Journal: Journal of Cell Science

Article Title: Transcriptional regulation of ferritin and antioxidant genes by HIPK2 under genotoxic stress

doi: 10.1242/jcs.073627

Figure Lengend Snippet: HIPK2 is an ATF1 binding protein. (A) PJ69-4A yeast clones transformed with indicated combinations of bait and prey (pGBDATF1 and pAct2HIPK2) were tested for their growth on a histidine-deficient agar plate. (B) Indicated combinations of pHA-ATF1 and pFlagHIPK2 [wild type (wt) or kinase dead (kd)] were transfected into HEK293 cells, and cell lysates were immunoprecipitated with anti-HIPK2 followed by western blotting with anti-HA antibody (top). The right-end lane was loaded with 10 μg of HEK293 whole-cell lysate transiently transfected with pHA-ATF1. The same set of cell lysates were subjected to western blotting with anti-HA (middle) or anti-HIPK2 (bottom). Of note is that wt HIPK2 (but not kd HIPK2) and HA-ATF1-transfected cell lysate exhibited a slower migrated ATF1 band (middle, lanes 5 and 6).

Article Snippet: Cell culture The HepG2 human hepatocarcinoma cells and HEK293 human embryonic kidney cells (ATCC) were cultured in minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids and 10% fetal bovine serum (FBS; Mediatech).

Techniques: Binding Assay, Clone Assay, Transformation Assay, Transfection, Immunoprecipitation, Western Blot

Journal: Journal of Cell Science

Article Title: Transcriptional regulation of ferritin and antioxidant genes by HIPK2 under genotoxic stress

doi: 10.1242/jcs.073627

Figure Lengend Snippet: HIPK2 phosphorylates ATF1. (A) Forty micrograms of HEK293 cell lysates transfected in duplicate with pCMVHAATF1 along with pCMVFlag (empty vector) or pCMVHIPK2-K221R kd or pCMVHIPK2 wt were independently separated on SDS-PAGE and analyzed by western blotting with anti-HA antibody. The HAATF1 and retarded HAATF1 bands were indicated by an arrowhead and arrow, respectively (top). The expression level of HIPK2 representing each duplicate was analyzed by western blotting with anti-HIPK2 antibody (bottom). (B) Forty micrograms of one of duplicated cell lysates in A were untreated or treated with λ-phosphatase for 37°C for 20 minutes prior to SDS-PAGE and western blotting with anti-HA antibody.

Article Snippet: Cell culture The HepG2 human hepatocarcinoma cells and HEK293 human embryonic kidney cells (ATCC) were cultured in minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids and 10% fetal bovine serum (FBS; Mediatech).

Techniques: Transfection, Plasmid Preparation, SDS Page, Western Blot, Expressing

Journal: Journal of Cell Science

Article Title: Transcriptional regulation of ferritin and antioxidant genes by HIPK2 under genotoxic stress

doi: 10.1242/jcs.073627

Figure Lengend Snippet: HIPK2 phosphorylates ATF1 at Ser198. (A) Wild-type Flag-HIPK2 and various ATF1 mutants (Ser63, Thr99, Ser164, Ser198 to Ala) were co-transfected into HEK293 cells and nuclear fractions were subjected to western blotting with anti-ATF1 antibody, anti-HIPK2 antibody or anti-lamin B antibody. ATF1 and retarded ATF1 are indicated with arrowheads. (B) Ten nanograms of His-tagged recombinant ATF1 wt, Ser198Ala (198A) or Ser63Ala (63A) was incubated with 4 ng recombinant HIPK2 (kinase domain aa 165–564) or PKA in the presence of 5 μCi γ-32P-ATP at 30°C for 20 minutes. Samples were loaded on 10% SDS-PAGE and phosphorylated bands were detected by autoradiograph. Comparable loading of the proteins was verified by gel Silver staining (bottom).

Article Snippet: Cell culture The HepG2 human hepatocarcinoma cells and HEK293 human embryonic kidney cells (ATCC) were cultured in minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids and 10% fetal bovine serum (FBS; Mediatech).

