Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Schematic of CRISPR/Cas9-mediated PLXNB2 knockout (KO) with small guide (sg) RNA targeting second coding exon. (B) Western blots show Plexin-B2 expression in different SD2 GSCs, with β-actin as loading control. Note Plexin-B2 precursor at 240 kDa and mature form at 170 kDa. (C) IF images show Plexin-B2 expression in different SD2 GSCs, with Hoechst nuclear counterstain. (D) Left, schematic of atomic force microscopy (AFM) indentation method to probe cell stiffness by cantilever deflection. Middle, AFM indentation curves of different SD2 GSCs; right, box plots of cell stiffness, showing 25– 75% quartiles, median (line), and mean (plus sign). n= 6 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (E) Left, depiction of membrane tension measurement with optical tweezers. Middle, force measurements during tether extrusion (shaded box). Right, quantifications of tether extrusion forces. n=5 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (F) Left, schematic of FLIM of cell membranes labeled with Flipper-TR membrane dye, with low and high membrane tension associated with shorter and longer lifetimes, respectively. Middle top, representative FLIM images, with lifetime heatmap shown on right. Middle bottom, images show similar fluorescence intensities of Flipper-TR dye in WT and PB2 KO cells. Right top, violin plots show fluorescence lifetime from 3 images per group. Two-sided unpaired t-test. Right bottom, phasor plots of FLIM image data, with arrow indicating a shift to shorter lifetime values for PB2 KO cells. (G) Model of Plexin-B2 regulation of cortical contractility and membrane tension. Phalloidin staining show differences of F-actin network in WT and PB2 KO SD2 GSCs. DAPI for nuclear staining. Arrows point to stress fibers and spread-out contours of the WT GSCs.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: CRISPR, Knock-Out, Western Blot, Expressing, Control, Microscopy, Membrane, Labeling, Fluorescence, Staining

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Top, timeline for dextran uptake assay. Bottom, live-cell imaging of WT and PB2 KO SD2 GSCs labeled with SPY-Actin and exposed to dextran-Alexa488. Enlarged images of boxed areas are shown below. Quantification of the areas of dextran + clusters per cell are shown in box plots, with 25–75% quartiles, median (line), and mean (plus sign). n=85 cells for WT, n=44 cells for PB2 KO. Mann–Whitney–Wilcoxon test. (B) Top, live cell confocal plane images of WT and PB2 KO GSCs with side views of z-stacks showing intracellular localization of diffuse dextran-Alexa 488 signals in PB2 KO cells in addition to dextran endosome signals. In contrast, WT cells contained only dextran + endosomes. Bottom, histograms show fluorescence profiles showing bimodal distribution of dextran-Alexa 488 fluorescence intensities in PB2 KO GSCs (blue and brown arrows). n=177 cells for WT, n=161 cells for PB2 KO. Mann–Whitney–Wilcoxon test. (C, D) Left, schematic of myr-palm-GFP or -CFP attached to inner membrane leaflet. Right, live cell fluorescence imaging at 72 hr after transfection shows internalization of myr-palm-GFP or -CPF on endomembranes (arrow) in WT GSCs, in contrast to membrane retention of the probes (arrowhead) in PB2 KO GSCs. (E) Left, schematic of TauSTED super-resolution microscopy of GSCs labeled with MemGlow. Middle, TauSTED live-cell images show reduced endosomes (arrowheads) in PB2 KO cells compared to WT. Right, box plots show areas of MemGlow clusters in each cell. n=26 cells for WT, n=13 cells for PB2 KO. Two-sided unpaired t-test. (F) Working model of regulation of cortical and membrane tension by Plexin-B2, affecting endocytosis and membrane permeability in GSCs.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Live Cell Imaging, Labeling, MANN-WHITNEY, Fluorescence, Membrane, Imaging, Transfection, Super-Resolution Microscopy, Permeability

