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    Cedarlane mice hypothalamic embryonic cells n1
    FIGURE 2 | CREB upregulates Klf10 promoter activity in embryonic <t>hypothalamic</t> cells. (A) Plasmids containing different fragments of the mouse Klf10 promoter region were cloned into the pGL3-Basic one. In some constructs, CREB binding sites were mutated at positions −241 and/ or −1840 bp as shown. (B) <t>mHypoE-N1</t> cells were transiently transfected with 800 ng of pKlf10-1977 or pKlf10-516, or the mutated version p1977, p516, together with 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (C) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p516, p916, p1511, p1839 or p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (D) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p1977, 2p1977, or, 3p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. 24 h after transfection, firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induction was calculated relative to basal levels (without CREB overexpression). The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. (E) Phospho-CREB (pCREB) is recruited to the Klf10 gene promoter in vivo and in vitro at −241 and −1840 sites. Chromatin immunoprecipitation assays were performed from 17-day-old embryos from wild-type mice hypothalamus (Hyp-E17) and from mHypoE-N1 cells. PCR products derived from ChIP-enriched genomic DNA showing CREB binding sites at positions −241 and −1840 bp in the pro- moter region of Klf10. Input; α-pCREB: Anti-phosphoCREB; Ig: IgG immunoprecipitation. A representative gel of 3 replicates in three independent experiments is shown. (F) Normalized densitometry values of bound pCREB (pCREB/input) at positions −241 and −1840 bp in the Klf10 promoter region. The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. A. U.: Arbitrary Units.
    Mice Hypothalamic Embryonic Cells N1, supplied by Cedarlane, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Klf10 Regulates the Emergence of Glial Phenotypes During Hypothalamic Development."

    Article Title: Klf10 Regulates the Emergence of Glial Phenotypes During Hypothalamic Development.

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.70020

    FIGURE 2 | CREB upregulates Klf10 promoter activity in embryonic hypothalamic cells. (A) Plasmids containing different fragments of the mouse Klf10 promoter region were cloned into the pGL3-Basic one. In some constructs, CREB binding sites were mutated at positions −241 and/ or −1840 bp as shown. (B) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or pKlf10-516, or the mutated version p1977, p516, together with 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (C) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p516, p916, p1511, p1839 or p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (D) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p1977, 2p1977, or, 3p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. 24 h after transfection, firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induction was calculated relative to basal levels (without CREB overexpression). The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. (E) Phospho-CREB (pCREB) is recruited to the Klf10 gene promoter in vivo and in vitro at −241 and −1840 sites. Chromatin immunoprecipitation assays were performed from 17-day-old embryos from wild-type mice hypothalamus (Hyp-E17) and from mHypoE-N1 cells. PCR products derived from ChIP-enriched genomic DNA showing CREB binding sites at positions −241 and −1840 bp in the pro- moter region of Klf10. Input; α-pCREB: Anti-phosphoCREB; Ig: IgG immunoprecipitation. A representative gel of 3 replicates in three independent experiments is shown. (F) Normalized densitometry values of bound pCREB (pCREB/input) at positions −241 and −1840 bp in the Klf10 promoter region. The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. A. U.: Arbitrary Units.
    Figure Legend Snippet: FIGURE 2 | CREB upregulates Klf10 promoter activity in embryonic hypothalamic cells. (A) Plasmids containing different fragments of the mouse Klf10 promoter region were cloned into the pGL3-Basic one. In some constructs, CREB binding sites were mutated at positions −241 and/ or −1840 bp as shown. (B) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or pKlf10-516, or the mutated version p1977, p516, together with 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (C) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p516, p916, p1511, p1839 or p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (D) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p1977, 2p1977, or, 3p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. 24 h after transfection, firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induction was calculated relative to basal levels (without CREB overexpression). The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. (E) Phospho-CREB (pCREB) is recruited to the Klf10 gene promoter in vivo and in vitro at −241 and −1840 sites. Chromatin immunoprecipitation assays were performed from 17-day-old embryos from wild-type mice hypothalamus (Hyp-E17) and from mHypoE-N1 cells. PCR products derived from ChIP-enriched genomic DNA showing CREB binding sites at positions −241 and −1840 bp in the pro- moter region of Klf10. Input; α-pCREB: Anti-phosphoCREB; Ig: IgG immunoprecipitation. A representative gel of 3 replicates in three independent experiments is shown. (F) Normalized densitometry values of bound pCREB (pCREB/input) at positions −241 and −1840 bp in the Klf10 promoter region. The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. A. U.: Arbitrary Units.

    Techniques Used: Activity Assay, Clone Assay, Construct, Binding Assay, Transfection, Plasmid Preparation, Concentration Assay, Luciferase, Over Expression, In Vivo, In Vitro, Chromatin Immunoprecipitation, Derivative Assay, Immunoprecipitation

