Journal: Cell reports

Article Title: TRPM4 mediates a subthreshold membrane potential oscillation in respiratory chemoreceptor neurons that drives pacemaker firing and breathing

doi: 10.1016/j.celrep.2021.108714

Figure Lengend Snippet:

Article Snippet: GFP was visualized using chicken anti-GFP (1:1000, Aves Labs, Davis, CA, Cat # GFP-1020, RRID: AB_10000240) and Alexa Fluor 488 AffiniPure Donkey Anti-Chicken (1:500, Jackson Immunoresearch, West Grove, PA, Cat # 703–545-155, RRID:AB_2340375).

Techniques: Recombinant, RNAscope, Multiplex Assay, Transgenic Assay, Software

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: International journal of molecular sciences

Article Title: Drp1 Overexpression Decreases Insulin Content in Pancreatic MIN6 Cells.

doi: 10.3390/ijms232012338

Figure Lengend Snippet: Figure 1. Insulin secretion of Drp1-KD MIN6 cells overexpressing Drp1. Drp1-KD MIN6 cells were transiently transfected with Drp1-containing plasmid using electroporation. (A) Immunoblot and densitometric quantification of overexpressed Drp1. Data are represented as mean ± SEM (n = 3) (B) Insulin release is expressed as nanograms of insulin per microgram of DNA. Data are presented as mean ± SEM (n = 4), and n-values represent independent experiments. The statistical significance

Article Snippet: Plasmid DNA extraction and transient transfection: The empty vector pcDNA3.1 (Plasmid #138209) and pcDNA3.1 (+) Drp1 (the latter was gifted by David Chan plasmid # 34706) were purchased from Addgene.

Techniques: Transfection, Plasmid Preparation, Electroporation, Western Blot

Journal: International journal of molecular sciences

Article Title: Drp1 Overexpression Decreases Insulin Content in Pancreatic MIN6 Cells.

doi: 10.3390/ijms232012338

Figure Lengend Snippet: Figure 2. Alteration of mitochondrial morphology but not respiratory machinery by Drp1 overexpres- sion in MIN6 cells. MIN6 cells were transiently transfected using Drp1 plasmid by the electroporation technique to overexpress Drp1: control (black bars) and overexpressed Drp1 (white bars). (A) The overexpression efficiency of Drp1 was confirmed by qPCR. HPRT was used as a control. (B) Repre- sentative immunoblot and densitometric quantification of Drp1 protein content. Tubulin was used as a loading control. (C) Relative levels of Mfn1, Mfn2, and Opa1 mRNA were measured by qPCR, using HPRT as the housekeeper gene. (D) Live confocal imaging of transiently transfected MIN6 cells that were stained with MitoTracker Red FM for 30 min. Representative confocal images of the control and Drp1-overexpressed cells. (E) Immunoblot and densitometric quantification of OXPHOS complexes. Data are represented as mean ± SEM (n = 6), and n-values represent independent experiments. The statistical significance of mean differences was tested by an unpaired two-tailed Student’s t-test. p < 0.05 (*), p < 0.0001 (****).

Article Snippet: Plasmid DNA extraction and transient transfection: The empty vector pcDNA3.1 (Plasmid #138209) and pcDNA3.1 (+) Drp1 (the latter was gifted by David Chan plasmid # 34706) were purchased from Addgene.

Techniques: Transfection, Plasmid Preparation, Electroporation, Control, Over Expression, Western Blot, Imaging, Staining, Two Tailed Test

Journal: International journal of molecular sciences

Article Title: Drp1 Overexpression Decreases Insulin Content in Pancreatic MIN6 Cells.

doi: 10.3390/ijms232012338

Figure Lengend Snippet: Figure 3. Insulin content and GSIS of Drp1-overexpressed MIN6 cells. MIN6 cells were transiently transfected with the Drp1 plasmid by the electroporation technique to overexpress Drp1; control (black bars) and overexpressed Drp1 (white bars). (A) Insulin content expressed as a % decrease. (B) Relative levels of Ins1 and Ins2 mRNA were measured by qPCR. HPRT was used as a control. Data are represented as mean ± SEM (n = 3). (C) Insulin release is expressed as nanograms per microgram of DNA. (D) Insulin release is expressed as a percentage of content. Data are represented as mean ± SEM (n = 4). (E) Averaged, time-resolved oxygen consumption traces measured with the

Article Snippet: Plasmid DNA extraction and transient transfection: The empty vector pcDNA3.1 (Plasmid #138209) and pcDNA3.1 (+) Drp1 (the latter was gifted by David Chan plasmid # 34706) were purchased from Addgene.

