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: 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

Journal: Science Advances

Article Title: Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function

doi: 10.1126/sciadv.aaw7935

Figure Lengend Snippet: ( A ) Side view of TRPC5’s pore region with chains A and C compared with TRPC4 (gray). The ion conduction pathway is shown as dots and mapped using HOLE with key amino acid residues labeled. ( B ) Pore radius along the central axis. The side chains of glycine form a narrow constriction at the selectivity filter. “INQ” motif forms the lower gate. ( C ) Side view of TRPC5 (blue) monomer subunit compared with TRPC4 (gray). Differences in the organizations of a linker (red arrowheads) of S5 and the small loop above the pore helix of the extracellular pore domain are enlarged. ( D ) Sequence of the mouse TRPC5 aligned to TRPC4 and other TRPC subfamily members (Clustal Omega) between S5 and S6 including the linker, pore helix, and pore loop. Regions corresponding to different extracellular pore domains are indicated by the red arrowheads. The two cysteines forming disulfide bonds, a conserved “LFW” motif, and the selectivity filter are highlighted. ( E to G ) Patch clamp recordings of TRPC5 mutants in response to channel activators Gd 3+ and EA.

Article Snippet: Primary antibodies against TRPC5 and TRPC4 were purchased from Sangon Biotech (Shanghai, China) and Proteintech (Wuhan, China), respectively.

Techniques: Labeling, Sequencing, Patch Clamp

Journal: Science Advances

Article Title: Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function

doi: 10.1126/sciadv.aaw7935

Figure Lengend Snippet: ( A ) Superimposed side views of mouse TRPC5 subunit (blue) compared with other TRPC family members, including mouse TRPC4 [Protein Data Bank (PDB) ID code 5Z96, gray] , human TRPC3 (PDB ID 5ZBG, pink) , and human TRPC6 (PDB ID 5YX9, yellow) . ( B ) The conserved LFW motif in the pore helix from a π-π interaction that stabilizes the pore region. ( C ) Key pore loop disulfide bond between Cys 553 and Cys 548 in TRPC5 (black arrowhead) and the corresponding pore loop disulfide bond in TRPC4 that close to a linker (red arrowheads) of S5 and loop above the pore helix. This disulfide bond is not present in TRPC3 or TRPC6. ( D ) Differences in the organization of the S3 helix between the TRPC4/5 and TRPC3/6. The S3 helices of TRPC3 and TRPC6 are longer than those of TRPC5 and TRPC4. ( E ) Patch clamp recordings of wild-type (WT) TRPC5 and cysteine mutants in response to the reducing agent [dithiothreitol (DTT)] and channel activator (EA). ( F ) Localization of enhanced yellow fluorescent protein–tagged WT TRPC5 and cysteine mutants in HEK293 cells. ( G ) Quantification of cell surface expression of TRPC5 cysteine mutants ( n = 3). Mean values are shown as gray bars. By analysis of variance (ANOVA), there is no statistically significant difference. a.u., arbitrary units.

Article Snippet: Primary antibodies against TRPC5 and TRPC4 were purchased from Sangon Biotech (Shanghai, China) and Proteintech (Wuhan, China), respectively.

Techniques: Patch Clamp, Expressing

Journal: Science Advances

Article Title: Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function

doi: 10.1126/sciadv.aaw7935

Figure Lengend Snippet: ( A to F and I ) The time course of whole-cell currents measured at +80 and −80 mV and representative I-V relationships in different conditions as labeled. TRPC5-C4motif, TRPC5 mutant carrying the TRPC4 motif ETKGLS; TRPC4-C5motif, TRPC4 mutant with the TRPC5 motif TRAIDEPNN; TRPC5-C4motif-C553A-C558A, TRPC5 mutant bearing the TRPC4 motif and double-cysteine mutations. ( G and H ) Time constants (τ) of channel activation and inactivation by 100 nM EA. τ values from each recording are plotted as dots. “∞” indicates that the current did not decay in the recording time frame. Mean τ values of each mutant are shown as gray bars. There is no significant difference by ANOVA in (G) ( n = 8 to 11).