    FIGURE 3 | Klf10 expression is upregulated by BDNF in embryonic hypothalamic cells. (A) Left panel. Expression of the Trk neurotrophin re- ceptors at 17.5-day-old embryos from wild-type mice hypothalamus (Hyp-E17.5) and in mHypoE-N1 cells was measured by RT-PCR assays. Right panel. Normalized densitometry values of Trk receptors (NTRs) expression (NTRs cDNA/actin cDNA) in the Hyp-E17.5 and mHypoE-N1 cells. (B) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977, pKlf10-516, or the mutated version p1977, p516. After transfection, cells were cultured for 12 h in DMEM with 5% FBS and then were cultured for 12 h in DMEM with 0.5% FBS. Subsequently, the p38 MAP kinase inhibitor (SB203580) or CREB inhibitor (666–15) was added at a final concentration of 10 μM for 30 min or 5 μM for 60 min, respectively, before the addition of 50 ng/mL BDNF; cells were then cultured for 24 h. Firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induc- tion was calculated relative to untreated or BDNF-treated conditions. (C) mHypoE-N1 cells cultured with 0.5% fetal bovine serum were treated with 50 ng/mL of BDNF for 24 h. The p38 (SB203580, 10 μM) or CREB (666–15, 5 μM) inhibitors were added to the culture 30 or 60 min, respectively, be- fore BDNF treatment. Klf10 mRNA levels were determined by qPCR and calculated as the ratio of Klf10 cDNA/Ubc cDNA signal. Results are plotted and expressed as relative fold change compared to untreated cells. The solid line represents the mean ± SEM of three replicates in three independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001.
    Figure Legend Snippet: FIGURE 3 | Klf10 expression is upregulated by BDNF in embryonic hypothalamic cells. (A) Left panel. Expression of the Trk neurotrophin re- ceptors at 17.5-day-old embryos from wild-type mice hypothalamus (Hyp-E17.5) and in mHypoE-N1 cells was measured by RT-PCR assays. Right panel. Normalized densitometry values of Trk receptors (NTRs) expression (NTRs cDNA/actin cDNA) in the Hyp-E17.5 and mHypoE-N1 cells. (B) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977, pKlf10-516, or the mutated version p1977, p516. After transfection, cells were cultured for 12 h in DMEM with 5% FBS and then were cultured for 12 h in DMEM with 0.5% FBS. Subsequently, the p38 MAP kinase inhibitor (SB203580) or CREB inhibitor (666–15) was added at a final concentration of 10 μM for 30 min or 5 μM for 60 min, respectively, before the addition of 50 ng/mL BDNF; cells were then cultured for 24 h. Firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induc- tion was calculated relative to untreated or BDNF-treated conditions. (C) mHypoE-N1 cells cultured with 0.5% fetal bovine serum were treated with 50 ng/mL of BDNF for 24 h. The p38 (SB203580, 10 μM) or CREB (666–15, 5 μM) inhibitors were added to the culture 30 or 60 min, respectively, be- fore BDNF treatment. Klf10 mRNA levels were determined by qPCR and calculated as the ratio of Klf10 cDNA/Ubc cDNA signal. Results are plotted and expressed as relative fold change compared to untreated cells. The solid line represents the mean ± SEM of three replicates in three independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Cell Culture, Concentration Assay, Luciferase, Activity Assay



