similarity analysis pcr-positive Search Results


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  • 99
    Thermo Fisher pcr reaction buffer
    Revealed results of <t>PCR</t> in 2% agarose gel stained with ethidium bromide under UV. Above: Samples from sixteen tsetse flies submitted to PCR using GmTub primers as DNA quality control. Expected band size ∼380 bp. Middle: Molecular diagnosis of samples from fifteen tsetse flies for T. b. <t>gambiense</t> . First sample corresponds to the one positive tsetse fly. The band at right is the positive control. Expected band size 270 bp. Below: Detection of T. brucei s.l. in animal samples. 5–8 and 11 are positives, 12 is the positive control. Expected band size 177 bp.
    Pcr Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 733 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcr reaction buffer - by Bioz Stars, 2020-04
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    99
    Millipore anti beta actin antibody
    HCMV induces secretion of IL-6 by HepG2 cells and PHH. (A) ELISA quantification of IL-6 levels in the culture supernatants of HepG2 cells and PHH left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). Where specified, cells were treated with ganciclovir (5 microg/ml). Results are representative of two independent experiments. (B) Decreased IL-6 production in culture supernatants of HepG2 cells treated with UV-inactivated HCMV in comparison cells infected with live HCMV . HCMV strain AD169 was used at a MOI = 1 and IL-6 production measured at day 3 post-infection was expressed arbitrarily as 100% in cell cultures infected with live virus. Results represent means (± SD) of two independent experiments. (C) Decreased IE1 transcript expression in HepG2 cells treated with UV-inactivated HCMV in comparison with live HCMV . HCMV strain AD169 was used at a MOI = 1 and IE1 and US28 transcripts were amplified by RT-PCR. Difference in DeltaCt values of two independent experiments is shown. Results represent means (± SD) of two independent experiments. (D) Decreased HCMV replication and IE1 protein expression in MRC-5 cells infected with UV-inactivated HCMV in comparison with cells infected with live HCMV . HCMV strain AD169 was used at a MOI = 1. Viral titers were determined in the culture supernatants at the indicated times post-infection by real-time PCR. IE1 pp72 antigen expression was measured up to day 3 postinfection by Western blotting, as described in the Materials and Methods section. <t>beta-actin</t> was used as control. Results are representative of two independent experiments.
    Anti Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti beta actin antibody/product/Millipore
    Average 99 stars, based on 885 article reviews
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    anti beta actin antibody - by Bioz Stars, 2020-04
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    85
    SLIT2 LTD e12 5
    Reduced number of GnRH neurons in the forebrain of Slit2 –/– mice. ( A ) Schematic of the mouse head area displayed in B,C. ( B , C ) Sagittal sections of <t>E12.5</t> mouse heads were immunolabelled for GnRH to reveal neurons migrating in the NC and FB. The FB boundary is delineated by a dotted line. Arrows (B,C) indicate areas displayed at higher magnification in insets. Arrow in C also points to abnormal accumulation of cells in the NC of Slit2 –/– mice as compared with wild type (Δ in B). ( D ) Counts of GnRH neurons in the FB of E12.5 wild-type and Slit2 –/– mice revealed a significant reduction in the mutant. ( E-G ′) Schematic (E) and sagittal sections (F,G) of E14.5 mouse heads immunolabelled for GnRH to reveal neurons migrating in the NC and FB. Arrows indicate areas displayed at high magnification in F′,G′. ( H-J ) Schematic (H) and representative images of sagittal sections (I,J) showing migrating GnRH neurons in the basal FB of E14.5 wild-type and Slit2 –/– mice. There is a reduction in cells in the FB of the mutant (Δ in J). Arrowheads indicate examples of migrating GnRH neurons. ( K ) Counts of GnRH neurons in the FB of E14.5 wild-type and Slit2 –/– mice revealed a statistically significant reduction in the mutant. ( L-N , P-R ) Schematic drawings (L,P) and coronal sections (M,N,Q,R) of E18.5 mouse brains immunolabelled for GnRH to reveal neurons projecting to the MPOA (M,N) and extending their axons to the ME (Q,R). Arrowheads indicate examples of migrating GnRH neurons (M,N) or GnRH + fibres projecting to the ME (Q,R). The paucity of such projections in the Slit2 –/– mice is indicated by Δ. ( O ) Counts of GnRH neurons in the brain of E18.5 wild-type and Slit2 –/– mice revealed a statistically significant reduction in the mutant. ( S ) Optical density measurement of GnRH neuron fibres projecting to the ME of E18.5 wild-type and Slit2 –/– mice revealed a statistically significant reduction in the mutant as compared with wild-type littermates. Mean ± s.e.m. * P