Techniques: Transfection, Western Blot, Recombinant, Incubation, SDS Page, Autoradiography, Silver Staining

Journal: Journal of Cell Science

Article Title: Transcriptional regulation of ferritin and antioxidant genes by HIPK2 under genotoxic stress

doi: 10.1242/jcs.073627

Figure Lengend Snippet: HIPK2 directly phosphorylates a novel ATF1 site, not Ser63. (A) Increasing amounts of recombinant HIPK2 (amino acids 165-564) was mixed with 2 μg of bacterially expressed His-ATF1 or His-ATF1 Ser63Ala mutant and incubated in the presence of γ-32P-ATP at 30°C for 20 minutes. Samples were separated on SDS-PAGE and visualized by autoradiography. PKA was used as a control. Asterisk indicates autophosphorylated HIPK2. Coomassie Blue staining of the same gel is shown (bottom). The stained band below the 76 kDa marker is BSA added to recombinant HIPK2 solution as a carrier protein. The molecular weight of the recombinant HIPK2 (a.a.165–564) is approximately 55 kDa. (B) Wt HIPK2 or kd HIPK2 (K221R) plasmid DNA were co-expressed with wt ATF1 or Ser63Ala-mutant ATF1 in HEK293 cells. Twenty-four hours after transfection, total cell lysates were collected and 40 μg of samples were separated on SDS-PAGE and then analyzed by western blotting using anti-phospho ATF1 Ser63 antibody (top), anti-ATF1 antibody (middle) or anti-HIPK2 antibody (bottom). Cell lysate with co-expression of PKA and ATF1 was used as a positive control of ATF1 Ser63 phosphorylation (lane 8).

Article Snippet: Cell culture The HepG2 human hepatocarcinoma cells and HEK293 human embryonic kidney cells (ATCC) were cultured in minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids and 10% fetal bovine serum (FBS; Mediatech).

Techniques: Recombinant, Mutagenesis, Incubation, SDS Page, Autoradiography, Control, Staining, Marker, Molecular Weight, Plasmid Preparation, Transfection, Western Blot, Expressing, Positive Control, Phospho-proteomics

Journal: Journal of Cell Science

Article Title: Transcriptional regulation of ferritin and antioxidant genes by HIPK2 under genotoxic stress

doi: 10.1242/jcs.073627

Figure Lengend Snippet: HIPK2 activates ATF1 Transcription function. GAL4-DBD-luciferase plasmid was transfected into HEK293 cells together with GAL4 or GAL4-ATF1 (1–228) plus HIPK2 or PKA expression plasmid. The luciferase assay was carried out 48 hours after transfection. GAL4-ATF1 plus empty-vector-transfected sample was set as 1.0. DNA transfection was carried out in duplicate in each experiment and the result of four independent experiments and standard errors are shown.

Article Snippet: Cell culture The HepG2 human hepatocarcinoma cells and HEK293 human embryonic kidney cells (ATCC) were cultured in minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids and 10% fetal bovine serum (FBS; Mediatech).

Techniques: Luciferase, Plasmid Preparation, Transfection, Expressing

Journal: Advanced Science

Article Title: TRAF3IP3 Induces ER Stress‐Mediated Apoptosis with Protective Autophagy to Inhibit Lung Adenocarcinoma Proliferation

doi: 10.1002/advs.202411020

Figure Lengend Snippet: TRAF3IP3 interacts with STRN3 and promotes STRN3 localization of endoplasmic reticulum. A) STRN3 was identified as the binding partner of TRAF3IP3 by two replicate co‐IP mass spectrometry experiments. B) Molecular docking of TRAF3IP3 and STRN3. The purple molecule stands for TRAF3IP3, and the sky‐blue molecule stands for STRN3. C) Exogenous reciprocal coimmunoprecipitation (co‐IP) of TRAF3IP3 and STRN3 in 293T cells overexpressing Flag‐TRAF3IP3 and/or HA‐STRN3. D) Immunofluorescence staining of DAPI (blue), TRAF3IP3 (red) and STRN3 (green) in LUAD cell lines. E) The colocalization curves showed the colocalization level of STRN3 and TRAF3IP3 in the representative image. F) Schematic representation of wild‐type TRAF3IP3 and TRAF3IP3 deletion truncations. G Lysates from HEK293T cells transfected with HA‐tagged STRN3 and Flag‐tagged wild‐type TRAF3IP3 or TRAF3IP3 deletion truncations (1‐235, 236–450, 451–520, 521–551) were collected for coimmunoprecipitation and immunoblotting. H) A549 cell was transfected with indicated expression vectors, and STRN3, GRP78, ATF4, and Bax levels were detected by western blotting. I) Wild‐type TRAF3IP3 or deletion truncations were transfected into A549 cells, and confocal microscopy analysis of GRP78, STRN3 and DAPI was conducted. J) The colocalization curves showed the colocalization level of GRP78 and STRN3 in the representative image. K,L) A549 cell was transfected with control vector, wild‐type TRAF3IP3, specific containing transmembrane domain, and transmembrane domain‐deleted (rTM) plasmids; cell lysates were subjected to immunoblot with indicated antibodies.

Article Snippet: LUAD cell lines (PC9, A549 and H1299), bronchial epithelioid cells (BEAS‐2B), and Human embryonic kidney (HEK) 293T cells were all purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Mass Spectrometry, Immunofluorescence, Staining, Transfection, Western Blot, Expressing, Confocal Microscopy, Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody

doi: 10.1074/jbc.M116.765313

Figure Lengend Snippet: Genes cloned for overexpression in a HEK293T system Single cells identified as astrocytes on the basis of established marker genes ( 19 ) were analyzed to identify transcripts fulfilling the following criteria. First, transcripts had to be present in ≥90% of identified astrocytes. Second, transcripts had to encode proteins containing a predicted transmembrane region. Third, only proteins localizing to the plasma membrane were considered. Only proteins fulfilling these criteria could realistically be used in the immunoisolation of live (non-fixed) astrocytes. This table lists identified transcripts fulfilling these criteria and the corresponding PubMed accession numbers of the transcripts used to design subsequent cloning steps. Essentially, PCR primers were designed to flank ORFs of genes of interest (capital letters) as well as to incorporate unique restriction sites on both ends (lowercase letters). Sequences were then amplified by PCR before ligation into the pCAGIG plasmid. Plasmids were verified by sequencing and checked against the deposited amino acid sequence for the respective proteins. Single-nucleotide polymorphisms that did not affect the amino acid sequence were tolerated. In addition to encoding genes of interest, these constructs also encoded cytosolic GFP to act as a marker for cell transfection.

Article Snippet: Overexpression of ORF-encoding plasmids, ACSA-2 staining, and flow cytometry Heterologous expression of identified genes in a HEK293T system HEK293T (human embryonic kidney) cells (sourced from ATCC) were maintained according to standard protocols ( 49 ) and were mycoplasma-free.

Techniques: Clone Assay, Over Expression, Marker, Clinical Proteomics, Membrane, Cloning, Amplification, Ligation, Plasmid Preparation, Sequencing, Construct, Transfection

Journal: The Journal of Biological Chemistry

Article Title: An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody

doi: 10.1074/jbc.M116.765313

Figure Lengend Snippet: Flow cytometry-based experiments identify ATP1B2 as a target for ACSA-2. HEK293T cells (which do not bind ACSA-2 under normal conditions) were transfected with plasmids encoding for proteins identified by our bioinformatic screen (see Table 3). These plasmids also expressed soluble GFP as a marker for successful plasmid transfection. Cells were then stained with an ACSA-2-APC conjugate and analyzed by flow cytometry. From the list of proteins identified in Table 3, only cells expressing ATP1B2 showed strong co-labeling for ACSA-2 and GFP (28.2% of cells). A representative experiment is presented in the figure. This experiment was repeated twice using independent samples (effectively 1 technical replicate per sample) on separate days with the same results. At least 60,000 cells were analyzed per sample. Lines in each plot delineate gates; numbers represent the proportion of cells in each particular gate.

Article Snippet: Overexpression of ORF-encoding plasmids, ACSA-2 staining, and flow cytometry Heterologous expression of identified genes in a HEK293T system HEK293T (human embryonic kidney) cells (sourced from ATCC) were maintained according to standard protocols ( 49 ) and were mycoplasma-free.

Techniques: Flow Cytometry, Transfection, Marker, Plasmid Preparation, Staining, Expressing, Labeling

Journal: Journal of Virology

Article Title: Griffithsin Protects Mice from Genital Herpes by Preventing Cell-to-Cell Spread

doi: 10.1128/JVI.00012-13

Figure Lengend Snippet: Griffithsin inhibits HSV-2 infection post entry. (A) CaSki, VK2/E6E7, and 3T3 cells were exposed to GRFT or PRO 2000 for 15 min, followed by challenge with HSV-2(4674) for 1 h. The cells were washed and then overlaid with medium, and plaques were counted after 24 to 48 h; results are presented as PFU/well as percentages of control (no drug) and are means + SEM from 2 to 4 experiments conducted in duplicate. (B) Immature moDCs were exposed to GRFT overnight and then challenged with HSV-2(333)ZAG (MOI of 1 PFU/cell) for 1 h at 37°C. Infection was assessed by quantifying GFP expression (10,000 live event acquisition) by flow cytometry 4 hpi. Results are presented as percentage of GFP+ cells and are means + SEM from six (pretreat) independent experiments. To monitor effects of GRFT on the release of viral progeny, the DCs were exposed to GRFT either before or after initial entry, and supernatants were harvested 24 hpi and overlaid onto Vero cells; viral plaques were counted after Giemsa staining and are presented as percentages relative to DCs infected in the absence of GRFT from 2 independent experiments, each conducted in duplicate. (C) CaSki or VK2/E6E7 cells were first exposed to HSV-2 for 1 h, washed with PBS, and then cultured in medium supplemented with the indicated concentration of GRFT; results are presented as percentages of the control and are the means + SEM from 3 experiments performed in duplicate. (D) Representative photograph of viral plaques in VK2/E6E7 cells when GRFT was added postentry. (E) EC50 and EC90 (μg/ml) were calculated for GRFT using data from panels A to C and Fig. 2A. Asterisks indicate P values of <0.05 compared to no drug (t test).

Article Snippet: CaSki (human cervical epithelial), HEC-1-A (human endometrial), VK2/E6E7 (human papillomavirus [HPV] E6/E7 immortalized vaginal epithelial), Vero (monkey kidney), and 3T3 (murine fibroblast) cells were obtained from the American Tissue Culture Collection.

Techniques: Infection, Control, Expressing, Flow Cytometry, Staining, Cell Culture, Concentration Assay

Journal: Cell

Article Title: TMEM41B Is a Pan-flavivirus Host Factor

doi: 10.1016/j.cell.2020.12.005

Figure Lengend Snippet:

Article Snippet: Human: HEK293T/17 (embryonic kidney epithelial) , ATCC , Cat.#CRL-11268 ; RRID: CVCL_1926.

Techniques: Virus, Subcloning, Western Blot, Recombinant, Infection, Transfection, Protease Inhibitor, Staining, Bicinchoninic Acid Protein Assay, Immunoprecipitation, SYBR Green Assay, cDNA Synthesis, Sequencing, Derivative Assay, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: The dengue virus non-structural protein 1 (NS1) is secreted from mosquito cells in association with the intracellular cholesterol transporter chaperone caveolin complex

doi: 10.1101/370932

Figure Lengend Snippet: A Inhibition of classical secretory pathway in YFV infected C6/36 and Vero E6 cells at 72hpi. B Inhibition of classical secretory pathway in ZIKV infected C6/36 and Vero E6 cells at 48hpi. C ZIKV NS1 secretion is reduced in C6/36 cells treated with CsA 9 μM. D Co-localization between YFV NS1 or ZIKV NS1 and CAV-1 in mosquito and VeroE6 cells. C6/36, Aag2 and VeroE6 cells were infected at MOI of 1 for 48 h. Cells were probed for CAV-1 (shown in red), viral NS1 (shown in green) and nuclei stained with DAPI (blue). E Pearson correlation coefficients (PCC) for CAV1-NS1 were measured in at least 20 confocal independent images with 0.47 μM laser sections. The bars represent means ± standard error. Data was evaluated using the 2way ANOVA test and significant differences are denoted by *(P ≤0.05). F Protein sequence alignment of caveolin binding domain (CBD) in Flavivirus NS1. Symbols: Φ, aromatic amino acid (F/Y/W) and X, any amino acid. A , B and C ; Cells were infected with YFV or ZIKV at MOI of 1 for 1 h. The cells were washed 3 times and after treated with DMSO (control), 7μM BFA, 27μM GCA or 9μM CsA. For ZIKV infection supernatants were harvested at 48 hpi and for YFV infection supernatants were collected 72 hpi. Secreted ZIKV NS1 was measured using Platelia NS1 Ag (Bio-Rad) and represented as percentage of secreted NS1 compared to DMSO. Secreted YFV NS1 was evaluated by western blot. The experiments were performed in triplicate, and the bars represent means ± standard error. Data was evaluated using the 2way ANOVA test and significant differences compared with DMSO treatment group, respectively, are denoted by *(P ≤0.05).

Article Snippet: Monkey epithelial kidney cells Vero E6 (ATCC ® CRL-1586TM) were grown at 37°C and cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC ® ) supplemented with 10% FBS and 100 U/ml penicillin-streptomycin.

Techniques: Inhibition, Infection, Staining, Sequencing, Binding Assay, Control, Western Blot

Journal: Journal of nanomedicine & nanotechnology

Article Title: Biocompatible Gold Nanorod Conjugates for Preclinical Biomedical Research

doi: 10.4172/2157-7439.S2-001

Figure Lengend Snippet: Fraction of dead cells for the cell lines (BT 474, MCF 7, MCF 10 and MDCK) after incubation with GNR HER2 conjugates for 48 h, 250 pM (or 1.5 × 1011 GNR/ml). The number of dead cells was counted after staining with Trypan Blue. Control groups of cells received treatment with PBS or GNR after PEGylation (PEG). Synthesis steps for protocols 1, 2 and 3 are the same as in Figure 4 (mean ± SD, n=6 independent measures for each conjugate). Results indicate significantly higher level of cell death for cells with HER2/neu expression, BT 474 and MCF7, and no significant changes without HER2/neu expression: MCF 10 and MDCK.

Article Snippet: Cell culture, viability and cell proliferation Cell lines BT 474 (Human breast distal carcinoma with HER2 receptor overexpression ), MCF 7 (Human breast adenocarcinoma with HER2 receptor overexpression ), MCF 10A (Human breast fibrocystic disease, no HER2 receptor overexpression ), MDCK (normal dog kidney epithelial cells), and Human lung normal fibroblast were obtained from American Type Culture Collection (ATCC) and cultured in essential media with 10% fetal bovine serum.

Techniques: Incubation, Staining, Control, Expressing

Journal: Scientific Reports

Article Title: Protective effects of miR-29a on diabetic glomerular dysfunction by modulation of DKK1/Wnt/β-catenin signaling

doi: 10.1038/srep30575

Figure Lengend Snippet: ( a ) Effects of high glucose, miR-29a precursor and miR-29a inhibitor on cell apoptosis. Renal mesangial cells were treated with high glucose, miR-29a precursor, miR-29a inhibitor, or a combination of high glucose and miR-29a precursor. TUNEL staining was used to detect apoptotic cells (red). Experiments were performed at least three times and representative results are shown. NC, normal glucose control; HG, high glucose. ( b – d ) Changes in the levels of TGF-β1, fibronectin and DKK1 mRNAs in mesangial cells under the above-mentioned conditions. All the experiments were repeated at least three times. Data are indicated as mean ± SEM. Symbol * represents significant difference vs. the normal glucose group, and symbol # represents significant difference vs. the high glucose group (P < 0.05). ( e ) Western blot analysis of GSK-3β phosphorylation and nuclear β-catenin in mesangial cells. Representative blots from three experiments are shown.

Article Snippet: Mouse SV40 MES-13 glomerular mesangial cells were obtained from American Type Culture Collection, Manassas, VA.

Techniques: TUNEL Assay, Staining, Control, Western Blot, Phospho-proteomics

Journal: Scientific Reports

Article Title: Protective effects of miR-29a on diabetic glomerular dysfunction by modulation of DKK1/Wnt/β-catenin signaling

doi: 10.1038/srep30575

Figure Lengend Snippet: ( a ) Western blot analysis of GSK-3β phosphorylation and nuclear β-catenin in mesangial cells that were treated with high glucose (HG) or transfected with β-catenin (Δ45) mutant. Experiments were repeated three times and representative blots are shown. ( b , c ) Stabilization of β-catenin by transfecting the β-catenin (Δ45) mutant significantly enhanced miR-29a, but reduced DKK1, in mesangial cells in either normal or high glucose conditions. ( d ) Treatment of mesangial cells with a GSK-3β inhibitor, BIO or LiCL, increased miR-29a expression. All quantitative RT-PCR experiments shown above were independently repeated at least three times. Symbol * indicates significant difference vs. the normal glucose group, and symbol # indicates significant difference vs. the high glucose group (P < 0.05). ( e ) Proposed model for the reciprocal regulation of miR-29a and Wnt/β-catenin signaling.