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Left, schematic of PH(PLCδ1)-GFP PIP2 probe. Right, live-cell imaging at 72 hr post transfection reveals that the PH(PLCδ1)-GFP probes were largely internalized in WT GSCs (arrow), but retained on membrane of PB2 KO GSCs (arrowhead). (B) Left, still images of videography show accumulation of the PH(PLCδ1)-GFP probes (arrow) in front of the nucleus (NucSpot) of migrating WT SD2 GSCs in tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Dashed lines delineate cell boundary. Long arrow denotes direction of migration. Right, quantifications of the ratio of PH(PLCδ1)-GFP fluorescence intensity at front vs. rear of GSCs during passage. n=13-16 cells per condition. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (C) Left, schematic of R(+8)-pre-GFP probe for negative surface charge of inner plasma membrane. Right, live-cell imaging at 72 hr post-transfection shows internalization of the probes (arrow) in WT GSCs, in contrast to the predominant membrane localization in PB2 KO GSCs (arrowhead). (D) Left, still images of videography show accumulation of the R(+8)-pre-GFP probes (arrow) at front zone of WT GSCs when traversing the 3 µm tunnel, but not in PB2 KO cells. Right, bar graphs show the ratio of R-pre-GFP fluorescence intensity at rear vs. front of GSCs when passing through tunnels. n=22 cells for WT, n=27 cells for PB2 KO. Mann–Whitney–Wilcoxon test. Data represent mean ± SEM. (E) Diagram illustrating voltage sensitive FluoVolt membrane dye, with fluorescent intensity quenched by voltage-sensitive electron transfer from electron-rich donor mediated by “molecular wire” in plasma membrane. (F) Left, FluoVolt live-cell imaging shows reduced FluoVolt fluorescent intensity in cell membrane of Plexin-B2 KO cells, consistent with higher negative charges of inner membrane. Right, box plots of membrane FluoVolt intensity. n=25 cells for WT, n=27 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. (G) Left, still images from videography show higher FluoVolt fluorescent signals at rear zone (arrowhead) of WT GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Migration direction is denoted by long arrow. Right, bar graphs show the ratio of FluoVolt intensity at rear vs. front during confined migration. n=15 cells per group. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (H) Live-cell images and quantifications show the effects of constitutive active (CA) RAP1B-V12 or dominant-negative (DN) RAP1B-N17 on FluoVolt intensity in WT or PB2 KO GSCs. n=25 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (I) Left, still images capture calcium localization (Fluo4-AM fluorescence, arrowhead) at the rear of WT GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Migration direction is denoted by long arrow. Right, bar graphs showing Fluo4-AM intensity ratio at rear vs. front in GSC during passage through tunnels. n=15-16 cells. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (J) Left, still images from videography show that calcium chelator BAPTA-AM disrupted the pattern of high FluoVolt signals at the rear of WT GSCs (arrowhead) during confined migration. Right, bar graphs show FluoVolt intensity ratio at rear vs. front of GSCs when traversing tunnels. n=21 cells for WT, n=16 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. (K) Model of Plexin-B2 signaling affecting membrane surface charge and electric field during polarized confined migration, with PIP2 enrichment at cell front and Ca 2+ at rear zone, leading to asymmetry of FluoVolt and R(+8)-pre-GFP.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Live Cell Imaging, Transfection, Membrane, Migration, Fluorescence, Comparison, Clinical Proteomics, MANN-WHITNEY, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Structure model of the extracellular domain of human Plexin-B2 show the locations of lock1 and lock2 mutations predicted to form disulfide bridges that lock the ring structure. (B) Western blots show absence of mature Plexin-B2 (170 kDa) in PB2 KO GSC, and expression of lock mutants in PB2 KO SD2 and SD3 GSCs. β-actin serves as a loading control. (C) Still images from videography show passage of GSCs (nuclei visualized by NucSpot) through microchannels with PB2 wildtype rescue construct but not lock mutants, nor PB2 with deletion of extracellular domain (dECTO). Chevrons point to 3 µm constrictions. (D) Box plots show velocity through constrictions, stalling time at constrictions, and sum of forward and backward movements, with 25–75% quartiles, minimal and maximal values (whiskers), median (line), and mean (cross). For velocity and sum of movements: n=17-20 cells per condition. For stalling time at constriction: n=14-28 cells per condition. One-way ANOVA followed by Dunnett’s multiple comparisons test. (E) Still images from videography show F-actin assembly (SPY-actin, arrowhead) at cell rear and MemGlow + endosomes (arrow) at cell front of SD3 GSCs with Plexin-B2 WT rescue but not mutant rescues when traversing 3 µm constrictions (chevrons). (F) Bar graphs showing fluorescence intensity ratio of SPY-actin and MemGlow at rear vs. front of GSCs during confined migration. n=10-18 cells per condition. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Data represent mean ± SEM. (G) Model of Plexin-B2 signaling and mechano-electrical regulation of membrane tension and membrane surface charge during polarized confined migration. Regionalized enrichment of endocytosis/PIP2 at cell front and F-actin/Ca 2+ at rear zone lead to asymmetry of FluoVolt and R(+8)-pre-GFP membrane probes.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Western Blot, Expressing, Control, Construct, Mutagenesis, Fluorescence, Migration, Membrane