    E12 5, supplied by SLIT2 LTD, used in various techniques. Bioz Stars score: 85/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    SABiosciences cdna array
    SCF FBXW7 interacts and targets ZEB2 for degradation in a GSK-3β phosphorylation-dependent manner. a Left, 2DE and MALDI-MS-based identification of novel Fbxw7-associated proteins using crypts (upper panel) isolated from 3-week <t>fbxw</t> 7 fl/fl and fbxw 7 ΔG mice. Yellow circles in the lower panel denote potential Fbxw7-associated proteins. a Right, WB analysis (upper panels), and RT-PCR analysis (lower panels) of fbxw 7 fl/fl vs. fbxw 7 ΔG derived crypts and intestinal proteins and mRNA expression for ZEB2 and β-actin control. Experiments were performed on at least three independent occasions. b Left, schematic representation of the modified yeast two-hybrid reverse Ras Recruitment Screening (rRRS) system identifying proteins interacting with Fbxw7 in a GSK-3β phosphorylation-dependent manner. GSK-3β under the control of the methionine-regulated MET 3 promoter induces phosphorylation of encoded myristoylated proteins through a <t>cDNA</t> library plus positive control expressing FLAG-β-catenin (B—Middle) which only rescued the growth of cdc25–2 mutant yeast by Fbxw7-associated protein(s), if they interact with RasV12-FBXW7ΔF (i.e. human FBXW7α isoform mutant lacking F-box domain; therefore, interaction with Skp1 is lost and degradation of SCF Fbxw7 substrates will not occur in yeast) used as a bait at the restrictive temperature 37 °C, in a methionine-dependent manner. In the FBXW7ΔF mutant, both the N-terminal F-box and Dim-domains are deleted to avoid any interactions with SKP1 and other FBXW7 isoform-associated proteins. Thus, cdc25–2 mutant yeasts can grow only at 37 °C, when a phosphorylation-dependent interaction between a protein target and RasV12-FBXW7ΔF takes place. The FBXW7ΔF(bait)-dependent growth of these clones was further analysed on galactose-containing medium at 37 °C (B—Right). Red circles show the GSK-3β-phosphorylation-dependent interactor, including the Zeb2-clone, green circles show the phosphorylation/non-phosphorylation-dependent interactor and blue circles show the revertant clones (B—Right). c Left, subcellular localisation of GFP-fused human ZEB2 in the absence (top; nuclear) and presence (bottom; nuclear spots indicative of protein degradation) of GSK-3β in HCT116 CRC cells. ( c— Middle and c— Right) WB analysis of total ZEB2 protein level following the inhibition of GSK-3β (e.g. WS119 or LiCl treatment, and siRNA against GSK-3β) and of UPS pathways (MG132) in SW620 CRC cells. d Direct binding and ubiquitin-dependent degradation of ZEB2 by FBXW7. Co-immunoprecipitation (IP) of ZEB2 upon pull-down of FBXW7 in HEK-293T cells (Left); co-IP of FBXW7 upon pull-down of ZEB2 using the TNT-coupled reticulocyte lysate (Middle), and ubiquitination assays with HA-tagged ubiquitin- (HA-Ub) expressing construct in HEK-293T cells (Right). The asterisk indicates a nonspecific band(s). e Co-IP of endogenous ZEB2 upon pull-down of FBXW7 in HCT116 cells with FBXW 7 deletion. f ZEB2 pulse-chase stability assays with 15 µg/ml cycloheximide (CHX) in HCT116 cells with or without FBXW 7 deletion
    Cdna Array, supplied by SABiosciences, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Revealed results of PCR in 2% agarose gel stained with ethidium bromide under UV. Above: Samples from sixteen tsetse flies submitted to PCR using GmTub primers as DNA quality control. Expected band size ∼380 bp. Middle: Molecular diagnosis of samples from fifteen tsetse flies for T. b. gambiense . First sample corresponds to the one positive tsetse fly. The band at right is the positive control. Expected band size 270 bp. Below: Detection of T. brucei s.l. in animal samples. 5–8 and 11 are positives, 12 is the positive control. Expected band size 177 bp.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Screening of Trypanosoma brucei gambiense in Domestic Livestock and Tsetse Flies from an Insular Endemic Focus (Luba, Equatorial Guinea)

