Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Differences in the response of UCP1 mRNA to hormonal stimulation between rat and mouse primary cultures of brown adipocytes.

doi: 10.1159/000335810

Figure Lengend Snippet: Fig. 2. A. B. Adrenergic stimulation of UCP-1 mRNA in mouse and rat brown adipocytes. Rat or mouse brown precursor cells were grown in standard conditions (10% NCS) until day 7. Cells were maintained during the last 20 hours in medium containing 1% NCS and NE (3 μM), 8Br-cAMP (BrcAMP, 1 mM) or Forskolin (F, 5 μM) were added during the last 6 hours before recollection. Fifteen μg of total RNA per lane were used. The specific mouse or rat UCP-1 cDNA probes were used to hybridize the respective mouse or rat samples. Hybridization with cyclophilin cDNA was used to correct for differences between lanes. A representative Northern and quantification of UCP-1 mRNA by qRT-PCR are shown (n=3 for mice and n=6 for rat). All increases were significant (P<0.05) vs basal values. C-F. Effect of triiodothyronine (T3) on the adrenergic stimulation of UCP-1 mRNA in mouse and rat brown adipocytes. Rat (C, E and F) or mouse (D) brown preadipocytes were grown in 10% NCS until day 7. Cells were maintained during the last 20 hours in medium containing 2% hypothyroid serum, supplemented or not with 5 nM T3. NE (1 μM) was added during the last 7.5 h before recollection. Twenty μg of total RNA (C, D) or 10 μg of Poly (A)+ RNA (E, F) were used per lane. Cyclophilin cDNA was used to correct for differences between lanes. A mouse (C, D and E) or a rat (F) UCP-1 cDNA was used. A representative experiment is shown (n=2). G. Time course increases in cellular cAMP levels in rat brown adipocytes. Rat brown adipocytes were grown in standard conditions until day 7. Cells were maintained during the last 20 hours in medium containing 1% hypothyroid serum, supplemented or not with 5 nM T3. NE (2 μM) was added and cells collected at 0.5, 1, 2 and 4 hours in 0.1 N HCl for cAMP determination. Data represent the mean ± SD of triplicate determinations in three different cultures.

Article Snippet: Recently, rat and mouse UCP-1 mRNAs were also quantified by qRT-PCR using specific Taqman probes for rat and mouse UCP-1 (Rn00562126m1 and Mm01244861m1; Gene expression assays, Applied Biosystems, Foster City, CA). cDNA was synthesized from 1 μg of RNA using iScript cDNA synthesis kit (BioRad, Hercules, CA).

Techniques: Hybridization, Northern Blot, Quantitative RT-PCR

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Differences in the response of UCP1 mRNA to hormonal stimulation between rat and mouse primary cultures of brown adipocytes.

doi: 10.1159/000335810

Figure Lengend Snippet: Fig. 3. A and B. Effect of T3 and insulin depletion on the adrenergic induction of UCP-1 mRNA in mouse and rat brown adipocytes. Rat or mouse brown preadipocytes were grown in 10% NCS until day 7. Half of the flasks from both cultures were depleted from insulin from day 4. Cells were maintained during the last 20 hours in medium containing 1% NCS supplemented or not with 2 nM T3. NE (3 μM) was added during the last 6 hours. Mouse or rat UCP-1 cDNA probes were used to hybridize the respective mouse or rat samples. Cyclophilin cDNA was used to correct for differences between lanes. A representative experiment is shown. Below, the figures show mouse or rat UCP-1 mRNA analysis using qRT-PCR and specific Taqman probes for rat or mouse UCP-1, using rat or mouse Cy as reference gene (n=2-7 points/bar). Results are means±SEM. *P<0.05 vs its respective T3. # P<0.05 vs +insulin. C. Direct effect of T3 on UCP-1 mRNA induction in rat brown adipocytes. The role of insulin. Cultures of rat brown preadipocytes were grown in standard conditions (10% NCS+ insulin) until day 7. Cells were maintained during the last 24 hours in 10% hypothyroid serum or in serum-free medium, with no insulin, supplemented as specified with 10 nM T3, 10 μM NE or 4 nM insulin. All treatments lasted 24 hours. A rat UCP-1 cDNA probe was used and rat cyclophilin cDNA was used to correct for differences between lanes. A representative experiment is shown and quantification of UCP-1 mRNA was done using qRT.PCR as described above. Results are means±SEM (n=4). *P<0.05 vs T3 in each of the conditions.

Article Snippet: Recently, rat and mouse UCP-1 mRNAs were also quantified by qRT-PCR using specific Taqman probes for rat and mouse UCP-1 (Rn00562126m1 and Mm01244861m1; Gene expression assays, Applied Biosystems, Foster City, CA). cDNA was synthesized from 1 μg of RNA using iScript cDNA synthesis kit (BioRad, Hercules, CA).

Techniques: Quantitative RT-PCR

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Differences in the response of UCP1 mRNA to hormonal stimulation between rat and mouse primary cultures of brown adipocytes.

doi: 10.1159/000335810

Figure Lengend Snippet: Fig. 4. A and B. Effect of RA and T3 on the adrenergic stimulation of UCP-1 mRNA in mouse and rat brown adipocytes. Effect of insulin depletion. Rat or mouse brown preadipocytes were grown in 10% NCS until day 7. Half of the flasks from both cultures were depleted from insulin from day 4. Cells were maintained during the last 20 hours in medium containing 1% NCS, supplemented or not with 2 nM T3 and/or 1 μM RA. NE (3 μM) was added during the last 6 hours. The specific mouse or rat UCP-1 cDNA probes were used to hybridize the respective mouse or rat cultures. Cyclophilin cDNA was used to correct for differences between lanes. A representative experiment is shown and quantification of UCP-1 mRNA was done using qRT-PCR and specific Taqman probes. Results are means±SEM (n=3-5 for mouse and n= 2-4 for rat). *P<0.05 vs RA, #P<0.05 vs +insulin. Fig. 4.C. Effect of RA on the stimulation of UCP-1 mRNA using rat brown adipocytes. Rat brown preadipocytes were grown in 10% NCS until day 7. Cells were maintained during the last 20 hours in medium containing 1% NCS supplemented with 0.1 nM, 10 nM or 1 μM RA for 6 or 20 hours. T3 was added during the last 20 hours as specified. NE (3 μM) was added during the last 6 hours. Rat UCP-1 cDNA probe was used for Northern analysis and Cyclophilin cDNA was used to correct for differences between lanes. A representative experiment is shown and quantification of UCP-1 mRNA was done using qRT-PCR (n=2).

Article Snippet: Recently, rat and mouse UCP-1 mRNAs were also quantified by qRT-PCR using specific Taqman probes for rat and mouse UCP-1 (Rn00562126m1 and Mm01244861m1; Gene expression assays, Applied Biosystems, Foster City, CA). cDNA was synthesized from 1 μg of RNA using iScript cDNA synthesis kit (BioRad, Hercules, CA).

Techniques: Quantitative RT-PCR, Northern Blot

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: CD4 + CD28 null T cell clones express CD158b/j, but do not express KARAP/DAP12. CD4 + CD28 null T cells were sorted from patients with RA, and clones were established by limiting dilution. Clones were analyzed by flow cytometry for expression of CD28 and CD158b/j. Four representative clones (#1 through #4) are shown. All clones expressed CD4 (unpublished data; A). RT-PCR was used to amplify transcripts for KARAP/DAP12 and β-actin from PBMCs (lane 1), Jurkat T cells (lane 2), and CD4 + CD28 null T cell clones #1–#4 (lanes 3–6, respectively). cDNA was omitted for the negative control (lane 7) (B). Western blotting was used to detect KARAP/DAP12 and β-actin protein (bottom panels) in Jurkat T cells (lane 1), Jurkat T cells transfected with KARAP/DAP12 + vaccinia virus (lane 2), and CD4 + CD28 null T cell clones (lanes 3–7) (C).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Clone Assay, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Transfection, Virus

