPA1 promotes overactive bladder progression by activating the NLRP3 inflammasome and driving pyroptosis

doi: 10.1038/s41419-026-08426-5

Figure Lengend Snippet: A Void spot assay was utilized to assess urination behavior, specifically measuring the frequency of urine points in mice (n = 14). B Urodynamic test was conducted to monitor key bladder parameters, including intervoid intervals and bladder compliance in mice ( n = 5). C Urodynamic test in rats to monitor key bladder parameters ( n = 5). D The volcano plot illustrates the DEGs in the bladders of Ctrl and CYP-induced OAB mice. E The Venn diagram depicts the overlap of genes associated with OAB, LUTS, and IC, as well as DEGs in our RNA-seq analysis. F Heatmap showcases the expression of common genes obtained from E in Ctrl and CYP-induced OAB mice. Colors represent Z-score of normalized expression. G , H RT-qPCR analyses were employed to quantify Trpa1 mRNA levels in the bladder tissues of mice and rats, respectively ( n = 6). I IHC staining was utilized to assess TRPA1 expression in bladder tissue of mice. J–M Western blot analysis quantified the protein levels of TRPA1 in the bladders of both mice and rats ( n = 6). N Immunofluorescence co-localization assay identified TRPA1 localization at the plasma membrane of 5637 cells, where Na/K-ATPase staining (plasma membrane) is shown in red, TRPA1 in green, and DAPI (nuclei) in blue. O RT-qPCR was utilized to determine TRPA1 mRNA levels in human urine sediment cells. P The correlations between TRPA1 mRNA levels and both the total OABSS and its individual components (including urge urinary incontinence, urgency, and nocturia scores) were analyzed using nonparametric Spearman’s correlation. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 or ns (not significant) using unpaired t test.

Article Snippet: After treatment of antigen retrieval, add 3% hydrogen peroxide (Sigma, USA) solution to the bladder sections (25 min, room temperature), blocked (3% BSA, Solarbio Life Sciences, China, 30 min, room temperature) and incubated overnight at 4 °C with the primary antibody against TRPA1 (1:200, Novus Biologicals, USA).

Techniques: Spot Test, RNA Sequencing, Expressing, Quantitative RT-PCR, Immunohistochemistry, Western Blot, Immunofluorescence, Clinical Proteomics, Membrane, Staining

Journal: Cell Death & Disease

Article Title: TRPA1 promotes overactive bladder progression by activating the NLRP3 inflammasome and driving pyroptosis

doi: 10.1038/s41419-026-08426-5

Figure Lengend Snippet: A RT-qPCR analysis was conducted to quantify the mRNA levels of TRPA1 in 5637 cells ( n = 3). B Western blot analysis assessed TRPA1 protein expression in 5637 cells ( n = 3). (C) Immunofluorescence co-localization and line scan were performed to evaluate TRPA1 expression at the plasma membrane of cells, with red representing Na/K-ATPase staining, green indicating TRPA1 staining, and blue denoting nuclear staining ( n = 30). White lines indicate the locations used for line scan analysis. D–F RT-qPCR assays were carried out to measure the mRNA levels of IL6 , IL1A , and IL1B in cells ( n = 3). G Propidium iodide (PI) staining was utilized to assess the viability of cells. H LDH assay was performed to further evaluate cell viability. I RT-qPCR was conducted to quantify the mRNA levels of Trpa1 in mice’ bladder tissue ( n = 5). J IHC staining was performed to detect TRPA1 expression in mouse bladder. K Western blot analysis quantified the protein expression levels of TRPA1 in mice’ bladder tissue ( n = 6). L–N RT-qPCR was conducted to quantify the mRNA levels of Il6 , Il1a , Il1b in mice’ bladder tissue ( n = 5). O Viod spot assays were conducted to assess urination behavior in mice, specifically counting urine spots ( n = 5). P Urodynamic test was utilized to evaluate intervoid interval, and bladder compliance in mice ( n = 5). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 or ns using one-way ANOVA, followed by Tukey’s post hoc test.