Techniques: Transfection, Plasmid Preparation, Electroporation, Control

Journal: International journal of molecular sciences

Article Title: Drp1 Overexpression Decreases Insulin Content in Pancreatic MIN6 Cells.

doi: 10.3390/ijms232012338

Figure Lengend Snippet: Figure 4. Regulation of mRNA expression of Drp1-overexpressed MIN6 cells. MIN6 cells were transiently transfected with the Drp1 plasmid using the electroporation technique to overexpress Drp1; control (black bars) and overexpressed Drp1 (white bars). (A) Relative levels of BiP and Grp94 mRNA were measured by qPCR. (B) Relative levels of Chop, Hsp60, and Inadl mRNA were measured by qPCR. (C) Relative levels of Atpif1, Fgf21, and Atf4 mRNA were measured by qPCR. Data are represented as mean ± SEM (n = 3), and n-values represent independent experiments. (D) Schematic model of the impact of Drp1 in pancreatic β cells. The statistical significance of mean differences was tested by an unpaired two-tailed Student’s t-test to compare two variables. p < 0.05 (*), p < 0.01 (**).

Article Snippet: Plasmid DNA extraction and transient transfection: The empty vector pcDNA3.1 (Plasmid #138209) and pcDNA3.1 (+) Drp1 (the latter was gifted by David Chan plasmid # 34706) were purchased from Addgene.

Techniques: Expressing, Transfection, Plasmid Preparation, Electroporation, Control, Two Tailed Test

Journal: Journal of Lipid Research

Article Title: Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4α

doi: 10.1194/jlr.m500430-jlr200

Figure Lengend Snippet: Fig. 3. Circadian rhythm of the expression of cholesterol 7a-hydroxylase (CYP7A1) and sterol 12a-hydroxylase (CYP8B1) mRNAs in the livers of HNF4aDL and HNF4aF/F mice. A, B: Northern blot analysis of mRNA in livers from 45-day-old mice at 10 AM (A) and 10 PM (B). C: Real-time PCR of liver mRNA for CYP7A1 at 10 AM (left) and 10 PM (right). Data are means 6 SEM (n 5 8 for each group). Significant differences compared with the other three groups: * P , 0.01. D, E: Western blots of total liver proteins (100 mg) at 10 AM (D) and 10 PM (E). CYP7A1 and b-actin polyclonal antibodies were used to assess protein expression.

Article Snippet: The membrane was incubated for 1 h with PBS containing 0.1% Tween 20, 5% dry milk, 6% glycine, and 3% fetal bovine serum and then for 1 h with a 1:1,000 dilution of primary antibodies against mouse CYP7A1 (a generous gift from Dr. David W. Russell, University of Texas Southwestern Medical Center) and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Northern Blot, Real-time Polymerase Chain Reaction, Western Blot

Journal: Experimental eye research

Article Title: Loss of cone function without degeneration in a novel Gnat2 knock-out mouse

doi: 10.1016/j.exer.2018.02.024

Figure Lengend Snippet: Targeted deletion of Gnat2 decreases Gnat2 mRNA expression while Rho expression remains unchanged. (A) Targeted deletion strategy used to generate the Gnat2 −/− mouse line (adapted from KOMP). (B) Representative qRT-PCR results from wild-type (filled circles) and Gnat2 −/− (filled diamonds) littermates 3 months of age. Threshold cycle (C T ) values are shown as a function of cDNA dilutions used for Gnat2 (green filled) and Hprt (blue filled). Across 4 independent experiments, the C T values for Gnat2 in the knockout were dramatically increased, indicating that there was at least a 60,000-fold reduction in expression in the knockout (p-value < 0.001). (C) Relative rhodopsin expression was unchanged between the knockout and control littermate (p-value = 0.75; n = 4–7). Bar graph shows mean 2 −ΔΔCT ± SD.

Article Snippet: TaqMan Gene Expression Assays (ThermoFisher Scientific) were used to quantify mRNA expression of Gnat2 , Hprt , and Rho (Mm00492394_m1, Mm03024075_m1, and Mm01184405_m1 respectively) (UC Davis Real-Time PCR Research and Diagnostics Core Facility).

Techniques: Expressing, Quantitative RT-PCR, Knock-Out, Control

Journal: Experimental eye research

Article Title: Loss of cone function without degeneration in a novel Gnat2 knock-out mouse

doi: 10.1016/j.exer.2018.02.024

Figure Lengend Snippet: Antibodies and stains used for immunohistochemical staining of retinal sections and flat-mounts.

Article Snippet: TaqMan Gene Expression Assays (ThermoFisher Scientific) were used to quantify mRNA expression of Gnat2 , Hprt , and Rho (Mm00492394_m1, Mm03024075_m1, and Mm01184405_m1 respectively) (UC Davis Real-Time PCR Research and Diagnostics Core Facility).

Techniques: Immunohistochemical staining, Staining, CRAfT Assay

Journal: Experimental eye research

Article Title: Loss of cone function without degeneration in a novel Gnat2 knock-out mouse

doi: 10.1016/j.exer.2018.02.024

Figure Lengend Snippet: Knockout retinas display normal rod-driven a- and b-wave amplitudes. (A) Comparison of the average response of Gnat2 −/− (green) and wild-type (WT, blue) animals to a flash of 100 photons μm −2 . (B) Average bright flash responses of Gnat2 −/− and WT animals (2 × 10 6 photons μm −2 . (C–D) Peak electroretinogram a-wave (C) and b-wave (D) amplitudes of WT (blue circles) and Gnat2 −/− (green diamonds) mice in response to increasingly bright flashes of 510 nm light (32–1.22 × 10 6 photons μm −2 ).