Journal: Frontiers in Pharmacology

Article Title: Arctigenin Suppressed Epithelial-Mesenchymal Transition Through Wnt3a/β-Catenin Pathway in PQ-Induced Pulmonary Fibrosis

doi: 10.3389/fphar.2020.584098

Figure Lengend Snippet: ATG decreased PQ-induced EMT in mouse lung. (A) The locations and expressions of E-cadherin, Cytokeratin 18, α-SMA and Vimentin were determined by immunohistochemical staining in the lung sections. ** p < 0.01 versus NC group; <0.01 versus PQ-treated group. (B) The expressions of Vimentin, E-cadherin, Occludin, Wnt3a, β-Catenin, GSK-3β and pGSK-3β in the lung tissue of mice were evaluated by Western blot. β-actin served as an internal control. Data were presented as mean ± SD of three independent experiments. ** p < 0.01 versus NC group; p < 0.01 versus PQ-treated group.

Article Snippet: Wnt3a overexpression plasmid was constructed by inserting Wnt3a cDNA clone into a cytomegalovirus pCMV6-XL5 vector (OriGene Technologies, Rockville, MD, United States); the resulting plasmid was called pCMV6-XL5-Wnt3a.

Techniques: Immunohistochemical staining, Staining, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Arctigenin Suppressed Epithelial-Mesenchymal Transition Through Wnt3a/β-Catenin Pathway in PQ-Induced Pulmonary Fibrosis

doi: 10.3389/fphar.2020.584098

Figure Lengend Snippet: ATG reversed PQ-induced EMT in A549 cells. (A) Cell counting kit-8 (CCK8) was applied to measure the cytotoxicity of PQ on A549 cells at concentration of 0, 50, 100 μM for 24, 48, and 72 h, respectively. Values were represented as the means ± SD, * p < 0.05, versus 0 μM, n = 4. (B) Effect of ATG on cell proliferation was determined using CCK8 assay. A459 cells were treated with ATG at concentrations of 0, 25, 50, and 100 μM for 24, 48, and 72 h, respectively. Values were represented as the means ± SD, n = 4. (C) Phalloidin immunofluorescence was performed to show the effect of ATG on EMT in-vitro. (D) A549 cells were pre-treated with ATG (100 and 200 μM). Two hours later, the cells were co-incubated with 50 μM PQ for 48 h. The expression of Vimentin, E-cadherin, Occludin, Wnt3a, β-Catenin, GSK-3β and P-GSK-3β in A549 cells were evaluated by Western blot. β-actin served as an internal control. Data were presented as mean ± SD of three independent experiments. ** p < 0.01 versus NC group; p < 0.01 versus PQ-treated control group. (E) After A549 cells were treated with PQ with or without different doses of ATG for 72 h, confluent epithelial monolayers were wounded and incubated with serum-free DMSO. At 24 h after wounding, cell migration was assessed using a microscope equipped with a camera.

Article Snippet: Wnt3a overexpression plasmid was constructed by inserting Wnt3a cDNA clone into a cytomegalovirus pCMV6-XL5 vector (OriGene Technologies, Rockville, MD, United States); the resulting plasmid was called pCMV6-XL5-Wnt3a.