Article Snippet: Mouse SV40 MES-13 glomerular mesangial cells were obtained from American Type Culture Collection, Manassas, VA.

Techniques: Western Blot, Phospho-proteomics, Transfection, Mutagenesis, Expressing, Quantitative RT-PCR

Journal:

Article Title: Biochemical Characterization of Human Epidermal Retinol Dehydrogenase 2

doi: 10.1016/j.cbi.2008.09.019

Figure Lengend Snippet: HEK293 cells were transfected with either empty vector (A) or RDH-E2 expression vector (B) and incubated with anti-FLAG antibodies. RDH-E2 expression was visualized using FITC-conjugated antibodies. Endoplasmic reticulum of HEK293 cells was visualized by staining with FITC-conjugated concanavalin A (C).

Article Snippet: Human embryonic kidney 293 (HEK293) cells (American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium containing 10% horse serum and penicillin/streptomycin at 37 °C with 5% CO 2 .

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Staining

Journal:

Article Title: Biochemical Characterization of Human Epidermal Retinol Dehydrogenase 2

doi: 10.1016/j.cbi.2008.09.019

Figure Lengend Snippet: HEK293 cells transfected with expression vector for RDH-E2 (+) or empty vector (−) were incubated with tritiated androsterone (ADT), dihydrotestosterone (DHT), allopregnanolone (ALLO), dehydroepiandrosterone (DHEA); progesterone (PROG), or corticosterone (CORT).

Article Snippet: Human embryonic kidney 293 (HEK293) cells (American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium containing 10% horse serum and penicillin/streptomycin at 37 °C with 5% CO 2 .

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation

Journal: Molecular cell

Article Title: TEX264 is an ER-resident ATG8-interacting protein critical for endoplasmic reticulum remodeling during nutrient stress

doi: 10.1016/j.molcel.2019.03.034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell lines HEK293 (human embryonic kidney, fetus, ATCC CRL-1573, RRID: CVCL_0045), HCT116 (human colorectal carcinoma, male, ATCC CCL-247, RRID: CVCL_0291), HEK293T (human embryonic kidney, fetus, ATCC CCL-3216, RRID: CVCL_0063), and COS7 ( Cercopithecus aethiops kidney, ATCC CRL-1651, RRID: CVCL_0224) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), high glucose, pyruvate and supplemented with 10% fetal calf serum and maintained in a 5% CO 2 incubator at 37°C.

Techniques: Recombinant, Mutagenesis, Staining, Protease Inhibitor, Peptide Fractionation, Sequencing, Software, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions

doi: 10.3389/fimmu.2024.1303089

Figure Lengend Snippet: Differential mRNA expression levels for rabbit GBPs. (A) Heat map of RT-qPCR mRNA expression analysis of ocGBPs and ocFurin in several tissues, primary cells, cell lines and overexpression in RK13 cells: ΔCt values to the reference gene ActinB are displayed (CtGBP – CtActB). Tissues of four female New Zealand white rabbits and three primary cells, cell lines and overexpression were analyzed. Scale: from red (low ΔCt value, i.e., higher expression of target gene) to blue (higher ΔCt, i.e., lower expression of target gene). (B) Rabbit transcriptome was retrieved from and blasted for GBP mRNA expression using the BLAST tool from NCBI. Gray color means present, white means absent.

Article Snippet: SIRC (Cornea, ATCC CCL-60), RAB-9 (Skin, ATCC CRL-1414), RK13 (Kidney, ATCC CCL-37) and RL-33 (Lung, tebu-bio JCRB0131) cell lines were cultured in monolayers in MEM GlutaMAXTM (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma Aldrich) and 1% Penicillin/Streptomycin (P/S, Sigma Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Over Expression

Journal: Frontiers in Immunology

Article Title: Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions

doi: 10.3389/fimmu.2024.1303089

Figure Lengend Snippet: Protein expression of overexpressed rabbit GBPs in a rabbit cell line. (A) RK13 cells were transfected with ocGBP expression plasmids. Two days post-transfection, cells were permeabilized and protein expression was determined via flow cytometry. Shown are the mean fluorescence intensities (MFI ± SD) of HA-positive cells stained with PB-coupled antibodies (n = 3). (B) RK13 cells were transfected with rabbit GBP expression plasmids. Two days post-transfection, protein expression was determined using Western blot. Membranes were probed for HA tag (GBP) and Vinculin (housekeeping protein). Shown is a representative Western blot. PB, pacific blue.

