Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure 1. TRPC4 expression in adult mouse brain. A, B, In situ hybridization of TRPC4-mRNA in amygdala, hippocampus, somatosensory cortex, auditory thalamus, and auditory cortex. BLA, amygdala basolateral nucleus; S1, primary somatosensory cortex;S2,secondarysomatosensorycortex;AuT,auditorythalamus;AuD,secondaryauditorycortex,dorsal;Au1,primaryauditory cortex; AuV, secondary auditory cortex, ventral; Ect, ectorhinal cortex; PRh, perirhinal cortex. Scale bar, 1 mm. C, TRPC4 (left) and CaMKII(middle;amarkerofpyramidalneurons)colocalizeintheLA(right).D,CellsexpressingTRPC4(red)andGFAP(green;amarker ofglialcells)donotcolocalizeintheLA.E,CellsexpressingTRPC5(red)andGAD67(green;amarkerofinterneurons)donotcolocalizeinthe LA.Scalebars:C–E,10m.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Expressing, In Situ Hybridization

Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure2. GenerationandconfirmationofTRPC4/mouse.A,TargetingstrategyforthedisruptionoftheTRPC4gene.Afterhomologousrecombination,deletionofexon4regionwascatalyzed by Cre-recombinase in ES cells. B, Targeting of the TRPC4 locus is confirmed by PCR analysis of tail genomic DNA. C, RT-PCR analysis of whole-brain mRNA from control and TRPC4/ littermates confirms the absence of exon 4. D, IP of TRPC4 protein in brain microsomes from control and TRPC4/ mice reveals loss of TRPC4 protein in TRPC4/ mice (top). Western blotting of NKA- confirmsequalproteinloadingincontrolandTRPC4/mice(bottom).E,ImmunohistochemicalstainingofbrainsectionsfromcontrolandTRPC4/littermatesrevealsselectivelossofTRPC4 expression in mutant mice. Scale bar, 1 mm.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure 9. mGluR-EPSCs in LA neurons are suppressed in TRPC4/ mice. A, Synaptic responses in cortical input to the LA neuroninaslicefromacontrolmouseevokedbytrainsofhigh-frequencystimulationbeforeandduringadditionofCNQX(AMPA receptorantagonist;20M),NMDAreceptorantagonists,D-APV(50M),andMK-801(10m)andGABABRantagonistCGP35348 (300 M) recorded as described previously (Riccio et al., 2009). Stimulation trains consisted of 10 pulses at 100 Hz which were delivered once every 30 s. Inset shows synaptic responses recorded in current-clamp mode before (1) and after (2) the addition of antagonists to the external medium. The dashed line indicates the time point where the EPSP amplitude was measured. B, The experiment was identical to A, but the recording was obtained from a TRPC4/ mouse. C, Summary data for the experiments as in A and B, performed in both cortical and thalamic inputs to the LA. The amplitude of the residual component of the EPSP in the presence of antagonists was smaller in both pathways in slices from TRPC4/mice comparedwithcontrollittermates.D,EPSCsincorticalinputrecordedinvoltage-clampmodeatholdingpotentialsranging from –100 mV to 40 mV in slices from control (left) and TRPC4/ (right) mice in the presence of the antagonists (as in A). E, Current–voltage (I–V) plots of the peak current in cortical input (as in D) in slices from control and TRPC4/ mice. F, EPSCs in thalamic input recorded in voltage-clamp mode at holding potentials ranging from –100 mV to 40 mV in slices from control (left) and TRPC4/ (right) mice in the presence of the antagonists (as in A). G, Current–voltage (I–V) plots of the peak current in thalamic input (as in F) in slices from control and TRPC4/ mice. H, Left, Representative immunoblotshowssimilaramountsofTRPC5proteininbrainmicrosomesextractedfromcontrolorTRPC4/littermates. Western blotting of Na -K -ATPase indicates equal protein loading (bottom). Right, Quantification of three different blots. Results are shown as mean SEM.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Control, Western Blot

Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure 10. CCK-mediated increase in spike firing in LA neurons is diminished in TRPC4/ mice. A, Spikes evoked in LA neurons by current injection (150 pA) recorded in current-clamp mode under baseline conditions and in the presence of 3 M CCK4 in a slice from a control mouse. B, Summary plot showing CCK4-induced depolarization in LA neurons in slices from control and mutant mice. C, Spikes evoked in LA neuron under baseline conditions and in the presence of 3 M CCK4 in a slice from a TRPC4/ mouse. D, The percentage increase in spike frequency in the presence of CCK4 relative to the baseline frequency (taken as 100%) in slices from control and null mice. E, Left, Fluorescence double labeling for CCKergic fibers (green) and CaMKII (red) reveals no differences in innervation of the LA by CCK-containing fibers in brain sections from control and TRPC4/ littermates. Right, Mean CCK fluorescence after normalization to CaMKII fluorescence from six images per group taken from different stainings (two images from each of 3 control and 3 KO mice). Scale bar, 10 m. Results are shown as mean SEM.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Injection, Control, Mutagenesis, Fluorescence, Labeling

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: ( A ) Volcano plot of gene expression features. X- axis indicates fold change of feature in sensitive versus refractory cell lines and Y-axis indicates statistical significance. ( B ) Waterfall plot showing TRPC4 expression levels (affymetrix microarray units) in englerin A sensitive (red) and refractory (blue) tumor cell lines.

