gsk1702934a  (TargetMol)

Journal: Nature Immunology

Article Title: Lipid asymmetry disruption by XKR8 orchestrates neutrophil extracellular trap formation and inhibits fungal infection

doi: 10.1038/s41590-026-02456-z

Figure Lengend Snippet: a , b , Quantification of PMA-induced NET formation in WT and XKR8-deficient BMNs, treated with or without EGTA from the beginning (1 mM) ( n = 10) ( a ), or treated with EGTA (1 mM) at indicated time points after PMA stimulation ( n = 9) ( b ). c , Evaluation of membrane tension using Flipper-TR by FLIM imaging. Representative images of Flipper-TR and annexin V in PMA-induced NETs (left) and quantification of Flipper-TR lifetime ( τ ) in PS-exposed (annexin V + ) versus non-exposed (annexin V − ) cells (right) ( n = 50 in WT control, n = 57 in KO control, n = 59 in PMA-WT-annexin V + , n = 62 in PMA-WT-annexin V − , n = 53 in PMA-KO). Red indicates annexin V-AF594; green shows Flipper-TR. Scale bars, 5 μm. d , Schematic diagram showing calcium channels highly expressed on mouse neutrophils along with their respective inhibitors or agonists. e – i , Quantification of NET formation in WT and XKR8-deficient neutrophils treated with inhibitors and/or agonists listed in d . e , PMA-induced NET formation with inhibitors targeting TRPM2 (JNJ-28583113, 10 μM), TRPM4 (9-phenanthrol, 30 μM), TRPM7 (carvacrol, 100 μg ml −1 ), TRPV2 ( SKF96365 , 20 μM), TRPV4 (HC067047, 1 μM), Piezo1 (GsMTx-4, 10 μM), and TRPC3 (Pyr3, 5 μM) for 6 h ( n = 10). f , PMA-induced NETs in WT and XKR8-deficient neutrophils treated with the agonists targeting TRPM2 (bilirubin, 10 μM), TRPM7 (naltriben (NTB), 50 μM), TRPV2 (cannabidiol (CBD), 10 μM), TRPV2 (probenecid, 1 mM), TRPV4 (GSK1016790A, 100 nM), Piezo1 (Yoda1, 10 μM), and TRPC3 (GSK1702934A, 100 μM) for 6 h ( n = 10). g , PMA-induced NETs in WT and XKR8-deficient neutrophils with both inhibitors and agonists against TRPV2 ( SKF96365 and CBD), TRPV4 (HC067047 and GSK1016790A), Piezo1 (GsMTx-4 and Yoda1), TRPC3 (Pyr3 and GSK1702934A) for 6 h ( n = 9). h , i , As in f , quantification of LPS-induced NETs ( h ) or C. albicans -induced NETs ( i ) in WT and XKR8-deficient neutrophils treated with the agonists targeting TRPV2 (CBD, 10 μM), TRPV2 (probenecid, 1 mM), TRPV4 (GSK1016790A, 100 nM), Piezo1 (Yoda1, 10 μM) and TRPC3 (GSK1702934A, 100 μM) for 6 h ( n = 10). NETs were measured by the area of decondensed chromatin DNA ( a , b , e – i ). Each dot represents one individual field of view ( a , b , e – i ) or a single cell ( c ). Data are presented as mean ± s.e.m. from one representative experiment of three ( c , e – i ) and four ( a , b ) independent experiments. Statistical analysis was performed using a two-way ANOVA ( a , b , e – i ) and one-way ANOVA ( c ). P values are shown on the graphs.

Article Snippet: Additional inhibitors, including S62845 (HY-100741), emricasan (HY-10396), 2-APB (B6643), SKF96365 (HY-100001), HC067047 (HY-100208), Pyr3 (HY-108465), JNJ-28583113 (HY-149143), 9-phenanthrol (HY-108457), carvacrol (HY-N0711), GsMTx-4 (HY-P1410), probenecid (HY-B0545), GSK1016790A (HY-19608), GSK1702934A (HY-111098), bilirubin (HY-N0323), Yoda1 (HY-18723) and NTB (HY-101302) were obtained from MedChemExpress.