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    FIGURE 2 | CREB upregulates Klf10 promoter activity in embryonic <t>hypothalamic</t> cells. (A) Plasmids containing different fragments of the mouse Klf10 promoter region were cloned into the pGL3-Basic one. In some constructs, CREB binding sites were mutated at positions −241 and/ or −1840 bp as shown. (B) <t>mHypoE-N1</t> cells were transiently transfected with 800 ng of pKlf10-1977 or pKlf10-516, or the mutated version p1977, p516, together with 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (C) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p516, p916, p1511, p1839 or p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (D) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p1977, 2p1977, or, 3p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. 24 h after transfection, firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induction was calculated relative to basal levels (without CREB overexpression). The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. (E) Phospho-CREB (pCREB) is recruited to the Klf10 gene promoter in vivo and in vitro at −241 and −1840 sites. Chromatin immunoprecipitation assays were performed from 17-day-old embryos from wild-type mice hypothalamus (Hyp-E17) and from mHypoE-N1 cells. PCR products derived from ChIP-enriched genomic DNA showing CREB binding sites at positions −241 and −1840 bp in the pro- moter region of Klf10. Input; α-pCREB: Anti-phosphoCREB; Ig: IgG immunoprecipitation. A representative gel of 3 replicates in three independent experiments is shown. (F) Normalized densitometry values of bound pCREB (pCREB/input) at positions −241 and −1840 bp in the Klf10 promoter region. The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. A. U.: Arbitrary Units.
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    FIGURE 2 | CREB upregulates Klf10 promoter activity in embryonic <t>hypothalamic</t> cells. (A) Plasmids containing different fragments of the mouse Klf10 promoter region were cloned into the pGL3-Basic one. In some constructs, CREB binding sites were mutated at positions −241 and/ or −1840 bp as shown. (B) <t>mHypoE-N1</t> cells were transiently transfected with 800 ng of pKlf10-1977 or pKlf10-516, or the mutated version p1977, p516, together with 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (C) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p516, p916, p1511, p1839 or p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (D) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p1977, 2p1977, or, 3p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. 24 h after transfection, firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induction was calculated relative to basal levels (without CREB overexpression). The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. (E) Phospho-CREB (pCREB) is recruited to the Klf10 gene promoter in vivo and in vitro at −241 and −1840 sites. Chromatin immunoprecipitation assays were performed from 17-day-old embryos from wild-type mice hypothalamus (Hyp-E17) and from mHypoE-N1 cells. PCR products derived from ChIP-enriched genomic DNA showing CREB binding sites at positions −241 and −1840 bp in the pro- moter region of Klf10. Input; α-pCREB: Anti-phosphoCREB; Ig: IgG immunoprecipitation. A representative gel of 3 replicates in three independent experiments is shown. (F) Normalized densitometry values of bound pCREB (pCREB/input) at positions −241 and −1840 bp in the Klf10 promoter region. The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. A. U.: Arbitrary Units.
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    93
    Cedarlane n1 hypothalamic neuron cells
    a , b Effects of oleate or palmitate treatment (200 μM each for the indicated duration) on cellular NAD + contents in <t>N1</t> <t>hypothalamic</t> neuronal cells or primary cultured hypothalamic astrocytes ( n = 3 wells). c Schematic illustration depicting the various NAD + biosynthetic pathways. d Heatmaps showing the palmitate treatment (200 μM, 48 h)-induced alterations in expression levels of NAD + biosynthetic enzymes in hypothalamic neurons and astrocytes ( n = 3 wells). e , f Changes in nicotinamide phosphoribosyltransferase (NAMPT) protein expression and enzyme activity in hypothalamic astrocytes following palmitate treatment (200 μM, 48 h) ( n = 3 wells). g , h Effects of NAMPT inhibition using FK866 (500 nM, 48 h) or Nampt siRNA treatment on the palmitate-induced increase in the cellular NAD + levels of hypothalamic astrocytes ( n = 3 wells). i Palmitate treatment (200 μM) did not alter cellular NADH levels in primary hypothalamic astrocytes ( n = 3 wells). j Nampt fluorescence in situ hybridization (FISH) and GFAP dual staining showing the changes in Nampt mRNA expression in hypothalamic, cortical, and hippocampal astrocytes of 15-week-old C57 male mice fed either a CD or HFD for 4 weeks ( n = 120 astrocytes from 4 mice per group). Scale bars: 20 μm. arb. units: arbitrary unit. k qPCR analysis of Nampt expression in astrocytes isolated from different brain regions (hypothalamus, cortex, and hippocampus) of 15-week-old C57 male mice subjected to a CD or HFD for 4 weeks ( n = 3). l Palmitate treatment did not induce alterations in Nampt mRNA expression or cellular NAD + content in primary cortical astrocytes (Nampt, n = 6; NAD + , n = 3). One-way ANOVA followed by Fisher’s LSD test ( a , b , g , h- NAD + ) and two-sided unpaired t-test ( d – f , h- Nampt, i – l ). n.s.: not significant. Three independent replicates were performed for all studies and measurement were taken from distinct samples. Results are presented as the mea n ± SEM. Source data are provided as a Source Data file.
    N1 Hypothalamic Neuron Cells, supplied by Cedarlane, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
    n1 hypothalamic neuron cells - by Bioz Stars, 2026-06
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    mouse hypothalamic cell line n44 mhypoe  (Cedarlane)
    93
    Cedarlane mouse hypothalamic cell line n44 mhypoe
    a , b Effects of oleate or palmitate treatment (200 μM each for the indicated duration) on cellular NAD + contents in <t>N1</t> <t>hypothalamic</t> neuronal cells or primary cultured hypothalamic astrocytes ( n = 3 wells). c Schematic illustration depicting the various NAD + biosynthetic pathways. d Heatmaps showing the palmitate treatment (200 μM, 48 h)-induced alterations in expression levels of NAD + biosynthetic enzymes in hypothalamic neurons and astrocytes ( n = 3 wells). e , f Changes in nicotinamide phosphoribosyltransferase (NAMPT) protein expression and enzyme activity in hypothalamic astrocytes following palmitate treatment (200 μM, 48 h) ( n = 3 wells). g , h Effects of NAMPT inhibition using FK866 (500 nM, 48 h) or Nampt siRNA treatment on the palmitate-induced increase in the cellular NAD + levels of hypothalamic astrocytes ( n = 3 wells). i Palmitate treatment (200 μM) did not alter cellular NADH levels in primary hypothalamic astrocytes ( n = 3 wells). j Nampt fluorescence in situ hybridization (FISH) and GFAP dual staining showing the changes in Nampt mRNA expression in hypothalamic, cortical, and hippocampal astrocytes of 15-week-old C57 male mice fed either a CD or HFD for 4 weeks ( n = 120 astrocytes from 4 mice per group). Scale bars: 20 μm. arb. units: arbitrary unit. k qPCR analysis of Nampt expression in astrocytes isolated from different brain regions (hypothalamus, cortex, and hippocampus) of 15-week-old C57 male mice subjected to a CD or HFD for 4 weeks ( n = 3). l Palmitate treatment did not induce alterations in Nampt mRNA expression or cellular NAD + content in primary cortical astrocytes (Nampt, n = 6; NAD + , n = 3). One-way ANOVA followed by Fisher’s LSD test ( a , b , g , h- NAD + ) and two-sided unpaired t-test ( d – f , h- Nampt, i – l ). n.s.: not significant. Three independent replicates were performed for all studies and measurement were taken from distinct samples. Results are presented as the mea n ± SEM. Source data are provided as a Source Data file.
    Mouse Hypothalamic Cell Line N44 Mhypoe, supplied by Cedarlane, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    embryonic mouse hypothalamic cell line n38 mhypoe-38  (CELLutions Biosystems)
    90
    CELLutions Biosystems embryonic mouse hypothalamic cell line n38 mhypoe-38
    a , b Effects of oleate or palmitate treatment (200 μM each for the indicated duration) on cellular NAD + contents in <t>N1</t> <t>hypothalamic</t> neuronal cells or primary cultured hypothalamic astrocytes ( n = 3 wells). c Schematic illustration depicting the various NAD + biosynthetic pathways. d Heatmaps showing the palmitate treatment (200 μM, 48 h)-induced alterations in expression levels of NAD + biosynthetic enzymes in hypothalamic neurons and astrocytes ( n = 3 wells). e , f Changes in nicotinamide phosphoribosyltransferase (NAMPT) protein expression and enzyme activity in hypothalamic astrocytes following palmitate treatment (200 μM, 48 h) ( n = 3 wells). g , h Effects of NAMPT inhibition using FK866 (500 nM, 48 h) or Nampt siRNA treatment on the palmitate-induced increase in the cellular NAD + levels of hypothalamic astrocytes ( n = 3 wells). i Palmitate treatment (200 μM) did not alter cellular NADH levels in primary hypothalamic astrocytes ( n = 3 wells). j Nampt fluorescence in situ hybridization (FISH) and GFAP dual staining showing the changes in Nampt mRNA expression in hypothalamic, cortical, and hippocampal astrocytes of 15-week-old C57 male mice fed either a CD or HFD for 4 weeks ( n = 120 astrocytes from 4 mice per group). Scale bars: 20 μm. arb. units: arbitrary unit. k qPCR analysis of Nampt expression in astrocytes isolated from different brain regions (hypothalamus, cortex, and hippocampus) of 15-week-old C57 male mice subjected to a CD or HFD for 4 weeks ( n = 3). l Palmitate treatment did not induce alterations in Nampt mRNA expression or cellular NAD + content in primary cortical astrocytes (Nampt, n = 6; NAD + , n = 3). One-way ANOVA followed by Fisher’s LSD test ( a , b , g , h- NAD + ) and two-sided unpaired t-test ( d – f , h- Nampt, i – l ). n.s.: not significant. Three independent replicates were performed for all studies and measurement were taken from distinct samples. Results are presented as the mea n ± SEM. Source data are provided as a Source Data file.
    Embryonic Mouse Hypothalamic Cell Line N38 Mhypoe 38, supplied by CELLutions Biosystems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    FIGURE 2 | CREB upregulates Klf10 promoter activity in embryonic hypothalamic cells. (A) Plasmids containing different fragments of the mouse Klf10 promoter region were cloned into the pGL3-Basic one. In some constructs, CREB binding sites were mutated at positions −241 and/ or −1840 bp as shown. (B) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or pKlf10-516, or the mutated version p1977, p516, together with 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (C) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p516, p916, p1511, p1839 or p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (D) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p1977, 2p1977, or, 3p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. 24 h after transfection, firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induction was calculated relative to basal levels (without CREB overexpression). The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. (E) Phospho-CREB (pCREB) is recruited to the Klf10 gene promoter in vivo and in vitro at −241 and −1840 sites. Chromatin immunoprecipitation assays were performed from 17-day-old embryos from wild-type mice hypothalamus (Hyp-E17) and from mHypoE-N1 cells. PCR products derived from ChIP-enriched genomic DNA showing CREB binding sites at positions −241 and −1840 bp in the pro- moter region of Klf10. Input; α-pCREB: Anti-phosphoCREB; Ig: IgG immunoprecipitation. A representative gel of 3 replicates in three independent experiments is shown. (F) Normalized densitometry values of bound pCREB (pCREB/input) at positions −241 and −1840 bp in the Klf10 promoter region. The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. A. U.: Arbitrary Units.

    Journal: Journal of neuroscience research

    Article Title: Klf10 Regulates the Emergence of Glial Phenotypes During Hypothalamic Development.

    doi: 10.1002/jnr.70020

    Figure Lengend Snippet: FIGURE 2 | CREB upregulates Klf10 promoter activity in embryonic hypothalamic cells. (A) Plasmids containing different fragments of the mouse Klf10 promoter region were cloned into the pGL3-Basic one. In some constructs, CREB binding sites were mutated at positions −241 and/ or −1840 bp as shown. (B) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or pKlf10-516, or the mutated version p1977, p516, together with 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (C) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p516, p916, p1511, p1839 or p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. (D) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977 or the mutated version, p1977, 2p1977, or, 3p1977, and 800 ng of pSVCREB plasmid or an equivalent concentration of empty pGL3-basic vector. 24 h after transfection, firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induction was calculated relative to basal levels (without CREB overexpression). The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. (E) Phospho-CREB (pCREB) is recruited to the Klf10 gene promoter in vivo and in vitro at −241 and −1840 sites. Chromatin immunoprecipitation assays were performed from 17-day-old embryos from wild-type mice hypothalamus (Hyp-E17) and from mHypoE-N1 cells. PCR products derived from ChIP-enriched genomic DNA showing CREB binding sites at positions −241 and −1840 bp in the pro- moter region of Klf10. Input; α-pCREB: Anti-phosphoCREB; Ig: IgG immunoprecipitation. A representative gel of 3 replicates in three independent experiments is shown. (F) Normalized densitometry values of bound pCREB (pCREB/input) at positions −241 and −1840 bp in the Klf10 promoter region. The solid line represents the mean ± SEM of three replicates in five independent experiments. Comparisons were made by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001. A. U.: Arbitrary Units.