Article Snippet: After treatment of antigen retrieval, add 3% hydrogen peroxide (Sigma, USA) solution to the bladder sections (25 min, room temperature), blocked (3% BSA, Solarbio Life Sciences, China, 30 min, room temperature) and incubated overnight at 4 °C with the primary antibody against TRPA1 (1:200, Novus Biologicals, USA).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Clinical Proteomics, Membrane, Staining, Lactate Dehydrogenase Assay, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: TRPA1 promotes overactive bladder progression by activating the NLRP3 inflammasome and driving pyroptosis

doi: 10.1038/s41419-026-08426-5

Figure Lengend Snippet: A Western blot analyses were conducted to evaluate the protein expression levels of TRPA1, NLRP3, cleaved Caspase-1, and cleaved GSDMD in 5637 cells ( n = 3). B Immunofluorescence co-localization and line scan revealed TRPA1 expression at the plasma membrane of 5637 cells, with red indicating Na/K-ATPase staining, green representing TRPA1 staining, and blue denoting nuclear staining ( n = 30). White lines indicate the locations used for line scan analysis. C PI staining was used to assess the viability of cells. D The LDH assay was performed to further evaluate the viability of cells. E Western blot analyses were completed to determine the protein expression levels of TRPA1, NLRP3, cleaved Caspase-1, and cleaved GSDMD in mice bladders ( n = 6). F IHC staining was performed to assess TRPA1 expression in mice bladders. G Urodynamic test evaluated intervoid interval, and bladder compliance in mice ( n = 5). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001 or ns using one-way ANOVA, followed by Tukey’s post hoc test.

Article Snippet: After treatment of antigen retrieval, add 3% hydrogen peroxide (Sigma, USA) solution to the bladder sections (25 min, room temperature), blocked (3% BSA, Solarbio Life Sciences, China, 30 min, room temperature) and incubated overnight at 4 °C with the primary antibody against TRPA1 (1:200, Novus Biologicals, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Clinical Proteomics, Membrane, Staining, Lactate Dehydrogenase Assay, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: TRPA1 promotes overactive bladder progression by activating the NLRP3 inflammasome and driving pyroptosis

doi: 10.1038/s41419-026-08426-5

Figure Lengend Snippet: A Venn analysis identifying transcription factors associated with NLRP3 . B Prediction of binding sites for SP1, MAZ, and SMAD3 in the NLRP3 promoter. C , D Dual luciferase reporter assays were conducted to evaluate the transcriptional activity of NLRP3 ( n = 3). E ChIP assay was performed to quantify the binding of transcription factors to the NLRP3 promoter in 5637 cells ( n = 3). F Co-IP was employed to detect the binding levels of TRPA1 to transcription factors in 5637 cells. G GeneMANIA was utilized to predict interactions between TRPA1 and MAZ, SMAD3. Each node represents a gene, and its size corresponds to the gene’s significance. The colors of the edges between nodes denote the type of functional interaction, as detailed in the figure, while the thickness of the edges indicates the strength of the association. H , I Western blot analysis was conducted to assess the protein expression levels of NLRP3, cleaved Caspase-1, and cleaved GSDMD in cells ( n = 3). J , K RT-qPCR analyses were carried out to measure the mRNA levels of NLRP3 , IL18 in cells ( n = 3). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001 using one-way ANOVA, followed by Tukey’s post hoc test.

Article Snippet: After treatment of antigen retrieval, add 3% hydrogen peroxide (Sigma, USA) solution to the bladder sections (25 min, room temperature), blocked (3% BSA, Solarbio Life Sciences, China, 30 min, room temperature) and incubated overnight at 4 °C with the primary antibody against TRPA1 (1:200, Novus Biologicals, USA).

Techniques: Binding Assay, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Functional Assay, Western Blot, Expressing, Quantitative RT-PCR