Techniques: Membrane, Imaging, Control, Single Cell

Journal: Nature Immunology

Article Title: Lipid asymmetry disruption by XKR8 orchestrates neutrophil extracellular trap formation and inhibits fungal infection

doi: 10.1038/s41590-026-02456-z

Figure Lengend Snippet: a , b , Quantification of NETs in WT and XKR8-deficient BMNs or HL-60 cells treated with PMA (100 nM) in the presence of inhibitors listed in Fig. . a , PMA-induced NETs formation with inhibitors targeting TRPM2 (glutathione, GSH, 10 μM; JNJ-28583113, 10 μM), TRPM4 (9-phenanthrol, 30 μM), TRPM7 (Carvacrol, 100 μg/mL), SOCE (2-APB, 25 μM; CM-4620, 10 μM), TRPV2 ( SKF96365 , 20 μM), TRPV4 (HC067047, 1 μM), Piezo1 (GsMTx-4, 10 μM), TRPC3 (Pyr3, 5 μM), TRPC6 (SAR7334, 1 μM) ( n = 8-9). b , As in a , Quantification of PMA-induced NETs in WT and XKR8-deficient dHL-60 cells with inhibitors HC067047 (1 μM), Pyr3 (5 μM), SKF96365 (20 μM), 9-phenanthrol (30 μM), JNJ-28583113 (10 μM), Carvacrol (100 μg/mL) ( n = 8). c , As in a , quantification of NETs in human primary neutrophils isolated from healthy donors (HD, n = 8) treated with PMA (10 nM) in the presence of mechanosensitive Ca 2+ channel inhibitors. d , Calcium influx was evaluated in WT and XKR8 KO BMNs by Indo-1. Indo-1 signals were recorded immediately after adding the agonists targeting TRPV2 (Cannabidiol, CBD, 5 μM), TRPC3 (GSK1702934A, 100 μM), TRPV4 (GSK1016790A, 100 nM), Piezo1 (Yoda1, 10 μM) for 5 min. A23187 (1.25 μM) as a positive control. e , Quantification of intracellular Ca 2+ levels in WT and XKR8 KO BMNs by Indo-1. Indo-1 fluorescence signals were measured at 2 h after treatment with PMA (100 nM) and in the presence of the agonists targeting TRPV2 (CBD, 5 μM), TRPC3 (GSK1702934A, 100 μM), TRPV4 (GSK1016790A, 100 nM), Piezo1 (Yoda1, 10 μM) ( n = 3). f , Quantification of PMA-induced NETs in WT and XKR8-deficient dHL-60 cells with agonists targeting TRPV2 (CBD, 5 μM; Probenecid, 1 mM), TRPC3 (GSK1702934A, 100 μM), TRPV4 (GSK1016790A, 100 nM), TRPM7 (Naltriben Mesylate, NTB, 50 μM) ( n = 8). g , h , Quantification of NETs in human primary neutrophils ( g , n = 8) or mouse BMNs ( h , n = 10) treated with agonists in the absence of PMA. i , Quantification of nigericin-induced NETs in WT and XKR8-deficient BMNs in the presence of the agonists targeting TRPV2 (CBD, 5 μM), TRPC3 (GSK1702934A, 100 μM), TRPV4 (GSK1016790A, 100 nM), Piezo1 (Yoda1, 10 μM) ( n = 10). j , k , Quantification of PMA-induced NETs ( j ) and PS exposure ( k ) in TRPV2, TRPC3, TRPV4, and Piezo1-deficient dHL-60 cells ( n = 10-12). NETs were quantified by a nuclear area greater than 80 µm 2 for BMNs ( a , c , g – i ) or 100 µm 2 for HL-60 cells ( b , f , j ). Each dot represents one individual field of view ( a – c , f – j ) or a biological replicate ( e , k ). Data are presented as mean ± SEM from one representative experiment of three ( a – k ) independent experiments. Statistical analysis was performed using two-way ANOVA ( a , b , f , h – k ), two-sided unpaired Student’s t-test ( e ), or one-way ANOVA ( c , g ). P values are shown on the graphs.