Article Snippet: Human TRPC4 was ordered from Origene (Origene; RC218941).

Techniques: Gene Expression, Expressing, Microarray

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: (A ) Effect of TRPC4 siRNA knockdown on viability of A-498 cells in the presence of englerin A. An siRNA targeting luciferase was used as a control (mean+/- S.E.M.) Percent reduction of the TRPC4 mRNA levels are indicated in the legend, KD stands for knockdown. TRPC4 mRNA levels were normalized to peptidyl prolyl isomerase A (PPIA) mRNA levels. ( B ) Effect of TRPC4 siRNA knockdown on viability of A-673 cells in the presence of englerin A (mean +/- S.E.M.) ( C ) Effect of overexpression of TRPC4 by transient transfection on viability of HEK293T cells in the presence of englerin A. TRPC4 expression vector concentrations are indicated by different shapes ( D ) Effect of TRPC4 expression on cell viability in the presence of an englerin A in HEK293T cells engineered to express TRPC4 under control of a Doxycycline (Dox) regulated promoter (mean +/- standard deviation). 100 ng/ml Dox (black circles), 0 ng/ml Dox (open circles). ( E ) Western blot visualizing the levels of TRPC4 in the presence or absence of 100 ng/ml Dox. ( F ) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-498 cells. ( G ) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-673 cells.

Article Snippet: Human TRPC4 was ordered from Origene (Origene; RC218941).

Techniques: Knockdown, Luciferase, Control, Over Expression, Transfection, Expressing, Plasmid Preparation, Standard Deviation, Western Blot

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: ( A ) Expression of TRPC4 and TRPC5 in the cell lines from the CLiP experiment. Each circle represents a single cell line and expression levels were measured by microarray (TRPC4 probe 220818_s_at, TRPC5 probe 220552_at). The circle representing the DMS-79 cell line is indicated. ( B ) The effect of englerin A on cell viability in HEK293T cells transiently transfected with a vector expressing TRPC5 (mean +/- standard deviation).

Article Snippet: Human TRPC4 was ordered from Origene (Origene; RC218941).

Techniques: Expressing, Microarray, Transfection, Plasmid Preparation, Standard Deviation

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: ( A ) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). ( B ) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. ( C ) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. ( D ) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) ( E ) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).

Article Snippet: Human TRPC4 was ordered from Origene (Origene; RC218941).

Techniques: Standard Deviation, Transfection, Membrane, Saline, Activity Assay

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: ( A ) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). ( B ) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. ( C ) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. ( D ) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) ( E ) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).

Article Snippet: Cells were transfected with TRPC expressing plasmids (Origene: TRPC4beta, RC226709, TRPC6, RC210783, TRPC4 & 5 as above) using Fugene 6 according to the manufacturer’s protocol.

Techniques: Standard Deviation, Transfection, Membrane, Saline, Activity Assay

Journal: CNS neuroscience & therapeutics

Article Title: TRPC4 Mediates Trigeminal Neuropathic Pain via Ca 2+ -ERK/P38-ATF2 Pathway in the Trigeminal Ganglion of Mice.

doi: 10.1111/cns.70368

Figure Lengend Snippet: FIGURE 5 | The activation of the AKT and MAPK signaling pathway by Englerin A is dependent on the presence of TRPC4. (A, B) The levels of p- Akt, p-ERK, p-P38, p-JNK, p-PI3K, and p-mTOR in HEK293 cells treated with EA were measured and normalized to GAPDH. (*p < 0.05, **p < 0.01, ***p < 0.001, control vs. EA; multiple unpaired Mann–Whitney tests, n = 6). (C, D) The levels of p-Akt, p-ERK, p-P38, p-JNK, p-PI3K, and p-mTOR in the TRPC-KO cells and WT cells after treatment with Englerin A were measured and normalized to GAPDH. (Two-way ANOVA followed by Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, and ***p < 0.001; n = 3).

Article Snippet: After blocking with 5% non- fat milk (Epizyme, PS112L) for 1 h, the membranes were incubated with the corresponding primary antibodies at 4°C for 16 h. A range of primary antibodies was utilized, including TRPC4 (1:1500; Alomone Labs, #ACC- 018 and Abclonal, A6996), p- mTOR (1:1500; Abclonal, AP0115), mTOR (1:1500; Abclonal, A2445), p- Akt (1:1500; Abclonal, AP1208), Akt (1:1500; Abclonal, A18675), p- PI3K (1:1500; Cell Signaling Technology, 4292S), PI3K (1:1500; Abclonal, A4992), p- ERK1/2 (1:1500; Abclonal, 83010S), ERK1/2 (1:1500; Abclonal, A4782), p- P38 (1:1500; Abclonal, AP502), P38 (1:1500; Abclonal, A5049), p- JNK (1:1500; Abclonal, AP1337), JNK (1:1500; Abclonal, A4867), p- ATF2 (1:1500; Abclonal, AP1051), ATF2 (1:1500; Abclonal, A22718), and GAPDH (1:10000; Abcam, AB181602).