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Stimulation through CD158b/j results in an up-regulation of ATF-2 and HSP27 transcripts. The PathwayFinder cDNA Array is spotted in duplicate with 23 cDNAs. Represented on the membrane are the ERK (egr-1 and c-fos), JNK (ATF-2, hsf1, HSP27, and HSP90), NF-κB (iNos, NF-κB, and IκBα), NFAT (IL-2, Fas, and CD5), TGF-β (p16, p21, and p57 Kip2 ), Wnt (c-myc), p53 (p21, gadd45, pig7, pig8, mdm2, and bax), and CREB pathways (egr-1, CYP19, and c-fos). The membrane also included a negative control (pUC18) and two positive controls (β-actin and GAPDH) (A). A CD4 + CD28 null CD158b/j + T cell clone was stimulated with control mouse IgG or anti-CD158j mAb and cross-linked with rabbit anti–mouse IgG Ab. Total RNA was harvested and used to probe the PathwayFinder cDNA Array (B).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Membrane, Negative Control, Control

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Phosphorylation of JNK is initiated by stimulation specifically through CD158j. Two CD4 + CD28 null CD158j + T cell clones (top panels) and a CD4 + CD28 null CD158b1 + T cell clone (bottom panels) were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against total JNK (right panels; A). Jurkat T cells were infected with either wild-type vaccinia virus (WR) or vaccinia virus containing CD158j cDNA and were analyzed for expression of CD158j by flow cytometry (B). Jurkat T cells infected with WR vaccinia virus or CD158j + vaccinia virus were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (top left panels) and MKK4 (bottom left panel). The blots were stripped and reprobed with Abs against β-actin (top right panels) or MKK4 (bottom right panel) (C).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Phospho-proteomics, Clone Assay, SDS Page, Membrane, Infection, Virus, Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Mutation of transmembrane lysine residue in CD158j abolishes ability to induce JNK phosphorylation. Jurkat T cells were transiently transfected with constructs containing the CD158j cDNA or the CD158j233I cDNA. Cell-surface expression was confirmed by flow cytometry (A). Jurkat T cells transfected with either CD158j or CD158jK233I were stimulated with anti-CD3 or anti-CD158b/j mAb and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against total JNK (right panels) (B).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Mutagenesis, Residue, Phospho-proteomics, Transfection, Construct, Expressing, Flow Cytometry, SDS Page, Membrane

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by streptavidin-HRP (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Negative Control, SDS Page, Membrane, Phospho-proteomics, Expressing, Stable Transfection, Transfection, Flow Cytometry, Immunoprecipitation, Western Blot

Journal: Biophysical journal

Article Title: Targeting Tmem63b and Piezo2 in C-fiber low-threshold mechanoreceptors: Limitation of Vglut3 -IRES- Cre

doi: 10.1016/j.bpj.2025.10.043

Figure Lengend Snippet: Tmem63b and Piezo2 expression persists in Vglut3 Cre cKO. In situ hybridization and immunostaining of cultured DRG neurons from ( A ) Tmem63b wt and ( B ) Tmem63b fl for probe against Tmem63b -exon 5 ( white ) and tdTomato ( red ), respectively. Solid arrows denote tdTomato+ neurons with Tmem63b puncta. Open arrows denote tdTomato+ neurons without Tmem63b puncta. ( C ) Tmem63b expression measured by the percentage of tdTomato+ cells with a defined number of in situ hybridization puncta. Groups are binned by the following levels, 0–1 puncta no expression (mean ± SD %), 2–20 puncta medium expression (mean ± SD %), 21–104 puncta high expression (mean ± SD %). Tmem63b wt (blue), N = 3 animals, 1 male, 2 females vs. Tmem63b fl (red), N = 3 mice, 2 males, 1 female. Each dot represents a percentage of the total tdTomato+ cells imaged in a culture from Tmem63b wt −110, 164, 527 cells and Tmem63b fl −226, 316, 766. Chi-squared ( p < 0.0001 χ 2 = 22.76, df = 2). ( D ) Piezo2 expression (relative to Gapdh ) in tdTomato+ DRG neurons from Piezo2 wt ( blue , N = 3 animals, 2 males, 1 female) and Piezo2 fl ( yellow , N = 3 animals, 2 males, 1 female) mice (mean ± SD). Paired t -test p = 0.40.

Article Snippet: Synthesized cDNA was then used to perform qPCR using predesigned TaqMan gene expression assays (Thermo: mmGapdh -VIC, 4331182_Mm99999915_g1; mmPiezo2 -FAM, 4351372_Mm01262434_m1 probe that targets exon boundary 43–44) using a CFX Connect Real-Time System (Bio-Rad).

Techniques: Expressing, In Situ Hybridization, Immunostaining, Cell Culture