Article Snippet: Additional inhibitors, including S62845 (HY-100741), emricasan (HY-10396), 2-APB (B6643), SKF96365 (HY-100001), HC067047 (HY-100208), Pyr3 (HY-108465), JNJ-28583113 (HY-149143), 9-phenanthrol (HY-108457), carvacrol (HY-N0711), GsMTx-4 (HY-P1410), probenecid (HY-B0545), GSK1016790A (HY-19608), GSK1702934A (HY-111098), bilirubin (HY-N0323), Yoda1 (HY-18723) and NTB (HY-101302) were obtained from MedChemExpress.

Techniques: Isolation, Positive Control, Fluorescence

Journal: Nature Communications

Article Title: Structural mechanism of the agonist binding on human TRPC3 channel

doi: 10.1038/s41467-025-64435-6

Figure Lengend Snippet: a , b Cryo-EM density maps of GSK1702934A-bound state hTRPC3 shown in side view ( a ) and top view ( b ). Subunits A, B, C, and D are colored in yellow, blue, pink, and green, respectively. Ligand GSK1702934A is colored in gold. The approximate boundary of the cell membrane is indicated by gray lines. TMD transmembrane domain, ICD intracellular cytosolic domain. c Densities of GSK1702934A and nearby residues shown in side view. Densities are shown as grey surfaces. GSK1702934A is shown as sticks, and hTRPC3 is shown as cartoon. Both are colored the same as in ( a ). d , e Close-up view of the GSK1702934A-binding site. The GSK1702934A molecule and the side chains of its interacting residues are shown as sticks. f Cartoon representation of the interactions between GSK1702934A and hTRPC3. S6 of subunit A and S5, S6, pore helices of subunit B are represented as yellow ovals and blue ovals, respectively. Residues that interact with GSK1702934A are labeled inside the ovals. g Sequence alignment among TRPC1/4/5 and TRPC3/6/7 is shown. The residues that interact with GSK1702934A are marked by gold asterisks. Conserved residues are colored in gold. The corresponding domains are labeled above the sequence.

Article Snippet: The concentration of Carbachol (CCh, HarveyBio, C33526 ), 4n (Dizal Pharmaceutical), GSK1702934A (Shanghai Universal Biotech, 6508), 1-Oleoyl-2-acetyl-sn-glycerol (OAG, Sigma, O6754), 1-Oleoyl-rac-glycerol (1-MOG, Sigma, M7765), 2-Oleoylglycerol (2-MOG, TargetMol, T37526 ), BTDM (Dizal Pharmaceutical), and (-)-Englerin A (EA, Sigma, PHL82530 ) used in patch-clamp are marked out in the figures.

Techniques: Cryo-EM Sample Prep, Membrane, Binding Assay, Labeling, Sequencing

Journal: Nature Communications

Article Title: Structural mechanism of the agonist binding on human TRPC3 channel

doi: 10.1038/s41467-025-64435-6

Figure Lengend Snippet: a Close-up view of DAG at the L2 site. The mutated amino acids on hTRPC3 are shown as sticks. DAG is colored in green. TRPC3 is colored in blue. b The ratio of peak currents activated by CCh and GSK1702934A in wild-type, E603A, K607A, and V637A under whole-cell mode at −60 mV. The calculation method for the activation current is described in detail in Supplementary Fig. . The number of biological replicates in each group is indicated below the genotype. The data are shown as the mean ± SD. The data were analyzed using Brown–Forsythe and Welch ANOVA tests, and the multiple comparisons between each group were analyzed using Dunnett’s T3 multiple comparisons tests with p -values indicated above the corresponding groups. Source data are provided as a Source Data file. c The ratio of peak currents activated by OAG and GSK1702934A in wild-type, E603A, K607A, and V637A under whole-cell mode at −60 mV. The calculation, annotation, and statistical analyses were identical to those described in ( b ). Source data are provided as a Source Data file. d The ratio of peak currents activated by CCh and 4n in wild-type, E603A, K607A, and V637A under whole-cell mode at −60 mV. The calculation, annotation, and statistical analyses were identical to those described in ( b ). Source data are provided as a Source Data file. e The ratio of peak currents activated by OAG and 4n in wild-type, E603A, K607A, and V637A under whole-cell mode at −60 mV. The calculation, annotation, and statistical analyses were identical to those described in ( b ). Source data are provided as a Source Data file. f Chemical structures of DAG, OAG, 1-MOG, and 2-MOG. g – i . Macroscopic currents of wild-type hTRPC3/6/7 recorded in the whole-cell mode. Zero current is indicated by a dashed line. The duration of ligand application is indicated by a solid line above. Source data are provided as a Source Data file.

Article Snippet: The concentration of Carbachol (CCh, HarveyBio, C33526 ), 4n (Dizal Pharmaceutical), GSK1702934A (Shanghai Universal Biotech, 6508), 1-Oleoyl-2-acetyl-sn-glycerol (OAG, Sigma, O6754), 1-Oleoyl-rac-glycerol (1-MOG, Sigma, M7765), 2-Oleoylglycerol (2-MOG, TargetMol, T37526 ), BTDM (Dizal Pharmaceutical), and (-)-Englerin A (EA, Sigma, PHL82530 ) used in patch-clamp are marked out in the figures.

Techniques: Activation Assay

Journal: Cells

Article Title: Soluble β-Amyloid Oligomers Selectively Upregulate TRPC3 in Excitatory Neurons via Calcineurin-Coupled NFAT.

doi: 10.3390/cells14110843

Figure Lengend Snippet: Figure 1. TRPC3 expression is upregulated in human brains. (A,B) Representative Western blots of TRPC3 and TRPC6 protein expression from 9 AD and 7 non-AD control (NC) hippocampal tissue samples. ** p < 0.01. F: female; M: male. (C) Relative mRNA levels of Trpc3 and Trpc6 genes were determined by RT-qPCR based on 7 AD and 6 NC samples of human AD brain PFC regions; only RIN > 7 samples were used. * p < 0.05. (D) Representative microscopic images displaying immunostained amyloid pathology (with 4G8 antibody). (E) Representative TRPC3-immunostained microscopic images of AD and control brains. Red arrowheads indicate typical cells with morphologies of large pyramidal neurons, respectively. (F) Quantification of TRPC3 DAB immunosignals based on 5 AD and 4 NC cases. ** p < 0.01. Note: human brain samples used for panels (A-C) were from same cohort of patient and control individuals, and representative images shown in panels (D,E) were taken from same pair of AD and NC cases from different cohort of clinical samples.

Article Snippet: The TRPC3 agonist GSK1702934A (GSK) (cat. # 6508, Tocris, Ellisville, MI, USA) and fast voltage-gated sodium channel blocker tetrodotoxin citrate (TTX) (cat. # 1069, Tocris) were each bath-applied.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: Cells

Article Title: Soluble β-Amyloid Oligomers Selectively Upregulate TRPC3 in Excitatory Neurons via Calcineurin-Coupled NFAT.

doi: 10.3390/cells14110843

Figure Lengend Snippet: Figure 2. 7PA2 treatment upregulates TRPC3 and downregulates TRPC6 expression in cultured primary neurons (14 DIV). (A) Representative TRPC3 and TRPC6 immunostaining images of 7PA2- treated neurons. (B) Representative Western blots and quantification based on 3 independent experi- ments. * p < 0.05; ** p < 0.01. (C) Representative TRPC family immunoblots of cellular lysates from cultured neurons at 4 h after 7PA2 treatment.

Article Snippet: The TRPC3 agonist GSK1702934A (GSK) (cat. # 6508, Tocris, Ellisville, MI, USA) and fast voltage-gated sodium channel blocker tetrodotoxin citrate (TTX) (cat. # 1069, Tocris) were each bath-applied.

Techniques: Expressing, Cell Culture, Immunostaining, Western Blot

Journal: Cells

Article Title: Soluble β-Amyloid Oligomers Selectively Upregulate TRPC3 in Excitatory Neurons via Calcineurin-Coupled NFAT.

doi: 10.3390/cells14110843

Figure Lengend Snippet: Figure 3. 7PA2-induced TRPC3 upregulation is mediated by overactivated calcineurin (CaN). (A) Cal- cineurin’s phosphatase activity was induced by 2-fold, as determined 1 h after 7PA2 treatment. (n = 4). ** p < 0.01. (B) Representative TRPC3 immunocytochemistry showing preventive effects of two CaN inhibitors against 7PA2-upregulated TRPC3 expression (4 h time point). FK506 and CsA were used at 12.5 nM and 1 µM, respectively, in co-treatment with 7PA2. (C) Quantification of experiments of panel B (N = 3 independent experiments). * p < 0.05 and ** p < 0.01, respectively.