Article Snippet: Primary antibodies against TRPC5 and TRPC4 were purchased from Sangon Biotech (Shanghai, China) and Proteintech (Wuhan, China), respectively.

Techniques: Labeling, Mutagenesis, Activation Assay

Journal: Science Advances

Article Title: Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function

doi: 10.1126/sciadv.aaw7935

Figure Lengend Snippet: ( A ) A cation (purple sphere) on the cytosolic face is in the hydrophilic pocket of the S1-S4 domain, interacting with Glu 418 , Glu 421 , Asn 436 , and Asp 439 . Enlarged view of the cation binding site. ( B ) Comparable Ca 2+ (green sphere) binding site and an enlarged view of TRPM4 as “E/Q/N/D” (TRPC5, “E/E/N/D”). ( C ) Sequence of the mouse TRPC5 aligned to mTRPC4 and other representative TRP members (Clustal Omega). The key residues are highlighted as E/E/N/D, which are conserved in TRPC2/3/5/6/7 and TRPM7 but as E/Q/N/D in TRPC4 and TRPM4. ( D ) Patch clamp recordings of TRPC5 mutants in response to the channel activators Gd 3+ and EA. For the N436R mutant, 12 transfected cells showed no response to EA; weak activation was observed in three cells.

Article Snippet: Primary antibodies against TRPC5 and TRPC4 were purchased from Sangon Biotech (Shanghai, China) and Proteintech (Wuhan, China), respectively.

Techniques: Binding Assay, Sequencing, Patch Clamp, Mutagenesis, Transfection, Activation Assay

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Normalized fold change in TRPC1/4/5 mRNA (2 –ΔΔ CT ) in TBI mice compared to sham.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Cell-type specific TRPC1, TRPC4, and TRPC5 channel upregulation in the hippocampus and cortex after CCI-TBI. (A,B) Representative Western immunoblots using (A) TRPC4 (∼120 kDa) and (B) TRPC5 (∼110 kDa) antibodies in sham and TBI cortex and hippocampus. Blots were normalized to β-actin protein (42 kDa) as loading control. (C,D) Summarized data for Western blot quantification of TRPC4 ( C , n = 7–13 animals per group) and TRPC5 ( D , n = 5–7 animals per group) from microdissected brain regions in mice 7 days after TBI. (E,F) Shown are summarized plots of percent difference in TRPC4 (E) and TRPC5 (F) protein between ipsilateral and contralateral hemispheres of microdissected regions from data in Panels (C,D) . All data bars represent the mean ± SEM. * p < 0.05 vs. sham of same subregion. † p < 0.05 vs. contralateral hemisphere of same subregion.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Western Blot, Control

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Normalized TRPC4/TRPC5 protein in 7-day sham and TBI mice.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Surges in neuronal activity following CCI-TBI are TRPC4/TRPC5-mediated. (A) Representative images of parietal cortex from sham and TBI TRAP mice that were administered 4-OHT at t = 12 h before procedure. Prefix “ c ” denotes contralateral, prefix “ i ” denotes ipsilateral. Red = cFos-tdTomato; blue = DAPI. (B) Representative images of hippocampal subregions from sham and TBI TRAP mice that were administered 4-OHT at t = 12 h before procedure. (C) Summarized quantification of cFos+ neuron density (neurons/0.1 mm 3 ) in sham and TBI TRAP mice activated at the time of TBI, as in Panels (A,B) . (D) Representative images taken from sham mice, TBI mice, and TBI mice also administered M084 (10 mg/kg) (TBI + M084) that were administered 4-OHT t = 7 days after procedure. (E) Summarized quantification of cFos+ neurons in sham, TBI, and TBI + M084 mice 7 days after procedure, as in Panel (D) . All data bars represent the mean ± SEM. * p < 0.05 vs. sham. # p < 0.05 vs. TBI cDG. † p < 0.05 vs. TBI of same region. Scale bars: 100 μm.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Activity Assay

