J. Biol. Chem., Vol. 282, Issue 1, 72-80

Measurement of Relative Rates of Leptin Biosynthesis桝fter preincubation of adipose tissue fragments in minimal essential medium (without methionine and cysteine, Sigma) in the absence (basal) or presence of insulin (6 nM) for 1 h at 37 癈, the media were exchanged for minimal essential medium containing [35S]methionine/cysteine (500 礐i/ml, Easy-TagTM EXPRE35S35S protein labeling mix, PerkinElmer Life Sciences) under the same hormonal conditions, and the incubation proceeded for another 45 min for labeling. Preliminary experiments showed that the incorporation of [35S]methionine/cysteine into total protein is linear up to 90 min and that [35S]methionine/cysteine incorporation into leptin is linear up to 60 min in rat adipose tissue. [35S]Leptin was then immunoprecipitated from tissue homogenates as described previously (12) using a polyclonal human leptin antibody from rabbit (Biovender, Brno, Czech Republic) followed by 4?2% SDS-PAGE. [35S]Leptin bands were detected using PhosphorImager (Storm 860, GE Healthcare), and band intensities were quantified with ImageQuant software 5.0 (GE Healthcare). The completeness and specificity of immunoprecipitation was confirmed. Incorporation of the label into trichloroacetic acid-precipitable protein was determined (13), and rates of leptin biosynthesis under different conditions were normalized to this value to calculate relative rates of leptin biosynthesis. Lipoprotein lipase (LPL) was immunoprecipitated by adding 3 礸 of chicken anti-bovine LPL antibody (gift from Dr. J. Goers, California Polytechnic State University, San Luis Obispo, CA) as described previously (14).

PLoS ONE. 2008; 3(6): e2491.

Cloning, expression analysis of AHK5 and characterisation of T-DNA mutants The AHK5 Entry clone was constructed using Gateway⑩ technology (Invitrogen, UK). It was obtained through TOPO-reaction using the pENTR/D-TOPO vector (Invitrogen). PCR was performed using Phusion⑩ polymerase (Finnzymes, UK) and cDNA from Arabidopsis roots as template. Primers were as follow: 5∏-CACC-ATGGAGACTGATCAGATTGAGGAA-3∏, 5∏-GTGCAAATACTGTTGCAAACACTCTC-3∏. The AHK5 Entry clone was verified via restriction analysis and sequencing (GATC Biotech). The construct for the expression of the GFP fusion proteins under the control of 35S promoter (P35S::GFP:AHK5) was cloned via LR-reaction into the destination vector pK7WGF2.0 [54]. For complementation of the mutant line in the Ws4 background, the TAP-tag destination vector pYL436 [55] was used to transform ahk5-3 plants. The transformants were selected on BASTA and gentamycin and analysed by PCR. For the Col-0 mutant complementation the P35S::GFP-AHK5 construct in pK7WGF2.0 was used to transform ahk5-1 plants. The transformants were selected on BASTA and kanamycin and analysed by PCR as described below. For the AHK5 overexpressor in Ws4 background, LR reaction was used to clone the AHK5 cDNA under the control of 35S promoter into the destination vector pMDC32 [56]. All plant transformations were carried out using Agrobacterium-mediated transformation by floral dipping [57]. A four-step procedure was used to generate the PAHK5::GFP::AHK5 expression cassette. In step 1, a 7117 bp fragment containing the 3.2 kb upstream promoter region of AHK5, the full length genomic sequence of AHK5 and 217 bp of 3∏ region was amplified from Col-0 genomic DNA by PCR using KOD Hot Start DNA polymerase (Novagen, Germany) and the primers AHK.FOR.-3205 (5∏-CACC-TCTAGACCCTACACGGGATAGATTATCG-3∏) and AHK.REV.+219 (5∏-TTTGTCGACTCTGCTGGATTCGAATGGTGGG-3∏) and cloned into the pENTRD/TOPO entry vector (Invitrogen, UK) to generate pMKC101. The entire construct was verified by sequencing. In step 2, a hybrid DNA fragment containing 643 bp upstream promoter region of AHK5 from pMKC101 was joined to the GFP sequence and AHK5 exon 1 sequence (from the P35S::GFP::AHK5 construct) by single joint PCR [58]. Briefly, in the 1st round PCR stage, 2 separate PCR reactions were set up. In reaction 1, primer 1 (5∏-CCTTTTGCATCTCGAGACTTCATGATTAC-3∏) and primer 2 (5∏-GGTGAACAGCTCCTCGCCCTTGCTCACCATTTCACAGACCATTGATCAAGGTTTCTC-3∏) were used to incorporate a XhoI restriction site (underlined in primer 1) and a 27 bp of the 5∏ end of GFP sequence (underlined in primer 2) onto the 5∏- and 3∏-ends of the 643 bp AHK5 promoter region, respectively. In reaction 2, a fragment containing the entire GFP sequence and AHK5 exon 1 was amplified using primer 3 (5∏-ATGGTGAGCAAGGGCGAGGAGCTGTTCACC-3∏) and primer 4 (5∏-GATGAGTCGAATTCAATAGGTTTGGTAACC-3∏) from the P35S::GFP::AHK5 construct. Primer 4 contains an EcoRI site (underlined). The products from the two reactions were joined (via the 27 bp overlapping 5∏ GFP sequence common to both PCR products) in the 2nd round PCR stage to generate a hybrid DNA fragment. This fragment was then amplified with the primers 1 and 4 in the 3rd round PCR stage. In step three, the hybrid PCR product was digested with XhoI/EcoRI, and cloned into an XhoI/EcoRI cut pMKC101 plasmid to create the PAHK5::GFP::AHK5 cassette. The integrity of the hybrid DNA fragment was verified by sequencing. Finally, step 4; the PAHK5::GFP::AHK5 cassette was cloned into the Gateway destination vector pMDC99 [56] using the LR reaction. This binary vector was then transformed into the Agrobacterium tumefaciens strain GV3101, and used in tobacco transient expression studies as described above. For identification and characterisation of homozygous insertion mutants, genomic DNA isolated from appropriate wild type and mutant plants was used for PCR analysis, using various PCR combinations and primers. T-DNA primers used were those already described [29], [30]. Individuals were chosen from homozygote lines by selection on BASTA, Southern analysis confirmed the presence of single T-DNA insertions in these lines and at least 3 generations were followed through to get a homozygote population. For RT-PCR, total RNA from corresponding tissues and developmental stages of A. thaliana was isolated using RNAwiz⑩ (Ambion, UK) or TRIZOL reagent (Invitrogen, UK) and genomic DNA was removed using TURBO DNA-free⑩ (Ambion, UK). RNA was isolated from guard cell-enriched epidermal fragments and whole leaves as described previously [16]. Subsequently, 1.5 レg of total RNA was reverse transcribed using oligo-dT primer with SuperScript⑩ III Reverse Transcriptase (Invitrogen, UK) and the resulting cDNA was used as template for the PCR with HotStart Taq polymerase (Genaxxon, Germany). PCR products were separated via agarose gel electrophoresis after different number of PCR cycles for comparison with ACTIN2 or EF1 as described in [59]. The sequences of the primers shown in Figure 2 are in Supplementary data.

PLoS ONE. 2008; 3(6): e2491.

Measurement of H2O2 and NO using confocal/fluorescent microscopy Epidermal peels from mature leaves, prepared as described above, were incubated in Mes/KCl buffer for 2–3 h. Following this, the fragments were loaded by incubation in 50 μM of the H2O2-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, Leiden, The Netherlands) for 10 min. After washing in fresh buffer for a further 20 min, the fragments were challenged with various compounds as indicated in the figure legends. For dark treatments the peels were incubated in darkness for 30 min and microscopy performed. Confocal laser scanning microscopy was used to visualise fluorescence, using an excitation wavelength of 488 nm and an emission wavelength of 515–560 nm (Nikon PCM2000, Nikon Europe B.V. Badhoewvedorp, The Netherlands). Images were acquired and analysed using Scion Image software (Scion Corp., USA) to measure the relative fluorescence intensities in the cells following various treatments. For the data in Figure 7D, fluorescent microscopy (Zeiss Axioskop2, Zeiss, UK) was used with filter set 10 (excitation filter BP 450–490 nm, beam splitter FT 510 nm and emission filter BP 515–565 nm). Images were acquired and analysed using Image J software (NIH, USA). Data represent fluorescence intensities expressed as average fluorescence or as a percent of the control values, from several guard cells analysed in different experiments. For NO fluorescence, epidermal peels were loaded with 10 μM of the NO-sensitive dye diaminofluorescein diacetate (DAF2-DA, Calbiochem, UK) using exactly the same dye loading procedure, and images acquired using confocal microscopy as described above.

PLoS ONE. 2008; 3(6): e2491.