    doi: 10.1371/journal.pntd.0000704

    Figure Lengend Snippet: Revealed results of PCR in 2% agarose gel stained with ethidium bromide under UV. Above: Samples from sixteen tsetse flies submitted to PCR using GmTub primers as DNA quality control. Expected band size ∼380 bp. Middle: Molecular diagnosis of samples from fifteen tsetse flies for T. b. gambiense . First sample corresponds to the one positive tsetse fly. The band at right is the positive control. Expected band size 270 bp. Below: Detection of T. brucei s.l. in animal samples. 5–8 and 11 are positives, 12 is the positive control. Expected band size 177 bp.

    Article Snippet: DNA samples that displayed a positive amplification signal for the tsetse tubulin gene were further tested to detect T. brucei s.l. and T. b. gambiense with the same primers and similar conditions as above: 1× PCR reaction buffer (10 mM Tris-HCl, 50 mM KCl, pH 8.3), 2 mM MgCl2 , primers at 0.5 µM, 200 µM of each dNTP, 1 µl of DNA template and 1 U of AmpliTaq® Gold DNA Polymerase (Applied Biosystems, Branchburg, New Jersey, USA) reaching a final volume of 25 µl.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Positive Control

    HCMV induces secretion of IL-6 by HepG2 cells and PHH. (A) ELISA quantification of IL-6 levels in the culture supernatants of HepG2 cells and PHH left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). Where specified, cells were treated with ganciclovir (5 microg/ml). Results are representative of two independent experiments. (B) Decreased IL-6 production in culture supernatants of HepG2 cells treated with UV-inactivated HCMV in comparison cells infected with live HCMV . HCMV strain AD169 was used at a MOI = 1 and IL-6 production measured at day 3 post-infection was expressed arbitrarily as 100% in cell cultures infected with live virus. Results represent means (± SD) of two independent experiments. (C) Decreased IE1 transcript expression in HepG2 cells treated with UV-inactivated HCMV in comparison with live HCMV . HCMV strain AD169 was used at a MOI = 1 and IE1 and US28 transcripts were amplified by RT-PCR. Difference in DeltaCt values of two independent experiments is shown. Results represent means (± SD) of two independent experiments. (D) Decreased HCMV replication and IE1 protein expression in MRC-5 cells infected with UV-inactivated HCMV in comparison with cells infected with live HCMV . HCMV strain AD169 was used at a MOI = 1. Viral titers were determined in the culture supernatants at the indicated times post-infection by real-time PCR. IE1 pp72 antigen expression was measured up to day 3 postinfection by Western blotting, as described in the Materials and Methods section. beta-actin was used as control. Results are representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes

    doi: 10.1371/journal.pone.0059591

    Figure Lengend Snippet: HCMV induces secretion of IL-6 by HepG2 cells and PHH. (A) ELISA quantification of IL-6 levels in the culture supernatants of HepG2 cells and PHH left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). Where specified, cells were treated with ganciclovir (5 microg/ml). Results are representative of two independent experiments. (B) Decreased IL-6 production in culture supernatants of HepG2 cells treated with UV-inactivated HCMV in comparison cells infected with live HCMV . HCMV strain AD169 was used at a MOI = 1 and IL-6 production measured at day 3 post-infection was expressed arbitrarily as 100% in cell cultures infected with live virus. Results represent means (± SD) of two independent experiments. (C) Decreased IE1 transcript expression in HepG2 cells treated with UV-inactivated HCMV in comparison with live HCMV . HCMV strain AD169 was used at a MOI = 1 and IE1 and US28 transcripts were amplified by RT-PCR. Difference in DeltaCt values of two independent experiments is shown. Results represent means (± SD) of two independent experiments. (D) Decreased HCMV replication and IE1 protein expression in MRC-5 cells infected with UV-inactivated HCMV in comparison with cells infected with live HCMV . HCMV strain AD169 was used at a MOI = 1. Viral titers were determined in the culture supernatants at the indicated times post-infection by real-time PCR. IE1 pp72 antigen expression was measured up to day 3 postinfection by Western blotting, as described in the Materials and Methods section. beta-actin was used as control. Results are representative of two independent experiments.

    Article Snippet: Anti-beta-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Enzyme-linked Immunosorbent Assay, Infection, Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

    Up-regulation of cyclin D1 and survivin in HepG2 cells and PHH infected with HCMV. (A) Time course of the expression of cyclin-D1 and survivin in HepG2 cells infected with HCMV. HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV strains AD169 (MOI = 0.5) and HCMV-DB (MOI = 1.0). Cyclin D1 and survivin expression was measured by Western blotting as described in the Materials and Methods , and beta-actin was used as an internal control. (B) Expression of cyclin-D1 and survivin in PHH and HepG2 cells infected with live HCMV or UV-inactivated HCMV . HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV or UV-inactivated HCMV (AD169, MOI = 0.5). Cyclin D1 and survivin expression was measured by Western blotting as described in the Materials and Methods , and beta-actin was used as an internal control. (C) Expression of cyclin D1 and survivin is mediated primarily by HCMV in HepG2 cells and PHH. The histogram shows survivin and cyclin D1 expression at day 3 post-infection as quantified using Image J 1.40 software. Results of western-blots are representative of two independent experiments; histogram represents means (± SD) of two independent experiments.

    Journal: PLoS ONE

    Article Title: HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes

    doi: 10.1371/journal.pone.0059591

    Figure Lengend Snippet: Up-regulation of cyclin D1 and survivin in HepG2 cells and PHH infected with HCMV. (A) Time course of the expression of cyclin-D1 and survivin in HepG2 cells infected with HCMV. HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV strains AD169 (MOI = 0.5) and HCMV-DB (MOI = 1.0). Cyclin D1 and survivin expression was measured by Western blotting as described in the Materials and Methods , and beta-actin was used as an internal control. (B) Expression of cyclin-D1 and survivin in PHH and HepG2 cells infected with live HCMV or UV-inactivated HCMV . HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV or UV-inactivated HCMV (AD169, MOI = 0.5). