Journal: Immunology and Cell Biology

Article Title: A novel mechanism for ERK-dependent regulation of IL4 transcription during human Th2-cell differentiation

doi: 10.1038/icb.2011.87

Figure Lengend Snippet: ERK selectively regulates Th2-cell differentiation. ( a ) Results of a western blot analysis, at indicated times, for ERK-1/2 in naive CD4 + T cells transfected with ERK-specific siRNA. The effect of treatment of GFP-specific siRNA on ERK levels, at 36 h later, is also included, with GAPDH as an internal loading control. ( b , c ) Effect of treatment of naive CD4 + T cells with siRNA specific for either ERK or GFP on the generation of either IL-4 and IL-13 (panel b ) or IFN-γ (panel c ) cytokine-producing cells as measured by an ELISPOT assay at day 7 of culture. The stimulation conditions tested here were activation (TCR), Th2 polarization (TCR+IL-4) and Th1 polarization (TCR+IL-12). The corresponding profiles obtained in mock (GFP)-siRNA-treated cells cultured under similar conditions are also shown. * P ⩽0.05 (one-way analysis of variance (ANOVA) test); NS=not significant. Each bar represents the mean (±s.d.) of three biological replicates in which each replicate represented a pool of cells isolated from 3 to 5 individuals. In a separate experiment, we stimulated either mock- (red line) or ERK-silenced (blue line) cells under Th2-polarizing conditions for 7 days and then measured the extent of apoptotic cell death by staining with Annexin-V, followed by flow-cytometric analysis. The profiles obtained are shown in d and represents one of three separate experiments. For e , a similar experiment was performed, except that cells were first labeled with CFSE before stimulation and the extent of cell proliferation was determined 7 days later ( n =3). f compares the concentration of IL-2 present in the supernatant of these cultures at day 7. Here, U indicates cells that were not activated, whereas S denotes the stimulated groups. Values are the mean (±s.d.) of three separate experiments.

Article Snippet: Recombinant human IL-4, IL-12, IL-2 cytokines, anti-human IL-4 and IL-4R-neutralizing antibodies, human IL-4 and IFN-γ ELISPOT kits were procured from R&D Systems, Minneapolis, MN, USA.

Techniques: Cell Differentiation, Western Blot, Transfection, Enzyme-linked Immunospot, Activation Assay, Cell Culture, Isolation, Staining, Labeling, Concentration Assay

Journal: Immunology and Cell Biology

Article Title: A novel mechanism for ERK-dependent regulation of IL4 transcription during human Th2-cell differentiation

doi: 10.1038/icb.2011.87

Figure Lengend Snippet: ERK regulates IL4 gene transcription. ( a ) Kinetics of IL-4 mRNA induction in naive CD4 + T cells stimulated with TCR in the presence (TCR+IL-4) or absence (TCR) of IL-4. ( b ) Effects of treatment of cells with either GFP or ERK1/2-specific siRNA on induction of the IL-4 transcript in activated and Th2-polarizing cells. The time points at which transcript levels were determined by RT-PCR are indicated. Here, primers specific for β-actin were also included to provide an internal normalization control. Western blots in c confirm the silencing of STAT6 protein levels in the nuclear extract of cells treated for the indicated times with specific siRNA. Blots were also probed with anti-histone H3 antibodies to provide a loading control. The effects of STAT6- versus GFP-silencing on IL-4 mRNA levels as a function of time are shown in d . In e , naive CD4 + T cells were stimulated under Th2-polarizing conditions either in the absence (−) or presence (+) of U0126, and IL-4 transcript levels determined by RT-PCR 84 h later.

Article Snippet: Recombinant human IL-4, IL-12, IL-2 cytokines, anti-human IL-4 and IL-4R-neutralizing antibodies, human IL-4 and IFN-γ ELISPOT kits were procured from R&D Systems, Minneapolis, MN, USA.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Immunology and Cell Biology