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: TRPC4/TRPC5 channel activation artificially prolongs Ca 2+ influx in DGGCs after CCI-TBI. (A) Cumulative probability distribution of the peak amplitude of GCaMP6f fluorescence (ΔF/F) for each DGGC from sham (control) and iTBI slices during EA (1 μM, red) or EA + M084 (10 μM, gray) application. (B) Cumulative probability distribution of the Ca 2+ influx duration (in seconds) for each DGGC from control and iTBI slices during EA or EA + M084 application. (C,D) Histogram population distribution of control DGGC Ca 2+ influx events according to peak amplitude (C) and Ca 2+ event duration (D) . (E,F) Histogram population distribution of iTBI DGGC Ca 2+ influx events according to peak amplitude (E) and Ca 2+ event duration (F) . (G) Summarized means of peak fluorescence from data as in Panel (A) . (H) Summarized means of Ca 2+ influx duration from data as in Panel (B) . Red = EA alone, gray = EA + M084. All data bars represent the mean ± SEM. * p < 0.05 vs. EA alone from same procedure condition, † p < 0.05 vs. control of same drug condition.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Activation Assay, Fluorescence, Control

Journal: PloS one

Article Title: Short-chain 3-hydroxyacyl-coenzyme A dehydrogenase associates with a protein super-complex integrating multiple metabolic pathways.

doi: 10.1371/journal.pone.0035048

Figure Lengend Snippet: Figure 1. Western blot of SCHAD following pulldown with fructose bisphosphate aldolase. Lane 1, homogenized liver SCHAD control, Lane 2 albumin blank, Lanes 3 and 4 results from 2 separate pulldown experiments showing a normal sized mitochondrial SCHAD. doi:10.1371/journal.pone.0035048.g001

Article Snippet: According to the protocol supplied by OriGene, bait plasmid SCHAD 75–119 was tested for its autoactivation potential and ability to enter the nucleus and bind LexA operators.

Techniques: Western Blot, Control

Journal: Nature Communications

Article Title: A glucose-enriched lung pre-metastatic niche triggered by matrix stiffness-tuned exosomal miRNAs in hepatocellular carcinoma

doi: 10.1038/s41467-025-56878-8

Figure Lengend Snippet: a Predicted target proteins of miR-365a-5p using three publicly available bioinformatic tools. b Western blot analysis of TRPC4AP in vascular endothelial cells transfected with miR-365a-5p mimic or inhibitor. c Schematic illustration of luciferase reporter plasmids for TRPC4AP 3’UTR (upper panel) and relative luciferase activity determined after co-transfection of miRNA mimic and plasmids (lower panel) ( n = 3 biological replicates). d Intracellular Ca 2+ fluorescence signals in vascular endothelial cells transfected with miR-365a-5p mimic or inhibitor ( n = 5 biological replicates). Scale bars: 100 μm. e Effects of miR-365a-5p mimic or inhibitor on the activation state of CaMKII/ERK5/KLF2/4 pathway in vascular endothelial cells detected by western blot. The samples derive from the same experiment but different gels for p-CaMKII, another for CaMKII and KLF2, another for p-ERK5 and β-actin, and another for ERK5 and KLF4 were processed in parallel. f Effects of TRPC4AP overexpression on the activation state of CaMKII/ERK5/KLF2/4 pathway and the expression levels of ZO-1, VE-cadherin, and VEGFR2 in vascular endothelial cells transfected with miR-365a-5p mimic detected by western blot. The samples derive from the same experiment but different gels for p-CaMKII and p-ERK5, another for CaMKII, ERK5, and β-actin (left), another for TRPC4AP and VEGFR2, another for ZO-1, VE-cadherin and KLF2, and another for KLF4 and β-actin (right) were processed in parallel. g Effects of KLF4 silence on ZO-1 and VE-cadherin expressions in vascular endothelial cells transfected with miR-365a-5p inhibitor detected by western blot. The samples derive from the same experiment but different gels for ZO-1 and KLF4, and another for VE-cadherin and β-actin were processed in parallel. h Effects of KLF2 silence on VEGFR2 expression in vascular endothelial cells transfected with miR-365a-5p inhibitor detected by western blot. The samples derive from the same experiment but different gels for VEGFR2 and β-actin, and another for KLF2 were processed in parallel. Representative images are presented from indicated biologically independent experiments ( d ). Representative blot (20 μg protein per group) was shown from 3 biologically independent experiments ( b , e – h ), and β-actin was used to normalize protein quantification. Data are presented as mean ± SD, and P values were calculated using one-way ANOVA ( b – c , e – h ). WT, wild type; mut, mutant; Ca 2+ , calcium ions. Source data are provided as a Source Data file.