Techniques: Activation Assay, Control, MANN-WHITNEY

Journal: CNS neuroscience & therapeutics

Article Title: TRPC4 Mediates Trigeminal Neuropathic Pain via Ca 2+ -ERK/P38-ATF2 Pathway in the Trigeminal Ganglion of Mice.

doi: 10.1111/cns.70368

Figure Lengend Snippet: FIGURE 6 | The sustained activation of the AKT and MAPK pathways induced by TRPC4 overexpression is dependent on intracellular calcium ions. (A, B) After transfection of HEK293 cells with TRPC4 plasmids, the expression levels of p-mTOR, p-PI3K, p-Akt, p-ERK, p-P38, and p-JNK were measured and normalized to GAPDH. (*p < 0.05, **p < 0.01, ***p < 0.001, control plasmids vs. TRPC4 plasmids, n = 3, one-way ANOVA was used for statistical analysis, followed by post hoc Tukey test). (C, D) After transfecting HEK293 cells with TRPC4 plasmids and treating them with the calcium chelator BAPTA-AM, the expression levels of p-Akt, p-ERK, p-P38, and p-JNK were measured and normalized to GAPDH. (Two-way ANOVA followed by Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, and ***p < 0.001; n = 3).

Article Snippet: After blocking with 5% non- fat milk (Epizyme, PS112L) for 1 h, the membranes were incubated with the corresponding primary antibodies at 4°C for 16 h. A range of primary antibodies was utilized, including TRPC4 (1:1500; Alomone Labs, #ACC- 018 and Abclonal, A6996), p- mTOR (1:1500; Abclonal, AP0115), mTOR (1:1500; Abclonal, A2445), p- Akt (1:1500; Abclonal, AP1208), Akt (1:1500; Abclonal, A18675), p- PI3K (1:1500; Cell Signaling Technology, 4292S), PI3K (1:1500; Abclonal, A4992), p- ERK1/2 (1:1500; Abclonal, 83010S), ERK1/2 (1:1500; Abclonal, A4782), p- P38 (1:1500; Abclonal, AP502), P38 (1:1500; Abclonal, A5049), p- JNK (1:1500; Abclonal, AP1337), JNK (1:1500; Abclonal, A4867), p- ATF2 (1:1500; Abclonal, AP1051), ATF2 (1:1500; Abclonal, A22718), and GAPDH (1:10000; Abcam, AB181602).

Techniques: Activation Assay, Over Expression, Transfection, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: Inositol 1,4,5-Trisphosphate (IP3) Receptor Up-regulation in Hypertension Is Associated with Sensitization of Ca2+ Release and Vascular Smooth Muscle Contractility

doi: 10.1074/jbc.m113.496802

Figure Lengend Snippet: FIGURE 2. Screening of Ca2-handling proteins reveals coupled up-regu- lationofthevascularIP3RandLTCCinexperimentalmodelsofhyperten- sion. A, immunoblot analysis of IP3R1, 1C, TRPC1, TRPC4, Orai1, and STIM1 in lysates from mouse MA. MA lysate pooled from three SAL or AHT mice was added to each lane. Increased expression levels of IP3R1, 1C, and TRPC4 are detected in MA of AHT mice, whereas levels of TRPC1, Orai1, and STIM1 were similar. B, agarose gel analysis of products obtained after PCRs for IP3R1 (274 bp), 1C (185 bp), and TRPC4 (226 bp) using specific primers. -Actin (152 bp) and vWF (178 bp) were used as VSM and endothelial cell markers, respec- tively.IP3R,1C,and-actinweredetectedbothinintactMAandisolatedVSM cells as expected. In contrast, TRPC4 and vWF were detected in arteries but not isolated VSM cells, implying expression only in endothelium. C, immuno- blots comparing the expression of IP3R, 1C, PMCA, and -actin (as a loading control) between MA from normotensive Wistar Kyoto rats (WKY) and SHR and between MA from sham-operated and aortic-banded (Band) hyperten- sive rats. In each case, lysates were pooled from arteries of three or four rats. Only the IP3R and 1C proteins are up-regulated in MA from hypertensive SHR and aortic-banded rats. Blots are representative of three experiments.

Article Snippet: Forty g of protein was loaded per well, and expression of Ca2 -handling proteins was detected using specific antibodies against IP3R1 (1:1000; Millipore), 1C (1:1000; Millipore), TRPC1 (1:200; Santa Cruz Biotechnology), TRPC4 (1:200;NovusBiologicals), STIM1 (1:1000; Cell SignalingTechnology), Orai1 (1:1000; Sigma), and PMCA1 (1:500; Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Agarose Gel Electrophoresis, Isolation, Control