Article Title: A novel mechanism for ERK-dependent regulation of IL4 transcription during human Th2-cell differentiation

doi: 10.1038/icb.2011.87

Figure Lengend Snippet: ERK associates with the IL4 promoter in activated T cells. ( a ) IL-4 spot-forming cells (SFCs) from an ELISPOT assay in which naive CD4 + T cells were either transfected with GFP siRNA (Group 1), ERK siRNA (Group 2) or ERK siRNA, followed by supplementation of recombinant IL-4 (Group 3) in the culture. ‘Un' represents unstimulated cells, whereas TCR and TCR+IL-4 represent activation and polarizing conditions, respectively. * P ⩽0.0001. Each bar represents the data from three independent experiments (mean±s.d. value), performed on three biological replicates in which each replicate represented a pool of cells isolated from 3–5 individuals. ( b ) Naive CD4 + T cells were either left untreated (top panel) or treated with either ERK1/2 siRNA or U0126 (lower two panels). These cells were then stimulated through the TCR, and time-dependent recruitment of ERK to the IL4 promoter was monitored by ChIP analysis. The corresponding profiles obtained without anti-ERK antibody (no Ab) and input are also shown. ( c ) ERK-specific chromatin-immunoprecipitated samples from either TCR-activated (3 h) or unstimulated cells were hybridized against a tiled array that was generated around the 60-nucleotide long IL-4 promoter region from −1374 to −1315 nt. This region included the ERK-binding site. The first probe in this array was a 60 nt sequence spanning from nt positions −1404 to −1345. All subsequent probes—also 60 nt long—then represented successive one-nucleotide shifts in sequence, terminating with a probe extending from −1344 to −1285 nt. The fold enhancement in hybridization intensity—in terms of log 2 values—obtained for each of these probes with sample from TCR-activated cells, relative to that obtained with a parallel sample from unstimulated cells, is plotted here. The individual probes are identified on the X axis on the basis of the sequence position of the central nucleotide. ( d ) Parent 60 nt region where the optimal ERK-binding domain is highlighted in red.

Article Snippet: Recombinant human IL-4, IL-12, IL-2 cytokines, anti-human IL-4 and IL-4R-neutralizing antibodies, human IL-4 and IFN-γ ELISPOT kits were procured from R&D Systems, Minneapolis, MN, USA.

Techniques: Enzyme-linked Immunospot, Transfection, Recombinant, Activation Assay, Isolation, Immunoprecipitation, Generated, Binding Assay, Sequencing, Hybridization

Journal: Immunology and Cell Biology

Article Title: A novel mechanism for ERK-dependent regulation of IL4 transcription during human Th2-cell differentiation

doi: 10.1038/icb.2011.87

Figure Lengend Snippet: Association of ERK is necessary for IL4 promoter activity. ( a ) Jurkat cells were transfected with the pEGFP1 vector (vector, top two panels), with the pIL-4-EGFP construct (from second to the fourth row of panels) or with a derivative of the pIL-4-EGFP construct in which the ERK-binding region was partially deleted (Pdel-IL-4-EGFP, bottom panels). This included two additional experimental groups in which pIL-4-EGF-transfected cells were also treated with either ERK-specific siRNA (panels in the third row from top) or with the MEK inhibitor U0126 (panels in the fourth row from top) as indicated. Figure shows the level of fluorescence obtained in each of these groups of cells when they were either left unstimulated or stimulated through the TCR for 24 h. Values indicated in each panel are the mean (±s.e.m) intensity of fluorescence measured for at least 40 cells from different fields in 3 separate slides for each group. ( b ) Naive CD4 + T cells were either left untreated (top panel) or treated with either ERK1/2 siRNA (KD) or U0126 (lower two panels). These cells were then stimulated through the TCR, and time-dependent recruitment of RNA polymerase II (RNAP II) to the IL4 promoter was monitored by ChIP analysis. The corresponding profiles obtained without anti-ERK antibody (no Ab) and input are also shown. ( c ) Time-dependent recruitment of indicated transcription factors, as determined by ChIP, to the IL-4 promoter after stimulation of cells through the TCR either in the presence (+) or absence (−) of U0126.

Article Snippet: Recombinant human IL-4, IL-12, IL-2 cytokines, anti-human IL-4 and IL-4R-neutralizing antibodies, human IL-4 and IFN-γ ELISPOT kits were procured from R&D Systems, Minneapolis, MN, USA.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Construct, Binding Assay, Fluorescence

Journal: Immunology and Cell Biology

Article Title: A novel mechanism for ERK-dependent regulation of IL4 transcription during human Th2-cell differentiation

doi: 10.1038/icb.2011.87

Figure Lengend Snippet: The effect of ERK on IL4 gene expression is specific and involves both ERK-1 and ERK-2. ( a ) compares the distribution of three transcription factors (GATA3, NFATc1 and c-Jun) between the cytoplasmic and nuclear fractions in unstimulated (UNST), TCR-triggered (TCR, stimulation time of 30 min) and U0126-treated cells, followed by TCR triggering (U0126+TCR, stimulation time of 30 min). LC denotes the loading control, which was PLCγ2 for cytoplasmic fractions and Histone 1 for the nuclear fractions. ( b ) Effects of treatment of cells with either GFP or ERK1/2-specific siRNA on induction of the IL-13 transcript in activated cells. The time points at which transcript levels were determined by RT-PCR are indicated. Here, primers specific for β-actin were also included to provide an internal normalization control. ( c ) Results from a ChIP assay that monitored the recruitment of NFATc, c-jun and GATA3 to the region spanning −76 to −234 nt of the IL-13 promoter. Here, either untreated cells (U0126−) or cells treated with U0126 (U0126+) were stimulated through the TCR as indicated. Input and no Ab controls are also shown. ( d ) Results of experiments in which cells were activated through the TCR for indicated time and then subjected to a ChIP analysis using antibodies that were either specific only to ERK-1 or to ERK-2. For the purposes of comparison, an additional group was also included in which antibodies to both ERK-1 and ERK-2 were combined in the ChIP experiment (ERK1/2). Results shown are a representative of three separate experiments. ( e ) gives the results of a DNA pull-down experiment in which nuclear extracts from CD4 + T cells, stimulated through the TCR for 3 h, were incubated either with a 20-mer double-stranded DNA probe extracted from or with a control probe of similar length. In both cases, the probes were biotinylated. After incubation, the probes were separated by affinity chromatography (streptavidin-agarose), and the bound proteins eluted and analyzed for the presence of ERK-1 and ERK-2 by western blot analysis (see the ‘Methods' section). Results shown are from one of three separate experiments. ( f ) Results obtained in an RT-PCR experiment in which cells transfected separately with ERK1 siRNA (ERK1), ERK2 siRNA (ERK2) or with a combination of both (ERK1/2) were stimulated through the TCR. At the end of 84 h, cells were harvested, the total RNA was isolated and analyzed by RT-PCR for the IL-4 transcript. For the purposes of comparison, results obtained in cells that were either not treated with any siRNA (Control) or those treated with siRNA specific for GFP (Mock) are also shown. In all cases, primers specific for β-actin were also included to provide an internal normalization control.

Article Snippet: Recombinant human IL-4, IL-12, IL-2 cytokines, anti-human IL-4 and IL-4R-neutralizing antibodies, human IL-4 and IFN-γ ELISPOT kits were procured from R&D Systems, Minneapolis, MN, USA.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison, Incubation, Affinity Chromatography, Western Blot, Transfection, Isolation

Journal: Immunology and Cell Biology

Article Title: A novel mechanism for ERK-dependent regulation of IL4 transcription during human Th2-cell differentiation

doi: 10.1038/icb.2011.87

Figure Lengend Snippet: Stochastic variability in ERK levels influences individual-specific variations in susceptibility to Th2-cell differentiation. ( a ) Extent of variability in ERK1/2 protein levels in unstimulated naive CD4 + T cells. ERK1/2 levels were determined from western blot analysis using antibodies specific for both ERK-1 and ERK-2. Here, each bar depicts the results for one individual ( n =38). For the purposes of comparison, GAPDH levels were also monitored in each of these samples and the panel shows the comparison of the relative variability of total ERK1/2 levels, in comparison with that of GAPDH. The Y axis gives the normalized intensity of the protein band in each sample, after subtracting the mean value of the band intensities in all the samples (X−X mean ). The corresponding values for variance in X−X mean for ERK (V ERK ) and GAPDH (V GAPDH ) are indicated. ( b ) Results of a regression analysis between p-ERK/ERK (that is, ratio of phospho-ERK1/2 to ERK1/2 protein) and the frequency of IL-4-producing cells in naive CD4 + T cells obtained from different individuals ( n =20) as described in the text. Here, the frequency of IL-4-producing cells was determined by an ELISPOT assay. Each value represents the mean of triplicate sets.

Article Snippet: Recombinant human IL-4, IL-12, IL-2 cytokines, anti-human IL-4 and IL-4R-neutralizing antibodies, human IL-4 and IFN-γ ELISPOT kits were procured from R&D Systems, Minneapolis, MN, USA.

Techniques: Cell Differentiation, Western Blot, Comparison, Enzyme-linked Immunospot

Journal: JCI Insight

Article Title: Reciprocal regulation of Th2 and Th17 cells by PAD2-mediated citrullination

doi: 10.1172/jci.insight.129687

Figure Lengend Snippet: (A) Indicated C57BL/6 Th cells were left unstimulated (–) or restimulated with anti-CD3 (+) overnight. The level of cit-H3 was examined with Western blotting using anti–cit-H3. The normalized density of cit-H3 is shown in the bar graph (n = 3, 1-way ANOVA). (B and C) C57BL/6 effector Th cells were generated in the presence of Cl-am at indicated concentrations for 5 days. Whole Th2 extract was examined with Western blotting using indicated antibodies. Representative blots and normalized density of cit-H3 from 2 experiments are shown in B. The expression of indicated cytokines by the Th cells after restimulation with anti-CD3 is shown in C (n = 4, 1-way ANOVA). (D) Primary human Th cells from 5 healthy donors were differentiated in vitro into Th2 or Th17 cells in the presence or absence of Cl-am (100 μM). The production of IL-4 and IL-17A after restimulation with anti-CD3 was quantified with ELISA. Data points from the same donors are connected with lines (1-tailed paired Student’s t test). (E–I) Allergic airway inflammation was induced in C57BL/6 mice (n = 6 per group) in the absence or presence of Cl-am. Splenocytes were restimulated with ovalbumin for 72 hours. The levels of IL-4 and IL-17A in supernatant are shown in E. Imm, immunized; cha, challeneged. The levels of ovalbumin-specific (ova-specific) IgE and IgG1 in serum are shown in F. Representative H&E staining of the lung tissue is shown in G. Scale bars: 100 μm. The total number of cells (H) and the percentage of eosinophils (I) in bronchial lavage are also shown. Statistical analysis for E, F, H, and I was performed with 2-tailed Student’s t test.

Article Snippet: Sandwich ELISA was performed with the following antibodies: anti–mouse IFN-γ (catalog 551221)/biotin anti–mouse IFN-γ (catalog 554550) from BD Pharmingen; anti–mouse IL-4 (catalog 554434)/biotin anti–mouse IL-4 (catalog 554390), anti–mouse IL-5 (catalog 554393)/biotin anti–mouse IL-5 (catalog 554397), anti–mouse IL-13 (catalog 14-7133-81)/biotin anti–mouse IL-13 (catalog 13-7135-81), anti–mouse IL-17A (catalog DY421)/biotin anti–mouse IL-17A (catalog DY421), anti–human IL-4 (catalog 14-7049)/biotin anti–human IL-4 (catalog 13-7048), and anti–human IL-17A (catalog 14-7178)/biotin anti–human IL-17A (catalog 13-7179) from eBioscience.

Techniques: Western Blot, Generated, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Staining

Journal: Human Reproduction (Oxford, England)

Article Title: Aberrant expression of regulators of cell-fate found in eutopic endometrium is found in matched ectopic endometrium among women and in a baboon model of endometriosis

doi: 10.1093/humrep/deq248

Figure Lengend Snippet: Localization of the markers of cell fate studied in the ectopic endometrial lesions from women. Photomicrographs are representatives of the immunostaining for telomerase, nucleolin, proliferating cell nuclear antigen (PCNA) and histone γ-H2AX; positive staining = brown; scale bars = 100 µm (f, g and h) applicable to all panels. (a–d) External positive control tissues with inserts illustrating the immunologically negative controls: (a) telomerase in human tonsillar cortex, (b) nucleolin in human adeno-carcinoma of the colon, (c) PCNA in human tonsil and (d) histone γ-H2AX in human late-secretory endometrium; (e–h) ectopic blue/chocolate lesions from women with endometriosis stained with (e) telomerase (f) nucleolin, (g)PCNA and (h) γ-H2AX.

Article Snippet: The commercially obtained primary antibodies used were against human nucleolin (Novo Castra, Newcastle-upon-Tyne, UK Mouse monoclonal), human PCNA (Dako, UK Mouse monoclonal), human telomerase (Abcam, Cambridge, UK Rabbit polyclonal) and γ-H2AX (Abcam, UK Rabbit polyclonal).

Techniques: Immunostaining, Staining, Positive Control

Journal: Human Reproduction (Oxford, England)

Article Title: Aberrant expression of regulators of cell-fate found in eutopic endometrium is found in matched ectopic endometrium among women and in a baboon model of endometriosis

doi: 10.1093/humrep/deq248

Figure Lengend Snippet: Localization of the markers of cell fate studied in the ectopic endometrial lesions during the window of implantation (WOI) in baboons. Photomicrographs are representatives of the immunostaining for telomerase, nucleolin, proliferating cell nuclear antigen (PCNA) and histone γ-H2AX; positive staining = brown; scale bars = 100 µm (b and g) applicable to all panels. (a–l) Ectopic blue/chocolate lesions from animals with induced disease stained for telomerase, for nucleolin, for PCNA and for γ-H2AX at 6, 12 or 15 months post-inoculation. (m–p) White lesions from induced animals did not show positive immunoreactivity for telomerase, nucleolin, PCNA or γ-H2AX.

Article Snippet: The commercially obtained primary antibodies used were against human nucleolin (Novo Castra, Newcastle-upon-Tyne, UK Mouse monoclonal), human PCNA (Dako, UK Mouse monoclonal), human telomerase (Abcam, Cambridge, UK Rabbit polyclonal) and γ-H2AX (Abcam, UK Rabbit polyclonal).

Techniques: Immunostaining, Staining

Journal: Human Reproduction (Oxford, England)

Article Title: Aberrant expression of regulators of cell-fate found in eutopic endometrium is found in matched ectopic endometrium among women and in a baboon model of endometriosis

doi: 10.1093/humrep/deq248

Figure Lengend Snippet: Box plot showing the median semi-quantitative scores for telomerase, nucleolin and PCNA, and immune-staining and median percentage of γ-H2AX immune-staining positive cells in baboon ectopic endometrial glandular (a–d) and stromal (e–h) compartments. Ectopic blue/chocolate lesions of animals (n = 6) with induced disease at 6 months (6M, n = 6), 12 months (12M, n = 6) and 15 months (15M, n = 6) after inoculation. The P-value from Freidman's test shown under each graph. Y-axis semi-quantitative (SQ) scores or percentage (%) of positive cells.

Article Snippet: The commercially obtained primary antibodies used were against human nucleolin (Novo Castra, Newcastle-upon-Tyne, UK Mouse monoclonal), human PCNA (Dako, UK Mouse monoclonal), human telomerase (Abcam, Cambridge, UK Rabbit polyclonal) and γ-H2AX (Abcam, UK Rabbit polyclonal).

Techniques: Staining

Journal: Human Reproduction (Oxford, England)

Article Title: Aberrant expression of regulators of cell-fate found in eutopic endometrium is found in matched ectopic endometrium among women and in a baboon model of endometriosis

doi: 10.1093/humrep/deq248

Figure Lengend Snippet: Localization of the markers of cell fate studied in the eutopic endometrium collected during the WOI in baboons. Photomicrographs are representatives of the immunostaining for telomerase, nucleolin, proliferating cell nuclear antigen (PCNA) and histone γ-H2AX; positive staining = brown; scale bars = 100 µm (c–h and l) applicable to all panels. (a–d) Endometrium from baboons (n = 5) prior to inoculation at 0 M and stained for telomerase, nucleolin, PCNA and histone γ-H2AX. (e–p) Eutopic endometrium from animals with induced disease (6, 12 or 15 months post-induction tissue from six animals included at each time point) stained with telomerase, nucleolin, PCNA and γ-H2AX. (q–t) Eutopic endometrium from the control healthy fertile animals (n = 8) stained for telomerase, nucleolin, PCNA and γ-H2AX.

Article Snippet: The commercially obtained primary antibodies used were against human nucleolin (Novo Castra, Newcastle-upon-Tyne, UK Mouse monoclonal), human PCNA (Dako, UK Mouse monoclonal), human telomerase (Abcam, Cambridge, UK Rabbit polyclonal) and γ-H2AX (Abcam, UK Rabbit polyclonal).

Techniques: Immunostaining, Staining

Journal: Human Reproduction (Oxford, England)

Article Title: Aberrant expression of regulators of cell-fate found in eutopic endometrium is found in matched ectopic endometrium among women and in a baboon model of endometriosis

doi: 10.1093/humrep/deq248

Figure Lengend Snippet: Box plots showing the median semi-quantitative scores for nucleolin, PCNA and telomerase, and immune-staining and median percentage of γ-H2AX immune-staining positive cells in baboon eutopic endometrial glandular (a–d) and stromal (e–h) compartments. Eutopic endometrium of healthy fertile control (FC) animals (n = 8), eutopic endometrium collected prior to inoculation (n = 5) 0M, and eutopic endometrium of animals with induced disease (n = 6) at 6 months (6M), 12 months (12M) and 15 months (15M) after inoculation. The P-value from Mann–Whitney U-test comparing FC animals and experimental animals before the induction of the disease is shown under each graph. Y-axis = semi-quantitative (SQ) scores or percentage (%) of positive cells.

Article Snippet: The commercially obtained primary antibodies used were against human nucleolin (Novo Castra, Newcastle-upon-Tyne, UK Mouse monoclonal), human PCNA (Dako, UK Mouse monoclonal), human telomerase (Abcam, Cambridge, UK Rabbit polyclonal) and γ-H2AX (Abcam, UK Rabbit polyclonal).

Techniques: Staining, MANN-WHITNEY

Journal: The FASEB Journal

Article Title: Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development

doi: 10.1096/fj.201900179RR

Figure Lengend Snippet: Expression and localization of Yap1 protein in mouse ovarian cells. A–C) Fluorescent IHC and confocal microscopy showing expression and localization of Yap1 (A), Ki67 (B), and 3β-Hsd (C) in ovarian cells using series sections of mouse ovarian tissues. The green color in A–C represents expression and localization of Yap1, Ki67, and 3β-HSD in mouse ovarian cells, respectively. A1–C1) Corresponding high-resolution images. Actin filaments were stained with rhodamine-phalloidin. Nuclei were stained with DAPI. GC, granulosa cell; LC, luteal cell; P2, secondary follicle. Scale bars, 100 μm.

Article Snippet: The antibody against Ki67 (marker of proliferation Ki-67) was from Abcam (Cambridge, United Kingdom).

Techniques: Expressing, Confocal Microscopy, Staining

Journal: The FASEB Journal

Article Title: Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development

doi: 10.1096/fj.201900179RR

Figure Lengend Snippet: Pharmacological inhibition of Yap1 disrupts ovarian follicle development in vitro and in vivo. A) Representative images showing the morphology of postnatal d 12 mouse ovaries incubated for 7 d in the presence or absence of VP (Verteporfin, a selective YAP1 antagonist). Scale bar, 500 μm. B) Hematoxylin and eosin (H&E) stain showing histology of control (CTL) and VP-treated ovaries. Scale bar, 50 μm. The insert image shows the histology of ovaries in postnatal d 12 mice. Please note that only early stage growing follicles (<5 layers of GCs) are present in these ovaries. C, D) Representative IHC images showing expression of Ki67 and cleaved Caspase-3 (Clvd-Cas3) in serial sections of cultured CTL and VP-treated ovarian tissues. Scale bars, 50 μm. E) Representative images showing expression of Yap1, Yap1-targeting genes [connective tissue growth factor (Ctgf), Myc, and Areg], and 3β-Hsd in the CTL and VP-treated ovaries determined by RT-PCR and Western blot. F) TUNEL assay showing the apoptosis of granulosa cells and atresia of follicles in CTL and VP-treated ovarian tissues. Scale bar, 500 μm. The percentages of TUNEL-positive follicles in CTL and VP-treated groups are also presented in a graph next to images. ***P < 0.001 compared with CTL. G) Inhibition of Yap1 decreases the number of corpora lutea in the mouse ovary. 3β-Hsd staining was used to identify the corpus luteum (CL). Scale bar, 500 μm. *P < 0.05 compared with CTL.

Article Snippet: The antibody against Ki67 (marker of proliferation Ki-67) was from Abcam (Cambridge, United Kingdom).

Techniques: Inhibition, In Vitro, In Vivo, Incubation, Staining, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, TUNEL Assay

Journal: Cell reports

Article Title: Mitochondrial GTP Links Nutrient Sensing to β Cell Health, Mitochondrial Morphology, and Insulin Secretion Independent of OxPhos

doi: 10.1016/j.celrep.2019.06.058

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mice were genotyped by PCR using genotyping primers (listed in , Oligonucleotides). table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Invitrogen mouse anti-V5 Thermo Fisher Scientific Cat# R960–25; RRID: AB_2556564 Mouse anti-beta actin Abcam Cat# ab8226; RRID: AB_306371 Rabbit anti-GFP Abcam Cat# ab6556; RRID: AB_305564 Goat anti-VDAC (N18) Santa Cruz Biotechnology Cat# sc-8828; RRID: AB_793935 Primary rabbit polyclonal antibodies against rodent SCSα (peptide sequence: DAAKKAVASVAKK) This paper N/A Primary rabbit polyclonal antibodies against rodent SCS-ATPβ (peptide sequence: KEAHVDVKFQLPI) This paper N/A Primary rabbit polyclonal antibodies against rodent SCS-GTPβ (peptide sequence: DAAKKAVASVAKK) This paper N/A Guinea pig anti-insulin Sigma discontinued secondary Alexa Fluor 555 goat anti-guinea pig IgG Sigma Cat # SAB4600297 secondary Alexa Fluor 488 donkey anti-rabbit (H+L) Abcam Cat # ab150073; RRID: AB_2636877 Bacterial and Virus Strains Invitrogen One Shot® OmniMAX 2-T1R Chemically Competent E. coli Thermo Fisher Scientific C854003 Biological Samples TaB-ATP pancreatic islets This paper N/A TaB-GTP pancreatic islets This paper N/A Chemicals, Peptides, and Recombinant Proteins Lipofectamine 2000 (Life Technologies) Thermo Fisher Scientific 11668019 DMEM-Base Cell culture media Sigma Aldrich D5030 RPMI 1640 Cell culture media Sigma Aldrich SLM-240 Quantitect reverse transcriptase QIAGEN 205311 SYBR PCR reagent BioRad 1708880 OptiMEM I Thermo Fisher Scientific 31985–062 complete mini EDTA-free protease inhibitor cocktail (Roche) Sigma Aldrich 11836170001 Exendin-4 Tocris 1933 tolbutamide Fluka Analytical 46968 glucokinase activator Merck 346021 TMRE Molecular Probes T669 MitoTracker Green Molecular Probes M7514 [U- 13 C 6 ]glucose Cambridge Isotope Laboratories 110187–42-3 2 H 4 -taurine CDN Isotopes D-1971 Doxycycline Slow Release Pellets Innovative Research of America B-168 Critical Commercial Assays Micro BCA protein assay kit Thermo Fisher Scientific 23235 Rat High Range ELISA ALPCO 80-INSRTH-E01 Mouse Ultrasensitive ELISA ALPCO 80-INSMSU-E01 RNeasy Mini Kit QIAGEN 74106 DNeasy Blood & Tissue Kit QIAGEN 69504 BrdU Cell Proliferation Assay Chemicon International, Millipore 2750 HIGH range rodent insulin ELISA assay kit (colorimetric) ALPCO Discontinued Invitrogen Quant-iT PicoGreen dsDNA Reagent Thermo Fisher Scientific P7581 Qiaquick Gel Extraction Kit QIAGEN 28704 QIAGEN Plasmid Maxi Kit QIAGEN 12163 Experimental Models: Cell Lines Clonal INS-1 832/13 cell line overexpressing the human insulin gene (INS-1) C.B.

Techniques: Sequencing, Recombinant, Cell Culture, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, BrdU Cell Proliferation Assay, Gel Extraction, Plasmid Preparation, Stable Transfection, Transgenic Assay, Expressing, Over Expression, Software, Imaging, Transmission Assay, Microscopy, Flow Cytometry