Article Snippet: Diluted primary antibodies were as follows: β-actin (Proteintech, 20536-1-AP, 1:20000), Fibronectin (Proteintech, 15613-1-AP, 1:1000), MMP9 (Boster, PB9668, 1:1000), GLUT1 (Proteintech, 66290-1-Ig, 1:500), SGLT2 (Proteintech, 24654-1-AP, 1:500), PFKP (CST, 8164, 1:1000), and HK2 (CST, 2867, 1:1000), PKM2 (Proteintech, 60268-1-Ig, 1:5000), ZO-1 (CST, 5406, 1:1000), VE-cadherin (Abcam, ab33168, 1:1000), VEGFR2 (CST, 2479, 1:1000), TSG101 (Proteintech, 14497-1-AP, 1:1000), CD63 (Proteintech, 25682-1-AP, 1:500), Hsp70 (Abmart, T55496, 1:1000), ALIX (Abcam, ab275377, 1:1000), Albumin (Abcam, ab207327, 1:2000), Cytochrome C (Proteintech, 66264-1-Ig, 1:5000), HMGA2 (CST, 5269, 1:1000), Rb (Abcam, ab181616, 1:1000), E2F1 (Invitrogen, 32-1400, 1:500), HDAC1 (Proteintch, 66085-1-Ig, 1:5000), Acetylated-Lysine (CST, 9441, 1:1000), Histone H3 (Proteintech, 17168-1-AP, 1:2000), α-tublin (Proteintech, 11224-1-AP, 1:5000), Flag (Proteintech, 66008-3-Ig, 1:5000), TRPC4AP (Proteintech, 66138-1-Ig, 1:500), Phospho-CaMKII (Thr286) (CST, 12716, 1:1000), CaMKII (Proteintech, 13730-1-AP, 1:1000), Phospho-Erk5 (Thr218/Tyr220) (CST, 3371, 1:1000), ERK5 (Abcam, ab40809, 1:5000), KLF2 (Bioss, bs-2772R, 1:250), KLF4 (Proteintech, 11880-1-AP, 1:1000), integrin β1 (CST, 34971 T, 1:1000), SNX17 (Proteintech, 68256-1-Ig, 1:5000), AMPKα (CST, 5832 T, 1:1000), Phospho-AMPKα (Thr172) (CST, 2535 T, 1:1000), and Na + /K + -ATPase (CST, 3010, 1:1000).

Techniques: Western Blot, Transfection, Luciferase, Activity Assay, Cotransfection, Fluorescence, Activation Assay, Over Expression, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: A glucose-enriched lung pre-metastatic niche triggered by matrix stiffness-tuned exosomal miRNAs in hepatocellular carcinoma