Article Snippet: TaqMan Gene Expression Assays (ThermoFisher Scientific) were used to quantify mRNA expression of Gnat2 , Hprt , and Rho (Mm00492394_m1, Mm03024075_m1, and Mm01184405_m1 respectively) (UC Davis Real-Time PCR Research and Diagnostics Core Facility).

Techniques: Knock-Out, Comparison

Journal: Experimental eye research

Article Title: Loss of cone function without degeneration in a novel Gnat2 knock-out mouse

doi: 10.1016/j.exer.2018.02.024

Figure Lengend Snippet: Normal cone photoreceptor morphology and number in Gnat2 −/− retinas. (A) In retinal sections, PNA staining (orange) showed normal cone morphology and Gnat2 staining (green) confirmed loss of Gnat2 protein in the knockout. Shown here are representative wild-type and Gnat2 −/− littermates, 5 months of age. (B) M-opsin staining (green) was the same in wild-type and knockout littermates. (C) Representative 150 μm × 150 μm areas of control and knockout littermate retinal flat mounts stained with PNA to visualize cones. Quantification revealed no difference in the number of cones per unit area (p-value = 0.4817; n = 5 measurements per flatmount, 4 flatmounts from each strain). All scale bars are 25 μm.

Article Snippet: TaqMan Gene Expression Assays (ThermoFisher Scientific) were used to quantify mRNA expression of Gnat2 , Hprt , and Rho (Mm00492394_m1, Mm03024075_m1, and Mm01184405_m1 respectively) (UC Davis Real-Time PCR Research and Diagnostics Core Facility).

Techniques: Staining, Knock-Out, Control

Journal: Experimental eye research

Article Title: Loss of cone function without degeneration in a novel Gnat2 knock-out mouse

doi: 10.1016/j.exer.2018.02.024

Figure Lengend Snippet: Gnat2 −/− retinas do not exhibit retinal degeneration. (A) OCT B-scans through the optic nerve head show normal layering of WT and Gnat2 −/− retinas. Shown here are a representative 5-month-old WT retina (top) and a representative 9-month-old Gnat2 −/− retina (bottom). The optic nerve head is located at the far right of each image. Scale bars indicates 100 μm. (B) The outer retinal thickness (choroid to INL) was on average the same in 5-month-old (5 mo) WT and Gnat2 −/− mice, as well as 9 month old (9 mo) Gnat2 −/− mice. Data displayed are mean ± range (n = 4–6 eyes for each). (C) Maximum intensity projections (through 5 μm in z) of the outer plexiform layer of Gnat2 +/+ and Gnat2 −/− littermate flat mounts stained for Iba1 (green, microglia) and PNA (orange, cones) reveal normal ramified microglia at the photoreceptor synapses. Scale bars indicate 50 μm. (D) Representative images of retinal sections from C57BL/6J and Gnat2 −/− stained for CRALBP (orange) and GFAP (green) reveal no differences in reactive gliosis. Scale bars indicate 50 μm. IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium.

Article Snippet: TaqMan Gene Expression Assays (ThermoFisher Scientific) were used to quantify mRNA expression of Gnat2 , Hprt , and Rho (Mm00492394_m1, Mm03024075_m1, and Mm01184405_m1 respectively) (UC Davis Real-Time PCR Research and Diagnostics Core Facility).

Techniques: Staining

Journal: Experimental eye research

Article Title: Loss of cone function without degeneration in a novel Gnat2 knock-out mouse

doi: 10.1016/j.exer.2018.02.024

Figure Lengend Snippet: Cone-driven ERG responses are absent in Gnat2 −/− mice. Electroretinogram a-wave amplitudes of WT (blue circles) and Gnat2 −/− (green diamonds) mice in response to saturating flashes (365 nm; 1.22 × 10 6 photons μm −2 ) delivered on increasingly intense 510 nm backgrounds. Insets: (A) Average electroretinogram of WT (blue) and Gnat2 −/− (green) mice in response to a 1 ms saturating flash delivered on top of a rod-saturating background (510 nm; 1 × 10 5 photons μm −2 s −1 ). The small deflection in the Gnat2 −/− trace is an electrical artifact. (B) Expanded view of WT and Gnat2 −/− a-wave amplitudes at the brightest background intensities. Error bars (frequently smaller than the points themselves) represent SEM.

Article Snippet: TaqMan Gene Expression Assays (ThermoFisher Scientific) were used to quantify mRNA expression of Gnat2 , Hprt , and Rho (Mm00492394_m1, Mm03024075_m1, and Mm01184405_m1 respectively) (UC Davis Real-Time PCR Research and Diagnostics Core Facility).

Techniques: