Morphology of established hypothalamus-derived cell lines.

<p>a, b. Representative images of established hypothalamus-derived cell lines. c, d. Representative images of electron microscopy. A few secretory granules (c: arrow) and intermediate filaments (d: dotted circle) were observed. e-g. Cells (11–55) were stained with anti-AgRP and anti αMSH antibodies (e: AgRP, f: αMSH, g: merge). h. Western blot analysis after Tricine SDS page of the culture medium of the cell lines and AgRP control peptide (50 pmol) with anti-AgRP antibody.</p

Increased intracellular Ca2⁺ and cAMP concentration following addition of known peptide hormones or neurotransmitters.

<p>a, b. NPY (a), muscarine, adrenaline (or the α1 agonist methoxamine, but not the α2 agonist clonidine), and histamine (b) evoked profound elevation of [Ca²⁺]_i. AVP and TRH induced [Ca²⁺]_i elevation to a lesser extent (a). Data from clone 11–55 is presented as the difference between maximum and minimum fluorescence intensities (max—min). Detailed fluorescent curves are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148639#pone.0148639.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148639#pone.0148639.s003" target="_blank">S3</a> Figs. c-f. Acetylcholine, noradrenaline and GABA elevated intracellular cAMP (c), while glutamate, histamine and serotonin suppressed it when added with 5μM forskolin (d). Oxytocin also suppressed cAMP (f). n = 3, **: <i>p</i> < 0.01, **: <i>p</i> < 0.05 relative to control.</p

Socs3, Ptpn1, Ptpn2 and Ptprf mRNA levels by high leptin, insulin and palmitate containing medium.

<p>Socs3 (a), Ptpn1 (b), Ptpn2 (c) and Ptprf (d) mRNA levels of clone 55 after overnight incubation with 10 nM leptin, 100 nM insulin and 200 μM palmitate containing medium. **: <i>p</i> < 0.01 relative to a non-pretreated sample. n = 6. e. The effects of 1 mM octanoate (C8), palmitate (C16), and palmitoleate (C16:1) on Socs3 mRNA expression levels in clone 11–55 cells. f. TLR mRNA expressions in 11–55 and 11–59 cells. g, h. Palmitate significantly elevated TNFα and IL-6 mRNA levels in 11–55 cells. i. LPS significantly elevated Socs3 mRNA levels. *: <i>p</i> < 0.05, **: <i>p</i> < 0.01 relative to a non-pretreated sample. n = 6. j. k. Knockdown of TLR4 expression by siRNAs (j) abolished the palmitate-induced Socs3 mRNA elevation (k). *: <i>p</i> < 0.05, **: <i>p</i> < 0.01 relative to siRNA negative control. n = 6.</p

STAT3, ERK, and Akt phosphorylation induced by leptin treatment in the established hypothalamus-derived cell lines.

<p>a–c. Stat3 (a), Erk1, 2 (b), and Akt (c) phosphorylation was induced by addition of 100 nM leptin for 15 min in clone 55 of the hypothalamus-derived cell lines. d. Akt phosphorylation was induced by addition of 100 nM insulin for 5 min in the clone 55 of hypothalamus-derived cell lines.*: n = 3, <i>p</i> < 0.05, **: <i>p</i> < 0.01 compared to vehicle.</p

Establishment of hypothalamus-derived cell lines from chromogranin A promoter-CreERT2/CAG-promoter-lox-STOP-lox-SV40 Tag transgenic mice.

<p>a, b. DNA constructs for chromogranin A promoter-CreERT2 (a) and CAG-promoter-lox-STOP-lox-SV40 Tag transgenic mice (b). Cre-mediated recombination was confirmed by crossing chromogranin A promoter-CreERT2 mice with Cre-reporter mice (Rosa-CAG-LSL-ZsGreen1). c-k. Duodenum (c-e), pancreas (f-h) and hypothalamus (i-k) were stained with anti-chromogranin A (c: ZsGreen, d: chromogranin A, e: merge), anti-insulin (f: ZsGreen, g: insulin, h: merge), and anti-neuron specific enolase (NSE; i: ZsGreen, j: NSE, k: merge), respectively. l: Unstained hypothalamus.</p

Tissue suction devices used in this study.

<p>Tissue suction devices used in this study.</p

<em>In vivo</em> Site-Specific Transfection of Naked Plasmid DNA and siRNAs in Mice by Using a Tissue Suction Device

<div><p>We have developed an <em>in vivo</em> transfection method for naked plasmid DNA (pDNA) and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the <em>in vivo</em> transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids.</p> </div

<i>In vivo</i> transfection to various tissues by tissue suction.

<p><i>In vivo</i> transfection by tissue suction was applied to various tissues (including the kidney, heart, spleen, liver, duodenum, muscle, and stomach). A type III device was used for the muscle and stomach. A type IV device was used for the kidney, heart, spleen, liver, and duodenum. Each value represents means + SD (n  = 3 [the kidney, spleen, and muscle], n  = 4 [the liver, duodenum, and stomach], or n  = 5 [the heart]). All mice were alive at the end of the experiment.</p

<i>In vivo</i> transfection of naked pDNA by tissue suction.

<p>A) <i>In vivo</i> imaging of luciferase activity in a mouse liver that was suctioned once by the type I device just after intravenous injection of pCMV-Luc. B) <i>Ex vivo</i> imaging of luciferase activity in the liver suctioned by the type I device. C) Bright field image of (B). D) Luciferase levels of various tissues. The right kidney in mice was suctioned once by the type III device. Each value represents means + SD (n  = 4). All mice were alive at the end of the experiment.</p

Tissue suction by using the device.

<p>A) Representative photograph of the tissue suction device (type II). B) Schematic illustration of <i>in vivo</i> transfection by tissue suction. The surface of the target tissues was suctioned by the suction device just after intravenous injection of naked nucleic acids.</p