doi: 10.1038/s41467-025-56878-8

Figure Lengend Snippet: a FISH images of let-7d-5p and miR-365a-5p expressions in human HCC tissues with COL1 low /LOX low (low-stiffness group, n = 24 patients) and COL1 high /LOX high (high-stiffness group, n = 24 patients). Scale bars: white, 200 μm; red, 50 μm. b Quantification of let-7d-5p or miR-365a-5p expression in ( a ). c Survival curve analysis of HCC patients in the COL1 low /LOX low group ( n = 24 patients) and COL1 high /LOX high group ( n = 24 patients). d Survival curve analysis of HCC patients in the let-7d-5p low /miR-365a-5p low group ( n = 16 patients) and let-7d-5p high /miR-365a-5p high group ( n = 16 patients). e Mechanism by which matrix stiffness-tuned exosomal miRNAs modulate glucose enrichment during the formation of the lung pre-metastatic niche in HCC through inhibiting glucose uptake and consumption of lung fibroblasts and enhancing angiogenesis and vascular permeability. Schematic illustration was created in BioRender. Zhao, Y. (2025) https://BioRender.com/s25i400 . Specifically, two pathways including matrix stiffness-tuned exosomal let-7d-5p/HMGA2/E2F1 acetylation/GLUT1, PFKP, and HK2 in lung fibroblasts and matrix stiffness-tuned exosomal miR-365a-5p/TRPC4AP/Ca 2+ /CaMKII/ERK5/KLF2/4 in vascular endothelial cells synergistically promote glucose enrichment during lung pre-metastatic niche formation. Representative images are presented from indicated biologically independent experiments ( a ). Data are presented as mean ± SD ( b ), and P values were calculated using two-tailed unpaired Student’s t-test ( b ) or log-rank test ( c , d ). COL1 collagen I, LOX lysyloxidase, BMDCs bone marrow-derived cells, CTCs circulating tumor cells. Source data are provided as a Source Data file.

Article Snippet: Diluted primary antibodies were as follows: β-actin (Proteintech, 20536-1-AP, 1:20000), Fibronectin (Proteintech, 15613-1-AP, 1:1000), MMP9 (Boster, PB9668, 1:1000), GLUT1 (Proteintech, 66290-1-Ig, 1:500), SGLT2 (Proteintech, 24654-1-AP, 1:500), PFKP (CST, 8164, 1:1000), and HK2 (CST, 2867, 1:1000), PKM2 (Proteintech, 60268-1-Ig, 1:5000), ZO-1 (CST, 5406, 1:1000), VE-cadherin (Abcam, ab33168, 1:1000), VEGFR2 (CST, 2479, 1:1000), TSG101 (Proteintech, 14497-1-AP, 1:1000), CD63 (Proteintech, 25682-1-AP, 1:500), Hsp70 (Abmart, T55496, 1:1000), ALIX (Abcam, ab275377, 1:1000), Albumin (Abcam, ab207327, 1:2000), Cytochrome C (Proteintech, 66264-1-Ig, 1:5000), HMGA2 (CST, 5269, 1:1000), Rb (Abcam, ab181616, 1:1000), E2F1 (Invitrogen, 32-1400, 1:500), HDAC1 (Proteintch, 66085-1-Ig, 1:5000), Acetylated-Lysine (CST, 9441, 1:1000), Histone H3 (Proteintech, 17168-1-AP, 1:2000), α-tublin (Proteintech, 11224-1-AP, 1:5000), Flag (Proteintech, 66008-3-Ig, 1:5000), TRPC4AP (Proteintech, 66138-1-Ig, 1:500), Phospho-CaMKII (Thr286) (CST, 12716, 1:1000), CaMKII (Proteintech, 13730-1-AP, 1:1000), Phospho-Erk5 (Thr218/Tyr220) (CST, 3371, 1:1000), ERK5 (Abcam, ab40809, 1:5000), KLF2 (Bioss, bs-2772R, 1:250), KLF4 (Proteintech, 11880-1-AP, 1:1000), integrin β1 (CST, 34971 T, 1:1000), SNX17 (Proteintech, 68256-1-Ig, 1:5000), AMPKα (CST, 5832 T, 1:1000), Phospho-AMPKα (Thr172) (CST, 2535 T, 1:1000), and Na + /K + -ATPase (CST, 3010, 1:1000).

Techniques: Expressing, Permeability, Two Tailed Test, Derivative Assay