Article Snippet: The TRPC3 agonist GSK1702934A (GSK) (cat. # 6508, Tocris, Ellisville, MI, USA) and fast voltage-gated sodium channel blocker tetrodotoxin citrate (TTX) (cat. # 1069, Tocris) were each bath-applied.

Techniques: Activity Assay, Immunocytochemistry, Expressing

Journal: Cells

Article Title: Soluble β-Amyloid Oligomers Selectively Upregulate TRPC3 in Excitatory Neurons via Calcineurin-Coupled NFAT.

doi: 10.3390/cells14110843

Figure Lengend Snippet: Figure 4. 7PA2 treatment induces NFAT4c binding to Trpc3 gene promoter. (A,B) Schematic rep- resentation of 5′ upstream region of rat Trpc3 and Trpc6 genes. Sequences of rat Trpc3 gene (be- tween –1802 and –1217 bp) and rat Trip6 gene (between –812 and –771 bp) are aligned with cor- responding sequences of mouse and human genes. NFAT sites are shown in red. We identified two NFAT binding sites between positions –1802 and –1217 within Trpc3 promoter based on motif ([A/T]GGAAA[A/N][A/T/C]N) [57], as well as two NFAT binding sites from promoter Trpc6 [58]. These sites are relatively conserved among humans and rodents. Please note two differently spatially located sites within these two promoters. (C) 7PA2 treatment induces NFATc4 nuclear translocation as shown by immunocytochemistry. DAPI (4′,6-diamidino2-phenylindole, blue) staining is used to show colocalization of NFAT immunosignals at 2 h after 7PA2 treatment. (D) Representative (bottom) gel images of ChIP-PCR using NFATc4 antibody at 2h after 7PA2 treatment.

Article Snippet: The TRPC3 agonist GSK1702934A (GSK) (cat. # 6508, Tocris, Ellisville, MI, USA) and fast voltage-gated sodium channel blocker tetrodotoxin citrate (TTX) (cat. # 1069, Tocris) were each bath-applied.

Techniques: Binding Assay, Translocation Assay, Immunocytochemistry, Staining

Journal: Cells

Article Title: Soluble β-Amyloid Oligomers Selectively Upregulate TRPC3 in Excitatory Neurons via Calcineurin-Coupled NFAT.

doi: 10.3390/cells14110843

Figure Lengend Snippet: Figure 5. TRPC3 expression is upregulated in mature excitatory neurons upon 7PA2 treatment (4 h). (A,D) Representative immunocytochemical images of TRPC3 (A, green) and TRPC6 (D, green) co-stained with CaMKIIα (red) and DAPI (blue), taken at 4 h after 7PA2 or vehicle treatment. White arrowheads indicate excitatory neurons identified by high immunosignals of CaMKIIα, which are exclusively colocalized with strong TRPC3 immunosignals. (B) Quantification graphs based on number counts of cells expressing high and low levels of CaMKIIα co-localized with 7PA2- upregulated TRPC3. (C,E) These panels quantify cells of 7PA2-inuduced TRPC3 expression and 7PA2-reduced TRPC6 expression, respectively, in both high and low CaMKIIα populations (gray bars), compared to vehicle group (black bars). * p < 0.05; ** p < 0.01. (F) Representative fluorescent microscopic images of CaMKIIα and VGAT staining, revealing reciprocal staining patterns. White arrowheads indicate two neurons showing strong CaMKIIα immunosignals but weak VGAT, and yellow arrowheads indicate cells with opposite pattern.

Article Snippet: The TRPC3 agonist GSK1702934A (GSK) (cat. # 6508, Tocris, Ellisville, MI, USA) and fast voltage-gated sodium channel blocker tetrodotoxin citrate (TTX) (cat. # 1069, Tocris) were each bath-applied.

Techniques: Expressing, Staining

Journal: Cells

Article Title: Soluble β-Amyloid Oligomers Selectively Upregulate TRPC3 in Excitatory Neurons via Calcineurin-Coupled NFAT.

doi: 10.3390/cells14110843

Figure Lengend Snippet: Figure 6. Overactivated TRPC3 channel induces neuronal hyperexcitation in hippocampal CA1 region. (A) Representative microscopic image showing green fluorescent protein expression from the intra-CA1-injected AAV-eGFP viruses. (B) Western blot shows increased expression of TRPC3 from hippocampi-injected AAV-mTRPC3 and AAV-eGFP. Quantification was based on 3 independent experiments. * p < 0.05. (C) Increased TRPC3 expression and activation increase neuronal activities in CA1 region in brain slices (N = 5 mice). Left panels display example extracellular recordings of spontaneous neuronal activities in control side CA1 during baseline, during application of 5 µM GSK1702934A (GSK), and upon adding 1 µM tetrodotoxin (TTX). Data are quantified and summarized in bottom graph: each black dot is data point from one mouse, and red dots are the mean of black data points (5 mice, control side CA1). Right panels display same parameter measurements as left panel from AAV-mTRPC3-injected CA1 region. Scale bars apply to both panels. Before and after GSK frequency values in each sample for both groups were compared by paired t-test, whereas frequency values during GSK application in two groups were compared by unpaired t-test.

Article Snippet: The TRPC3 agonist GSK1702934A (GSK) (cat. # 6508, Tocris, Ellisville, MI, USA) and fast voltage-gated sodium channel blocker tetrodotoxin citrate (TTX) (cat. # 1069, Tocris) were each bath-applied.

Techniques: Expressing, Injection, Western Blot, Activation Assay, Control

Journal: Cells

Article Title: Soluble β-Amyloid Oligomers Selectively Upregulate TRPC3 in Excitatory Neurons via Calcineurin-Coupled NFAT.

doi: 10.3390/cells14110843

Figure Lengend Snippet: Figure 7. Selective blocking of TRPC3 alleviates 7PA2-induced synaptotoxicity. (A,B) Specificity of chosen siRNAs. (A) Quantified qRT-PCR results from neurons transfected with selected siRNAs against Trpc3 and Trpc6 genes. Neurons (5 DIV) were transfected with scrambled or specific siRNAs of Trpc3 and Trpc6 genes (sITRPC3 and siTRPC6). * p < 0.05 and ** p < 0.01. RT-qPCRs were performed in triplication using specific primers for detecting TRPC family genes. (B) Representative Western blot displays selectively downregulated expression of TRPC3 and TRPC6 proteins in transfected neurons (14 DIV). (* p < 0.05 and ** p < 0.01). (C) Representative MAP2 staining of neurons after being transfected with scrambled or specific siRNAs of Trpc3 and Trpc6 genes, without and with 7PA2 treatment. Quantification was based on 3 independent experiments. ** p < 0.01. (D) Co-treatment of neurons of 7PA2 with two TRPC3 selective antagonists (Pyr 3 and 10) significantly prevents dendritic loss. Quantification was based on 3 independent experiments. ** p < 0.01. (E) Co-treatment of neurons of 7PA2 with Pyr3 and 10 completely prevents synaptic Ca2+ flux induced by 7PA2. Quantification was based on over 20 neurons in each condition. Arrows indicate when compounds were perfused with and without 7PA2. “NS.” indicates non-significance and ** p < 0.01.

Article Snippet: The TRPC3 agonist GSK1702934A (GSK) (cat. # 6508, Tocris, Ellisville, MI, USA) and fast voltage-gated sodium channel blocker tetrodotoxin citrate (TTX) (cat. # 1069, Tocris) were each bath-applied.

Techniques: Blocking Assay, Quantitative RT-PCR, Transfection, Western Blot, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

doi: 10.1093/nar/gkae245

Figure Lengend Snippet: Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.

Article Snippet: The following TRPC3/C6/C7 activators were employed in the current study: L687 ( , WO/2022/118966) , Cannabidiol (CBD) (#Axon1234; Axon Medchem, VA, USA), and GSK1702934A (#6508; Tocris Bioscience, Bristol, UK).

Techniques: Knockdown, Fluorescence, Flow Cytometry, Transfection, Expressing, Western Blot, Cell Culture, Incubation, Imaging, Staining, Microscopy