    Article Snippet: Mice hypothalamic embryonic cells N1 (mHypoE- N1) (CEDARLANE, Ontario, CA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco- BRL, Gaithersburg, MD, USA) containing 5% fetal bovine serum (FBS) (Biowest International, Coinsins, Switzerland), 0.5 mM glutamine (Sigma), and 1% antibiotic–antimycotic (Gibco- BRL, Gaithersburg, MD, USA).

    Techniques: Activity Assay, Clone Assay, Construct, Binding Assay, Transfection, Plasmid Preparation, Concentration Assay, Luciferase, Over Expression, In Vivo, In Vitro, Chromatin Immunoprecipitation, Derivative Assay, Immunoprecipitation

    FIGURE 3 | Klf10 expression is upregulated by BDNF in embryonic hypothalamic cells. (A) Left panel. Expression of the Trk neurotrophin re- ceptors at 17.5-day-old embryos from wild-type mice hypothalamus (Hyp-E17.5) and in mHypoE-N1 cells was measured by RT-PCR assays. Right panel. Normalized densitometry values of Trk receptors (NTRs) expression (NTRs cDNA/actin cDNA) in the Hyp-E17.5 and mHypoE-N1 cells. (B) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977, pKlf10-516, or the mutated version p1977, p516. After transfection, cells were cultured for 12 h in DMEM with 5% FBS and then were cultured for 12 h in DMEM with 0.5% FBS. Subsequently, the p38 MAP kinase inhibitor (SB203580) or CREB inhibitor (666–15) was added at a final concentration of 10 μM for 30 min or 5 μM for 60 min, respectively, before the addition of 50 ng/mL BDNF; cells were then cultured for 24 h. Firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induc- tion was calculated relative to untreated or BDNF-treated conditions. (C) mHypoE-N1 cells cultured with 0.5% fetal bovine serum were treated with 50 ng/mL of BDNF for 24 h. The p38 (SB203580, 10 μM) or CREB (666–15, 5 μM) inhibitors were added to the culture 30 or 60 min, respectively, be- fore BDNF treatment. Klf10 mRNA levels were determined by qPCR and calculated as the ratio of Klf10 cDNA/Ubc cDNA signal. Results are plotted and expressed as relative fold change compared to untreated cells. The solid line represents the mean ± SEM of three replicates in three independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

    Journal: Journal of neuroscience research

    Article Title: Klf10 Regulates the Emergence of Glial Phenotypes During Hypothalamic Development.

    doi: 10.1002/jnr.70020

    Figure Lengend Snippet: FIGURE 3 | Klf10 expression is upregulated by BDNF in embryonic hypothalamic cells. (A) Left panel. Expression of the Trk neurotrophin re- ceptors at 17.5-day-old embryos from wild-type mice hypothalamus (Hyp-E17.5) and in mHypoE-N1 cells was measured by RT-PCR assays. Right panel. Normalized densitometry values of Trk receptors (NTRs) expression (NTRs cDNA/actin cDNA) in the Hyp-E17.5 and mHypoE-N1 cells. (B) mHypoE-N1 cells were transiently transfected with 800 ng of pKlf10-1977, pKlf10-516, or the mutated version p1977, p516. After transfection, cells were cultured for 12 h in DMEM with 5% FBS and then were cultured for 12 h in DMEM with 0.5% FBS. Subsequently, the p38 MAP kinase inhibitor (SB203580) or CREB inhibitor (666–15) was added at a final concentration of 10 μM for 30 min or 5 μM for 60 min, respectively, before the addition of 50 ng/mL BDNF; cells were then cultured for 24 h. Firefly luciferase activity was determined and normalized to Renilla luciferase values. Fold induc- tion was calculated relative to untreated or BDNF-treated conditions. (C) mHypoE-N1 cells cultured with 0.5% fetal bovine serum were treated with 50 ng/mL of BDNF for 24 h. The p38 (SB203580, 10 μM) or CREB (666–15, 5 μM) inhibitors were added to the culture 30 or 60 min, respectively, be- fore BDNF treatment. Klf10 mRNA levels were determined by qPCR and calculated as the ratio of Klf10 cDNA/Ubc cDNA signal. Results are plotted and expressed as relative fold change compared to untreated cells. The solid line represents the mean ± SEM of three replicates in three independent experiments. Comparisons were made by paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

    Article Snippet: Mice hypothalamic embryonic cells N1 (mHypoE- N1) (CEDARLANE, Ontario, CA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco- BRL, Gaithersburg, MD, USA) containing 5% fetal bovine serum (FBS) (Biowest International, Coinsins, Switzerland), 0.5 mM glutamine (Sigma), and 1% antibiotic–antimycotic (Gibco- BRL, Gaithersburg, MD, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Cell Culture, Concentration Assay, Luciferase, Activity Assay

    Induction of autophagy in hypothalamic neurons in response to hypoglycemic conditions. N39 cells were exposed to glucose concentrations of 4500, 2000, 900, 500, and 200 mg/L for 24, 48, and 72 h. The relative fold change in the gene expression levels of (a) LC3B and (c) LAMP2 in N39 cells as compared with the gene expression levels at the glucose concentration of 4500 mg/L, which is considered as the reference with a value of 1, and determined by using the quantitative real‐time polymerase chain reaction (qPCR) process. (b) Shows the representative estern blot indicating the protein expression of LC3BI (16KDa) and LC3BII (18KDa) at 24, 48, and 72 h across decreasing glucose concentrations # and the corresponding densitometric analysis of LC3B lipidation represented as the ratio of LC3BII to LC3BI as well as LC3BII normalized to β‐actin. (d) Shows the representative Western blot indicating the protein expression of LAMP2A (120 KDa) at 24, 48, and 72 h across decreasing glucose concentrations # and the corresponding densitometric analysis of LAMP2A expression. The protein expression of β‐actin (42 KDa) at 24, 48, and 72 h at decreasing glucose concentrations # was used as loading control. All experiments were repeated at least three times and the graphical data are represented as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, **** p < 0.0001 determined using two‐way ANOVA. # Glucose concentrations: L1 = 4500 mg/L, L2 = 2000 mg/L, L3 = 900 mg/L, L4 = 500 mg/L, L5 = 200 mg/L.

    Journal: Clinical and Translational Science

    Article Title: Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons

    doi: 10.1111/cts.13749

    Figure Lengend Snippet: Induction of autophagy in hypothalamic neurons in response to hypoglycemic conditions. N39 cells were exposed to glucose concentrations of 4500, 2000, 900, 500, and 200 mg/L for 24, 48, and 72 h. The relative fold change in the gene expression levels of (a) LC3B and (c) LAMP2 in N39 cells as compared with the gene expression levels at the glucose concentration of 4500 mg/L, which is considered as the reference with a value of 1, and determined by using the quantitative real‐time polymerase chain reaction (qPCR) process. (b) Shows the representative estern blot indicating the protein expression of LC3BI (16KDa) and LC3BII (18KDa) at 24, 48, and 72 h across decreasing glucose concentrations # and the corresponding densitometric analysis of LC3B lipidation represented as the ratio of LC3BII to LC3BI as well as LC3BII normalized to β‐actin. (d) Shows the representative Western blot indicating the protein expression of LAMP2A (120 KDa) at 24, 48, and 72 h across decreasing glucose concentrations # and the corresponding densitometric analysis of LAMP2A expression. The protein expression of β‐actin (42 KDa) at 24, 48, and 72 h at decreasing glucose concentrations # was used as loading control. All experiments were repeated at least three times and the graphical data are represented as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, **** p < 0.0001 determined using two‐way ANOVA. # Glucose concentrations: L1 = 4500 mg/L, L2 = 2000 mg/L, L3 = 900 mg/L, L4 = 500 mg/L, L5 = 200 mg/L.

    Article Snippet: Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE‐N39 or N39) was obtained from Cedarlane (Burlington, Ontario, Canada) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin‐streptomycin (P/S; Sigma Aldrich) in a humidified atmosphere of 5% CO 2 at 37°C.

    Techniques: Gene Expression, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

    Fluorescence microscopy images of live N39 cells exposed to various glucose concentrations for 24 and 48 h depicting the levels of autophagosomes (green) and nuclei (blue with DAPI stain). Scale bar 200 μM.

    Journal: Clinical and Translational Science

    Article Title: Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons

    doi: 10.1111/cts.13749

    Figure Lengend Snippet: Fluorescence microscopy images of live N39 cells exposed to various glucose concentrations for 24 and 48 h depicting the levels of autophagosomes (green) and nuclei (blue with DAPI stain). Scale bar 200 μM.

    Article Snippet: Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE‐N39 or N39) was obtained from Cedarlane (Burlington, Ontario, Canada) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin‐streptomycin (P/S; Sigma Aldrich) in a humidified atmosphere of 5% CO 2 at 37°C.

    Techniques: Fluorescence, Microscopy, Staining

    Effects of low glucose conditions on FOS and BECN1 gene expression in N39 cells with FOS and BECN1 gene knockdowns. N39 cells were treated with siRNA negative control, FOS siRNA, and BECN1 siRNA, and were exposed to various concentrations of glucose (4500, 900, and 200 mg/L) for 48 h post‐siRNA transfection. The relative fold change in the mRNA expression levels of (a) FOS and (b) BECN1 in FOS knockdown (FOS KD) and BECN1 knockdown (BECN1 KD) cells were compared with the mRNA expression of negative control cells at the corresponding glucose concentrations of 4500, 900, and 200 mg/L, as determined using the quantitative real‐time polymerase chain reaction (qPCR) process. All experiments were repeated at least three times and the graphical data are represented as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 determined using two‐way ANOVA.

    Journal: Clinical and Translational Science

    Article Title: Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons

    doi: 10.1111/cts.13749

    Figure Lengend Snippet: Effects of low glucose conditions on FOS and BECN1 gene expression in N39 cells with FOS and BECN1 gene knockdowns. N39 cells were treated with siRNA negative control, FOS siRNA, and BECN1 siRNA, and were exposed to various concentrations of glucose (4500, 900, and 200 mg/L) for 48 h post‐siRNA transfection. The relative fold change in the mRNA expression levels of (a) FOS and (b) BECN1 in FOS knockdown (FOS KD) and BECN1 knockdown (BECN1 KD) cells were compared with the mRNA expression of negative control cells at the corresponding glucose concentrations of 4500, 900, and 200 mg/L, as determined using the quantitative real‐time polymerase chain reaction (qPCR) process. All experiments were repeated at least three times and the graphical data are represented as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 determined using two‐way ANOVA.