Stomatal bioassaysStomatal assays were performed on leaves essentially as described in [15]. Leaves were floated for 2.5 h under continuous illumination (100–150 μE m−2 s−1) in Mes/KCl buffer (5 mM KCl/10 mM Mes/50 μM CaCl2, pH 6.15). Once the stomata were fully open, leaves were treated with various compounds for a further 2.5 h. The leaves were subsequently homogenised individually in a Waring blender for 30 s and the epidermal fragments collected on a 100 μm nylon mesh (SpectraMesh, BDH-Merck, UK). Stomatal apertures from epidermal fragments were then measured using a calibrated light microscope attached to an imaging system (Leica QWin software, Leica, UK). Flg22 and elf-26 peptides were a kind gift from J Mansfield (Imperial College London). Elicitors were added to the incubation buffer at 2.5 h and stomatal apertures measured after a further 3 h. For bacterial experiments, Pseudomonas syringae pv DC3000 or P. syringae hrpA− mutant were grown overnight in LB media and overnight cultures centrifuged, resuspended in 10 mM MgCl2 at an OD600=0.2 (equivalent to 2×108 cfu/ml). Silwet (0.002% v/v) was added to cultures or MgCl2 to act as a wetting agent. Bacteria were gently coated onto the abaxial side of leaves on intact plants (controls were MgCl2 with Silwet alone). Plants were left in the growth chambers with a covered lid (to increase humidity) for 3 h, inoculated leaves subsequently detached and stomatal apertures measured.

PLoS ONE. 2008; 3(6): e2491.

Growth and maintenance of plants Wild type and mutant seeds of Arabidopsis thaliana ecotype Columbia (Col-0) and Wassilewskijia (Ws4) were sown on Levington's F2 compost and grown under a 16 h photoperiod (100–150 μE m−2 s−1), 22°C and 65% relative humidity in controlled environment growth chambers (Sanyo Gallenkamp, UK). atrbohD/F seeds were obtained from J Jones (Sainsbury Laboratory, Norwich, UK).]. Details of the T-DNA insertion lines in AHK5 are as follows: ahk5-1 mutant seeds (SAIL 50_H11) were originally obtained from Syngenta (SAIL collection, now available at ABRC/NASC), and the ahk5-3 seeds (FLAG_271G11) were obtained from the INRA/FLAG-FST collection at Versailles [29], [30].

PLoS ONE. 2008; 3(6): e2491.

As demonstrated by the wild type behaviour of both ahk5 mutants to ABA, the AHK5-dependent signalling pathway does not contribute to the ABA response pathway in guard cells.

PLoS ONE. 2008; 3(6): e2491.

Figure 2 Expression pattern of AHK5 and characterisation of the T-DNA insertion sites in ahk5-1 and ahk5-3. (A) Steady-state levels of AHK5 transcript in different tissues and developmental stages of Arabidopsis detected by semi-quantitative RT-PCR using different cycle numbers. For the detection of AHK5, up to 40 cycles of PCR were performed using primers 3 and 10 (left panel) or primers 8 and 9 and 40 cycles of PCR (right panel). GC, cDNA from guard cell-enriched non-treated leaf sample; GC+H, cDNA from guard cell-enriched leaf samples treated with 0.5 mM H2O2 for 2 h; WL, cDNA from whole-leaf sample. ACTIN and EF1 were used as controls. Primer numbers indicated in panel C. (B) Expression of a PAHK5-GFP-AHK5genomic construct in transiently transformed tobacco leaf cells. Agrobacteria carrying the PAHK5-GFP-AHK5 construct were infiltrated into the abaxial side of the leaf and the GFP fluorescence analysed by CLSM 2 days later. Top panels = GFP images, lower panels = GFP image overlaying bright field image. The scale bar represents 20 μm. Images shown from repeat experiments. (C) AHK5 gene structure, position of primers used for genomic and RT-PCR and T-DNA insertions in ahk5-1 (Col-0) and ahk5-3 (Ws4). (D) Analysis of AHK5 expression in seedlings of wild type, ahk5-1 and ahk5-3 mutant, P35S-AHK5 complemented and AHK5 over-expressing plants using the indicated primer pairs (see Supplementary data for sequences). PLoS ONE. 2008; 3(6): e2491.

PLoS ONE. 2008; 3(6): e2491.

Figure 1 Subcellular localisation of AHK5 in plant cells. (A) Confocal images of Arabidopsis protoplasts and tobacco (Nicotiana benthamiana) leaf cells transiently transformed with a construct expressing P35S:GFP-AHK5 cDNA. Left panel, GFP fluorescence; right panel, bright field image. The bars represent 10 μm. (B) Western blot showing the expression of full-length GFP-AHK5 in transiently transformed tobacco leaf cells using anti-GFP antibody. Lane 1, protein standard; lane 2, extracts from cells transformed with a P35S-GFP-AHK5 construct; lane 3, extracts from cells transformed with the empty vector. (C) Cell fractionation of transiently transformed tobacco leaf cells expressing either GFP-AHK5, the microsomal marker BRI-GFP, the ER marker ERS1-GFP or the soluble marker ARR4-GFP.Two days after the infiltration of the Agrobacteria the leaf tissue was harvested and total protein extracted. The microsomal fraction (M) and the soluble fraction (S) were separated by ultracentrifugation. Equal cell equivalents were loaded per lane. (D) Fluorescence intensity images (upper panel) and the corresponding intensity profiles (lower diagram) of the indicated plasmalemma-cell wall section (blue bar in the magnification) of two adjacent, transiently transformed tobacco leaf cells co-expressing GFP-AHK5 (green dots) and the plasma membrane marker pm-rk-CD3-1007 (red dots). The red line represents the mono-peak Gauss fit of RFP fluorescence and the green line the multi-peak Gauss fit of GFP fluorescence (green). The single fits which compose the multi-peak Gauss fit of GFP, are shown in black. PLoS ONE. 2008; 3(6): e2491.

plos genet. 2006 november 2(11): e196

Functional Identification of Api5 as a Suppressor of E2F-Dependent Apoptosis In Vivo

J. Clin. Invest. 118(1): 89-99 (2007)

Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cellsJ. Clin. Invest. Takuma Aikawa, et al. 118:89 doi:10.1172/JCI32412 [Go to this article.] Figure 3Effects of GPC1 levels on tumor growth. (A) Representative tumors. GAS6 cells consistently formed smaller tumors than sham-transfected cells. The mice bearing GAS tumors appeared healthy. By contrast, the mice bearing tumors from sham-transfected cells had to be terminated due to their cachectic appearance, tumor size, and tendency of the tumors to develop surface ulcerations. (B) Tumor growth curves. Exponentially growing (2 ?106) sham-transfected PANC-1 cells (filled diamonds) and GAS7 (open triangles) and GAS6 (open squares) cells were injected subcutaneously in athymic mice, and tumor growth was measured weekly. Tumor volume was determined by the equation: volume = (l ?h ?w) ??/4, where l is length, h is height, and w is width of the tumor. Data are mean ?SEM from 9 tumors for sham-transfected PANC-1 cells and for each of the 2 GAS clones.

J. Biol. Chem., Vol. 282, Issue 1, 200-207

Cytosolic NADP+-dependent Malic Enzyme 1 Activity朏resh INS-1 cell extracts from the malic enzyme isoform knock-down studies were analyzed for malic enzyme activity. NADP+-dependent malic enzyme was measured spectrophotometrically (340 nm) at 32 癈 in a 96-well plate (250 祃/well). The assay was performed under the following reaction conditions: pH 7.4, 100 mM Tris/HCl, 1.0 mM MnCl2, 1.0 mM NH4Cl, 100 mM KCl, 1.25 mM NADP (freshly prepared), and 10 mM L-malate. All cell samples were run in duplicate with or without malate as substrate for test and control conditions, respectively. Cell extracts were added to all of the wells last, immediately shaken, and the absorbance read every 1 min for 40 min. Enzyme activity was determined by subtracting the activity of the control wells for each sample from the test wells run with L-malate. The resulting slopes of absorbance versus time were averaged and normalized to protein content.

J. Biol. Chem., Vol. 282, Issue 1, 287-293

Cell Death and Apoptosis桟ells were plated in 6-well plates and cultured until 80% confluent. Following transduction with -galactosidase or Foxo1 or treatment with 25 ?FONT size=-2>M H2O2 for 1 h to induce apoptosis (31), we measured caspase-3 activation and cell toxicity using the CaspACE and CytoTox 96 cytotoxicity (Promega) assay systems, respectively. The CytoTox 96 assay measures the release of cytoplasmic enzymes in the medium upon loss of membrane integrity as a result of cytotoxicity. Simultaneously, cells were harvested to measure apoptosis. After a brief centrifugation at 450 x g, cell pellets were resuspended in lysis buffer, and caspase-3 activity was determined by adding assay buffer containing the DEVD-p-nitroanilide substrate.

J. Biol. Chem., Vol. 282, Issue 1, 72-80

Transient Transfection of Reporter Constructs into Differentiated 3T3-L1 adipocytes and Luciferase Assay桹n day 4 of differentiation, 3T3-L1 adipocytes were transfected with 1 礸 of DNA using Lipofectamine Plus reagents (Invitrogen). Briefly, cells were washed with phosphate-buffered saline and incubated with reduced serum medium (Opti-MEM? modification of Eagle's minimal essential medium, Invitrogen) for 45 min at 37 癈. Pre-complex DNA-Plus-LipofectamineTM reagent was prepared according to the manufacturer's protocol and added into each well for transfection. After overnight transfection, cells were replenished with 10% fetal bovine serum Dulbecco's modified Eagle's medium and incubated for another 36?8 h for expression. For monitoring transfection efficiency, pRL-TK vector (containing Renilla luciferase, Promega) was co-transfected. Cells co-transfected with various UTR-pGL3 constructs and pRL-TK vectors were harvested in lysis buffer, and activities of firefly and Renilla luciferase were measured by using the Dual-Luciferase assay kit (Promega) in a luminometer (Turner Designs, Sunnyvale, CA). The reporter activity was expressed as arbitrary LUC units (firefly/Renilla).

PLoS ONE. 2008; 3(6): e2491.

Transient transformation of tobacco leaf cells and Arabidopsis protoplasts, GFP and RFP analyses The p19 protein from tomato bushy stunt virus cloned in pBIN61 [60] was used to suppress gene silencing in tobacco (Nicotiana benthamiana). All plasmids were transformed in Agrobacterium tumefaciens strain GV3101 pMP90, which was grown in YEB medium to OD600 1.0 and prior to infiltration resuspended in AS medium (10 mM MgCl2, 150 μM acetosyringone and 10 mM MES pH 5.7) to OD600=0.8. The Agrobacterium strains containing the GFP or p19 construct were mixed in a 11 relationship and co-infiltrated into leaves of 4-week-old tobacco plants as described in [60]. The abaxial epidermis of infiltrated tobacco leaves was assayed for fluorescence by CLSM (confocal laser-scanning microscopy) 2 to 3 days post infiltration according to [61]. Arabidopsis protoplasts were transformed using PEG mediated transformation procedure and assayed for fluorescence by CLSM after 20 h [61]. CLSM was performed using a Leica TCS SP2 confocal microscope (Leica Microsystems, Germany). These CLSM images were obtained using the Leica Confocal Software and the HCX PL APO 63×/1.2 W CORR water-immersion objective. For recording the RFP and GFP intensity profiles a homemade confocal laser-scanning microscope, based on a Zeiss Axiovert was used [62], [63]. The microscope was equipped with an avalanche photodiode (APD, SPCM-AQR-14, Perkin Elmer, USA) as a spectrally integrating detector. A pulsed 473 nm diode laser (Picoquant LDH-P-C470) operating at a repetition rate of 10 MHz served as excitation source. Fluorescence intensity images were obtained by raster scanning the sample and detecting emission intensity for every spot on the sampled area. The setup was equipped with a 480 nm long pass filter (Semrock Razor Edge LP02-473RU-25) to block back-scattered excitation light, a 500 nm bandpass filter (Semrock BrightLine BL500/24) to detect GFP-fluorescence and a 590 nm bandpass filter (Semrock FF01-590/20-25) to detect RFP-fluorescence in front of the APD. The processing of the obtained fluorescence intensity images was accomplished with the WSxM software [64].

J. Biol. Chem., Vol. 280, Issue 48, 40144-40151

Radioligand Binding桯EK293 cells (4 x 106 cells/100-mm dish) were transfected with 0.5 礸 of S138R or wild-type 5-HT2C receptor plasmid DNA using 20 祃 of Lipofectamine. For saturation experiments, HEK293 and S138R-expressing stable cells were transfected with 0.2 or 0.5 礸 of wild-type 5-HT2C receptor plasmid DNA. Membranes were prepared from transfected cells as described previously (23). Saturation experiments were performed using [3H]mesulergine (0.1-5 nM) in the presence and absence of 1 ?FONT size=-2>M mianserin in 0.5 ml of buffer (50 mM Tris-HCl, 10 mM MgSO4, 0.5 mM EDTA, and 0.1% ascorbate) with 0.5 ml of diluted cell membranes (10 礸 of protein/assay tube). The mixture was incubated at 37 癈 for 30 min, followed by filtration through glass fiber filters (Schleicher & Sch黮l) on a Brandel cell harvester and liquid scintillation counting in Ecoscint (National Diagnostics). Protein was determined using BCA (Pierce). Data were analyzed using GraphPad Prism software.