Techniques: Cell Counting, Concentration Assay, CCK-8 Assay, Immunofluorescence, In Vitro, Incubation, Expressing, Western Blot, Control, Migration, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Arctigenin Suppressed Epithelial-Mesenchymal Transition Through Wnt3a/β-Catenin Pathway in PQ-Induced Pulmonary Fibrosis

doi: 10.3389/fphar.2020.584098

Figure Lengend Snippet: Wnt3a overexpression rescued the inhibitory effect of ATG on PQ-induced EMT-like phenotypic changes in A549 cells. (A) After transfection of Wnt3a overexpression plasmids for 24 h, the cells were treated in the presence or absence of PQ (50 μM) or ATG (100 μM) for 48 h. After treatment, whole cell lysates were immunoblotted with antibodies against vimentin, E-cadherin, Occludin, Wnt3a, β-Catenin, GSK-3β, P-GSK-3β and β -actin. The experiments were repeated for three times with similar results, and a representative immunoblot was shown for each protein. ** p < 0.01 versus NC group; * p < 0.05 versus PQ-treated group, p < 0.01 versus PQ-treated group, $ p < 0.05 versus PQ+ATG group, $$ p < 0.01 versus PQ+ATG group. (B) The expression of E-cadherin and Vimentin were further analyzed by immunofluorescence.

Article Snippet: Wnt3a overexpression plasmid was constructed by inserting Wnt3a cDNA clone into a cytomegalovirus pCMV6-XL5 vector (OriGene Technologies, Rockville, MD, United States); the resulting plasmid was called pCMV6-XL5-Wnt3a.

Techniques: Over Expression, Transfection, Western Blot, Expressing, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 1. Generation of Nckx4/ knock-out mice. A, diagram illustrating the Nckx4 (slc24a4) locus, the targeting vector, and the targeted locus. The location of Nckx4 exons 5, 6, 7, and 8 is indicated. The neomycin (neo) gene driven by a pol II promoter replaced exons 6 and 7 in the targeted locus. The thymidine kinase gene driven by a herpes simplex virus promoter was used for negative selection. The location of the probe used for Southern analysis and the length of the two diagnostic NsiI fragments of 9.9 and 4.9 kb are illustrated at the top and bottom of the diagram. The position of the primers used for PCR analysis, P1, P2, and P3, which generate diagnostic fragments of 175or350bparealsoindicated.B,genotypeanalysisbySouthernblot(left)or PCR (right) of DNA from wild-type (/), heterozygous (/), or Nckx4 knock-out(/)animals.C,Northernblot(leftpanel)analysisof10goftotal RNA samples, or immunoblot (right panel) analysis of 20 g of membrane proteins samples, isolated from the indicated tissues of mice of different genotype.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Plasmid Preparation, Virus, Selection, Diagnostic Assay, Western Blot, Membrane, Isolation

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 2. Nckx4 knock-out does not change brain morphology or the expression of other Na/Ca2-exchangers. A, serial coronal sections through the hypothalamus of wild-type adult mice were labeled for Nckx4 transcripts with an antisense or a sense control probe, as indicated. B, hematoxylin and eosin-stained coronal paraffin sections through the PVN (wild-type, /; Nckx4 knock-out, /). No differences were observed between genotypes in this or in other brain regions examined in either coronal or parasagittal stained sections (data not shown). In both panels A and B, the right-hand images are magnified views of the area in the box in the corresponding left-hand images. Scale bars: left-hand images, 1 mm; right-hand images, 200 m. C, Northern blots of 10 g of total RNA samples isolated from adult brain of wild-type (/), heterozygous (/), or Nckx4 knock-out (/) mice were analyzed with probes for the indicated Na/Ca2-exchangers genes. The blots were then stripped and reprobed for Gapdh as a loading control. All images in this figure are representative of independent experiments from 3 different animals of each genotype.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Expressing, Labeling, Control, Staining, Northern Blot, Isolation

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 3. Nckx4/ mice are anorexic and hypophagic. Knock-out (/) and wild-type (/) and heterozygous (/) littermates, as indicated, were fed ad libitum either standard or enriched laboratory chow (4.5% or 9% fat). A, animals were weighed weekly from 4 weeks of age onwards (n 12–15 for each group; however, measurements were not made for all animals for all time points, all conditions or both sexes; therefore, some data points corre- spond to a smaller n value). B, animals were weighed at birth (P0; n 10) and then at weaning (n 63, 107, and 56 for wild-type, heterozygous, and knock- out, respectively). C, weight (n 8) and length (nose to anus; n 10) were measured on adult animals. D, food (n 8 for females and n 10 for males) and water consumption (n 6) were measured in adult animals. In all cases the mean S.E. is plotted. Statistical significance of differences between knock-out and wild-type is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 4. Body temperature and tissue weight. A, basal body temperature was determined rectally in free-fed or fasted adult animals (n 4–8). B, tissues were extracted from adult (5–6 months of age) animals (n 5–7), blotted dry and weighed. Fat corresponds to total abdominal cavity adipose tissue. Average values S.E. are plotted. Statistical significance of the differences between wild-type (/; WT) and Nckx4 knock-out (/; KO) is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. C, microscopic images of hematoxylin and eosin-labeled sections from paraffin-embedded samples of abdominal fat pad or liver are shown. Scale bar, 100 m. These images are representative of samples from at last 5 pairs of animals of each genotype.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Labeling

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 5. Health parameters, motivation and olfaction in Nckx4/ knock-out and wild-type (/) mice. A, a glucose tolerance test was performed on 5–6-month-old animals following overnight fasting. Glucose (2 mg/g body weight) was injected intraperitoneally, and blood glucose was measured just prior and at intervals thereafter. Average values S.E. are plotted (n 6). B, a Kaplan Meier survival curve is plotted for 18 wild-type (/) and 13 Nckx4 knock-out (/) animals. A log-rank (Mantel-Cox) test indicates a significant difference between genotypes, p 0.05. C, cumulative food consumption was measured in adult mice immediately following a 24-h fasting period (n 11). D, time taken to find buried urine-scented litter (left) or buried food pellet (right) was measured for adult mice (n 11–13). Statistical significance of the differences between genotypes determined by analysis of variance using the Bonferroni post-test is indicated where present: *, p 0.05; ***, p 0.001.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Injection

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 7. Neurons in the PVN are activated in Nckx4/ mice. c-Fos expression was detected by immunofluorescence in sections from hypothal- amus of adult mice. WT, wild-type; KO, Nckx4/. All animals were fasted for 48 h and subsequently treated for 30 min prior to sacrifice either with glucose injection (intraperitoneally; 2 mg/g body weight) or by perfusion with Ca2- free Krebs-Henseleit solution containing 1 mM EGTA, normal Krebs-Henseleit solution containing 100 nM insulin, or normal Krebs-Henseleit solution con- taining 1 M of the MC4R antagonist, SHU9119 (SHU). The total number of c-Fos positive nuclei contained within the PVN area were counted for each section, and the averaged data S.E. from 4 to 7 independent experiments are shown in the panel at the lower right. Statistical significance is indicated where present: compared with wild-type untreated: *, p 0.05; **, p 0.01; ***, p 0.001; compared with glucose-treated wild-type: #, p 0.05; com- pared with knock-out untreated: †††, p 0.001. Scale bar, 500 m.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Expressing, Immunofluorescence, Injection, Knock-Out

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 6. Blood hormones and hypothalamic mediators. A, serum sam- ples from overnight fasted female (F) or male (M) animals of wild-type (/) or Nckx4 knock-out (/) genotype (n 7–10) were tested for the indicated factors. Levels are plotted normalized to the wild-type female levels in each case. Statistical significance of differences between knock-out and wild-type for each gender is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. The values corresponding to 100% are: insulin, 1.51 ng/ml; leptin, 55.5 ng/ml; glucose, 261 mg/dl; triglycerides, 0.49 mg/ml; T4, 5.0 g/dl; cortisol, 4.1 g/dl; MSH, 110 pmol/liter. B, localization of MSH and AgRP was deter- mined by immunofluorescence in coronal sections through the hypotha- lamic region of adult female wild-type (/) or Nckx4 knock-out (/) mice. For illustrative purposes, bilaterally symmetric image halves are compared between Nckx4/ and Nckx4/ animals for sections taken at the level of the PVN and ARC. The PVN and ARC regions are circled. Scale bar, 500 m. The right-hand most image pairs are magnified views of the ARC regions from the central image pairs, illustrating the punctate nature of staining, consis- tent with packaging of peptide hormones in vesicles destined for secretion. Scale bar, 100 m. These images are representative of three independent experiments.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Immunofluorescence, Staining

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 9. MSH-induced Ca2 signals. A, the fura-2 340/380 fluorescent exci- tation ratios are shown for individual neurons, each plotted with a uniquely colored trace, from primary hypothalamic cultures from either wild-type (WT) or Nckx4 knock-out (KO) mice. Cells were continually perfused with Hepes- buffered saline, containing either 1 M MSH or 75 mM KCl where indicated. B, the number of cells in each microscopic field that responded with a change in fura-2 ratio, as shown in panel A, following MSH treatment is plotted. Aver- ages S.E. for 18–20 microscopic fields from six independent cultures are shown. ***, p 0.001. C, GT1–7 cells were infected with lentivirus expressing mouse NCKX4, or a deleted control construct, and subsequently assayed for Ca2 response 2 days later. The fura-2 340/380 excitation ratios for individual responding cells are shown, each plotted with a uniquely colored trace. D, the average number of responding cells per field is summarized. n 9; **, p 0.01. E, GT1–7 cells in Opti-MEM were treated with vehicle only (control) or with 10 M SKF96365 or 10 M U73122 at 37 °C for 15 min with the indicated concentration of MSH, followed by extraction and determination of cAMP levels (n 5–8).

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Saline, Infection, Expressing, Control, Construct, Concentration Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 8. The Nckx4/ phenotype is MC4R-dependent. Age-matched adult mice of different genotypes were compared: WT, wild-type; N4KO, Nckx4/; M4KO, Mc4r/; DKO, Nckx4/; Mc4r/. A, body weight (n 6–9); B, food consumption (n 10); C and D, c-Fos immunofluorescence in coronal hypothalamic sections of fasted animals, as described in the legend toFig.7(n4–7).InpanelsAandB,N4KO,M4KO,andDKOareallsignificantly different from WT, and in panels A, B, and D, M4KO and DKO are significantly different from N4KO, with p 0.001 in all cases. Scale bar, 500 m.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 11. Control of oxytocin expression in the hypothalamic paraven- tricular nucleus. Oxytocin expression was detected by immunofluorescence in adult mice. WT, wild-type; KO, Nckx4/. Animals were fasted for 48 h and then subjected to a 30-min transcardial perfusion with Krebs-Henseleit solu- tion containing 1 M MSH, 10 M SKF96365, or 10 M U73122, as indicated. The oxytocin-positive cells were scored as being of high or low staining inten- sity, and the numbers pooled across all sections from each of five animals per treatmentgroup.Thetotalnumberofcellsscoredvariedfrom522to1288per animal, but the averaged values were not different between groups (800 cells). The averaged ratios of high/low stained cells S.E. are shown in the panel at lower right. Statistical significance is indicated where present: com- pared with wild-type untreated: **, p 0.01; ***, p 0.001. Scale bar, 100 m.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Control, Expressing, Immunofluorescence, Staining

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 12. A model for MC4R-dependent Ca2 signaling and the role of NCKX4. MSH binding to the MC4R is shown to active Gq, via the exchange of GTP for GDP (upper row). It is important to note that MC4R may constitutively activate Gq even in the absence of ligand (54). So activated, Gq goes on to bind and stimulate PLC-, which subsequently cleaves phosphatidylinositol 1,4-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The membrane-bound diacylglycerol then activates the Ca2-selective entry channel, TRPC6, which results in an elevation of cytosolic [Ca2] and consequent neuronal activation, which may include the initiation of action potentials (15) and the release of oxytocin (16, 41) (lower row). Normally, NCKX4 acts locally to extrude the Ca2 that enters via TRPC6, thus moderating the signaling pathway so only strong signals drive neuronal activation. In the absence of this moderating influence, even weak, constitutive, signaling via MC4R can result in sufficient Ca2 entry to initiate neuronal activation.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Binding Assay, Membrane, Activation Assay