Article Snippet: SIRC (Cornea, ATCC CCL-60), RAB-9 (Skin, ATCC CRL-1414), RK13 (Kidney, ATCC CCL-37) and RL-33 (Lung, tebu-bio JCRB0131) cell lines were cultured in monolayers in MEM GlutaMAXTM (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma Aldrich) and 1% Penicillin/Streptomycin (P/S, Sigma Aldrich).

Techniques: Expressing, Transfection, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: Frontiers in Immunology

Article Title: Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions

doi: 10.3389/fimmu.2024.1303089

Figure Lengend Snippet: Intracellular localization of ocGBPs in RK13. RK13 cells were transfected with GBP expression plasmids. Two days post-transfection, localization was determined via immunofluorescence microscopy. The following colors were used: pink (phalloidin, actin filaments), yellow (TGN46, trans-Golgi network), indigo (Hoechst, Nucleus), green (HA-tag, GBPs). Shown are representative images out of 5 -10 imaged positions. 100x magnification, scale bars indicate 10 µm.

Article Snippet: SIRC (Cornea, ATCC CCL-60), RAB-9 (Skin, ATCC CRL-1414), RK13 (Kidney, ATCC CCL-37) and RL-33 (Lung, tebu-bio JCRB0131) cell lines were cultured in monolayers in MEM GlutaMAXTM (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma Aldrich) and 1% Penicillin/Streptomycin (P/S, Sigma Aldrich).

Techniques: Transfection, Expressing, Immunofluorescence, Microscopy

Journal: Molecular microbiology

Article Title: P fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses.

doi: 10.1046/j.1365-2958.1999.01513.x

Figure Lengend Snippet: Fig. 1. P ®mbriae augment epithelial cytokine response of A498 cells to E. coli (108 cfu mlÿ1). Supernatants were collected at 0, 2, 6 and 24 h. Concentrations of IL-6 (A) and IL-8 (B) were determined by ELISA. Numbers are means 6 s.e.m. of ®ve experiments.

Article Snippet: The human epithelial cell line A498 (ATCC HTB44, human kidney carcinoma) has previously been used as a model system in studies of bacterially induced epithelial cell responses to uropathogenic E. coli.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Molecular microbiology

Article Title: P fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses.

doi: 10.1046/j.1365-2958.1999.01513.x

Figure Lengend Snippet: Fig. 2. Top. msbB and pap genotypes of the E. coli strains used in the present study as shown by colony hybridization. The msbB gene was detected using the 1630 bp msbB DNA fragment as a probe. The pap sequences were detected by hybridization with the 5 kb HindIII fragment of the pap gene cluster from E. coli J96. Bottom. The E. coli strains JM83 (msbB) and M607 (msbB±) were transformed with pPIL110- 75. Con¯uent human kidney A498 cells were exposed to the ®mbriated strains as well as the non-®mbriated isogenic strains (108 cfu mlÿ1). IL-6 (A) and IL-8 (B) concentrations were measured by ELISA in supernatants after 0, 2, 6 and 24 h of stimulation. Numbers are means 6 s.e.m. of seven experiments. Transduction of P-®mbriated E. coli with the non-functional msbB gene from E. coli M607 does not alter epithelial cytokine responses to this strain. strr/spcr E. coli AD110 was used for cell activation experiments. Con¯uent layers of A498 cells were stimulated with E. coli AD110 (msbB) and E. coli AD110 (msbB±) (108 cfu mlÿ1). Samples collected at 0, 2, 6 and 24 h was used for IL-6 (C) and IL-8 (D) determinations. Numbers are means 6 s.e.m. of seven experiments.

Article Snippet: The human epithelial cell line A498 (ATCC HTB44, human kidney carcinoma) has previously been used as a model system in studies of bacterially induced epithelial cell responses to uropathogenic E. coli.

Techniques: Hybridization, Transformation Assay, Enzyme-linked Immunosorbent Assay, Transduction, Functional Assay, Activation Assay

Journal: Molecular microbiology

Article Title: P fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses.

doi: 10.1046/j.1365-2958.1999.01513.x

Figure Lengend Snippet: Fig. 3. The LPS inhibitor BPI does not reduce the cytokine response of A498 cells to E. coli. Con¯uent layers of A498 cells were exposed to E. coli HB101(pPIL110-75) (108 cfu mlÿ1) in the presence or absence of BPI (10 nM, 100 nM, 1 mM). Samples were collected after 0, 0.5, 2, 4, 6 and 24 h. IL-6 (A) and IL-8 (B) concentrations in the 6 h sample were determined by ELISA. Data represent means 6 s.e.m. of four experiments and triplicate samples.

Article Snippet: The human epithelial cell line A498 (ATCC HTB44, human kidney carcinoma) has previously been used as a model system in studies of bacterially induced epithelial cell responses to uropathogenic E. coli.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Molecular microbiology

Article Title: P fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses.

doi: 10.1046/j.1365-2958.1999.01513.x

Figure Lengend Snippet: Fig. 4. The anti-LPS drug Polymyxin B failed to reduce cytokine responses to whole bacteria. Con¯uent layers of A498 cells were exposed to E. coli HB101(pPIL110-75) (108 cfu mlÿ1) with or without Polymyxin B (50 ng mlÿ1, 500 ng mlÿ1, 5 mg mlÿ1, 50 mg mlÿ1). Samples were collected after 0, 0.5, 2, 4, 6 and 24 h. IL-6 (A) and IL-8 (B) concentrations in the 6 h samples are shown. The phorbol ester PMA was used as a non-bacterial stimulant to control non-speci®c effects of Polymyxin B. Data are means 6 s.e.m. of seven experiments and triplicate samples.

Article Snippet: The human epithelial cell line A498 (ATCC HTB44, human kidney carcinoma) has previously been used as a model system in studies of bacterially induced epithelial cell responses to uropathogenic E. coli.

Techniques: Bacteria, Control

Journal: Molecular microbiology

Article Title: P fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses.

doi: 10.1046/j.1365-2958.1999.01513.x

Figure Lengend Snippet: Fig. 5. Cytokine response of A498 cells to smooth Salmonella LPS. Supernatants were collected at 0, 2, 6 and 24 h, and IL-6 (A) and IL-8 (B) concentrations were determined by ELISA. Results are means 6 s.e.m. of four experiments and triplicate samples.

Article Snippet: The human epithelial cell line A498 (ATCC HTB44, human kidney carcinoma) has previously been used as a model system in studies of bacterially induced epithelial cell responses to uropathogenic E. coli.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Molecular microbiology

Article Title: P fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses.

doi: 10.1046/j.1365-2958.1999.01513.x

Figure Lengend Snippet: Fig. 6. Surface CD14 expression as shown by ¯ow cytometry after staining with anti-CD14 antibody. Vitamin D3-treated U937 cells stained with anti-CD14 antibody but did not react with the control antibody. A498 cells failed to express surface CD14.

Article Snippet: The human epithelial cell line A498 (ATCC HTB44, human kidney carcinoma) has previously been used as a model system in studies of bacterially induced epithelial cell responses to uropathogenic E. coli.

Techniques: Expressing, Cytometry, Staining, Control

Journal: Molecular microbiology

Article Title: P fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses.

doi: 10.1046/j.1365-2958.1999.01513.x

Figure Lengend Snippet: Fig. 7. RT-PCR of CD14 mRNA in A498 and U937 cells. A. CD14 mRNA was detected in U937 cells stimulated with vitamin D3 for 48 h but not in A498 cells before or after 24 h of stimulation with LPS or E. coli AD110. B. RT-PCR of actin mRNA in the A498 and the U937 cells. Actin mRNA was detected in all cells.

Article Snippet: The human epithelial cell line A498 (ATCC HTB44, human kidney carcinoma) has previously been used as a model system in studies of bacterially induced epithelial cell responses to uropathogenic E. coli.

Techniques: Reverse Transcription Polymerase Chain Reaction