    Article Snippet: Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE‐N39 or N39) was obtained from Cedarlane (Burlington, Ontario, Canada) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin‐streptomycin (P/S; Sigma Aldrich) in a humidified atmosphere of 5% CO 2 at 37°C.

    Techniques: Gene Expression, Negative Control, Transfection, Expressing, Knockdown, Real-time Polymerase Chain Reaction

    Effects of low glucose conditions on the expression of LC3B and LAMP2A in N39 cells with FOS and BECN1 gene knockdowns. N39 cells (Ctrl), N39 + siRNA negative control (−ve Ctrl), N39 + FOS siRNA (FOS KD), and N39 + BECN1 siRNA (BECN1 KD) cells were exposed to various concentrations of glucose (4500, 900, and 200 mg/L) for 48 h post‐siRNAs transfection. The relative fold change in the mRNA expression levels of (a) LC3B and (c) LAMP2 in these cells were compared with the mRNA expression of Ctrl cells at the glucose concentration of 4500 mg/L, which is considered as the reference with a value of 1, and determined using the quantitative real‐time polymerase chain (qPCR) reaction. (b) Representative Western blot indicating the protein expression of LC3BI (16 KDa) and LC3BII (18 KDa) in these cells at glucose concentrations of 4500 and 200 mg/L and the corresponding densitometric analysis of LC3B lipidation represented as the ratio of LC3BII to LC3BI. (d) Representative Western blots indicating the protein expression of LAMP2A (120 KDa) in these cells at glucose concentrations of 4500 and 200 mg/L and the corresponding densitometric analysis of LAMP2A expression. The protein expression of β‐actin (42KDa) was used as loading control. All experiments were repeated at least three times and the graphical data are represented as mean ± standard error of the mean (SEM). * p < 0.05, **** p < 0.0001 determined using two‐way ANOVA. Representation of lanes in images for Western blots: L1 = control (4500 mg/L glucose), L2 = control (200 mg/L glucose), L3 = negative control (4500 mg/L glucose), L4 = negative control (200 mg/L glucose), L5 = FOS KD (4500 mg/L glucose), L6 = FOS KD (200 mg/L glucose), L7 = BECN1 KD (4500 mg/L glucose), L8 = BECN1 KD (200 mg/L glucose). Glucose concentrations are represented in milligrams/liter (mg/L).

    Journal: Clinical and Translational Science

    Article Title: Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons

    doi: 10.1111/cts.13749

    Figure Lengend Snippet: Effects of low glucose conditions on the expression of LC3B and LAMP2A in N39 cells with FOS and BECN1 gene knockdowns. N39 cells (Ctrl), N39 + siRNA negative control (−ve Ctrl), N39 + FOS siRNA (FOS KD), and N39 + BECN1 siRNA (BECN1 KD) cells were exposed to various concentrations of glucose (4500, 900, and 200 mg/L) for 48 h post‐siRNAs transfection. The relative fold change in the mRNA expression levels of (a) LC3B and (c) LAMP2 in these cells were compared with the mRNA expression of Ctrl cells at the glucose concentration of 4500 mg/L, which is considered as the reference with a value of 1, and determined using the quantitative real‐time polymerase chain (qPCR) reaction. (b) Representative Western blot indicating the protein expression of LC3BI (16 KDa) and LC3BII (18 KDa) in these cells at glucose concentrations of 4500 and 200 mg/L and the corresponding densitometric analysis of LC3B lipidation represented as the ratio of LC3BII to LC3BI. (d) Representative Western blots indicating the protein expression of LAMP2A (120 KDa) in these cells at glucose concentrations of 4500 and 200 mg/L and the corresponding densitometric analysis of LAMP2A expression. The protein expression of β‐actin (42KDa) was used as loading control. All experiments were repeated at least three times and the graphical data are represented as mean ± standard error of the mean (SEM). * p < 0.05, **** p < 0.0001 determined using two‐way ANOVA. Representation of lanes in images for Western blots: L1 = control (4500 mg/L glucose), L2 = control (200 mg/L glucose), L3 = negative control (4500 mg/L glucose), L4 = negative control (200 mg/L glucose), L5 = FOS KD (4500 mg/L glucose), L6 = FOS KD (200 mg/L glucose), L7 = BECN1 KD (4500 mg/L glucose), L8 = BECN1 KD (200 mg/L glucose). Glucose concentrations are represented in milligrams/liter (mg/L).

    Article Snippet: Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE‐N39 or N39) was obtained from Cedarlane (Burlington, Ontario, Canada) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin‐streptomycin (P/S; Sigma Aldrich) in a humidified atmosphere of 5% CO 2 at 37°C.

    Techniques: Expressing, Negative Control, Transfection, Concentration Assay, Western Blot, Control

    Fluorescence microscopy images of (a) N39 cells without siRNA treatment (positive control), (b) N39 with siRNA negative control, (c) FOS knockdown (KD) N39 cells, and (d) BECN1 KD N39 cells at different glucose conditions (4500, 2000, 900, 500, and 200 mg/L). The levels of autophagosomes are depicted in green and nuclei in blue with DAPI stain. Scale bar 200 μM.

    Journal: Clinical and Translational Science

    Article Title: Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons

    doi: 10.1111/cts.13749

    Figure Lengend Snippet: Fluorescence microscopy images of (a) N39 cells without siRNA treatment (positive control), (b) N39 with siRNA negative control, (c) FOS knockdown (KD) N39 cells, and (d) BECN1 KD N39 cells at different glucose conditions (4500, 2000, 900, 500, and 200 mg/L). The levels of autophagosomes are depicted in green and nuclei in blue with DAPI stain. Scale bar 200 μM.

    Article Snippet: Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE‐N39 or N39) was obtained from Cedarlane (Burlington, Ontario, Canada) and was cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma Aldrich) and 1% penicillin‐streptomycin (P/S; Sigma Aldrich) in a humidified atmosphere of 5% CO 2 at 37°C.

    Techniques: Fluorescence, Microscopy, Positive Control, Negative Control, Knockdown, Staining

    a , b Effects of oleate or palmitate treatment (200 μM each for the indicated duration) on cellular NAD + contents in N1 hypothalamic neuronal cells or primary cultured hypothalamic astrocytes ( n = 3 wells). c Schematic illustration depicting the various NAD + biosynthetic pathways. d Heatmaps showing the palmitate treatment (200 μM, 48 h)-induced alterations in expression levels of NAD + biosynthetic enzymes in hypothalamic neurons and astrocytes ( n = 3 wells). e , f Changes in nicotinamide phosphoribosyltransferase (NAMPT) protein expression and enzyme activity in hypothalamic astrocytes following palmitate treatment (200 μM, 48 h) ( n = 3 wells). g , h Effects of NAMPT inhibition using FK866 (500 nM, 48 h) or Nampt siRNA treatment on the palmitate-induced increase in the cellular NAD + levels of hypothalamic astrocytes ( n = 3 wells). i Palmitate treatment (200 μM) did not alter cellular NADH levels in primary hypothalamic astrocytes ( n = 3 wells). j Nampt fluorescence in situ hybridization (FISH) and GFAP dual staining showing the changes in Nampt mRNA expression in hypothalamic, cortical, and hippocampal astrocytes of 15-week-old C57 male mice fed either a CD or HFD for 4 weeks ( n = 120 astrocytes from 4 mice per group). Scale bars: 20 μm. arb. units: arbitrary unit. k qPCR analysis of Nampt expression in astrocytes isolated from different brain regions (hypothalamus, cortex, and hippocampus) of 15-week-old C57 male mice subjected to a CD or HFD for 4 weeks ( n = 3). l Palmitate treatment did not induce alterations in Nampt mRNA expression or cellular NAD + content in primary cortical astrocytes (Nampt, n = 6; NAD + , n = 3). One-way ANOVA followed by Fisher’s LSD test ( a , b , g , h- NAD + ) and two-sided unpaired t-test ( d – f , h- Nampt, i – l ). n.s.: not significant. Three independent replicates were performed for all studies and measurement were taken from distinct samples. Results are presented as the mea n ± SEM. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Hypothalamic astrocyte NAD + salvage pathway mediates the coupling of dietary fat overconsumption in a mouse model of obesity

    doi: 10.1038/s41467-024-46009-0

    Figure Lengend Snippet: a , b Effects of oleate or palmitate treatment (200 μM each for the indicated duration) on cellular NAD + contents in N1 hypothalamic neuronal cells or primary cultured hypothalamic astrocytes ( n = 3 wells). c Schematic illustration depicting the various NAD + biosynthetic pathways. d Heatmaps showing the palmitate treatment (200 μM, 48 h)-induced alterations in expression levels of NAD + biosynthetic enzymes in hypothalamic neurons and astrocytes ( n = 3 wells). e , f Changes in nicotinamide phosphoribosyltransferase (NAMPT) protein expression and enzyme activity in hypothalamic astrocytes following palmitate treatment (200 μM, 48 h) ( n = 3 wells). g , h Effects of NAMPT inhibition using FK866 (500 nM, 48 h) or Nampt siRNA treatment on the palmitate-induced increase in the cellular NAD + levels of hypothalamic astrocytes ( n = 3 wells). i Palmitate treatment (200 μM) did not alter cellular NADH levels in primary hypothalamic astrocytes ( n = 3 wells). j Nampt fluorescence in situ hybridization (FISH) and GFAP dual staining showing the changes in Nampt mRNA expression in hypothalamic, cortical, and hippocampal astrocytes of 15-week-old C57 male mice fed either a CD or HFD for 4 weeks ( n = 120 astrocytes from 4 mice per group). Scale bars: 20 μm. arb. units: arbitrary unit. k qPCR analysis of Nampt expression in astrocytes isolated from different brain regions (hypothalamus, cortex, and hippocampus) of 15-week-old C57 male mice subjected to a CD or HFD for 4 weeks ( n = 3). l Palmitate treatment did not induce alterations in Nampt mRNA expression or cellular NAD + content in primary cortical astrocytes (Nampt, n = 6; NAD + , n = 3). One-way ANOVA followed by Fisher’s LSD test ( a , b , g , h- NAD + ) and two-sided unpaired t-test ( d – f , h- Nampt, i – l ). n.s.: not significant. Three independent replicates were performed for all studies and measurement were taken from distinct samples. Results are presented as the mea n ± SEM. Source data are provided as a Source Data file.

    Article Snippet: N1 hypothalamic neuron cells were obtained from Cedarlane (CED-CLU101) and cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.

    Techniques: Cell Culture, Expressing, Activity Assay, Inhibition, Fluorescence, In Situ Hybridization, Staining, Isolation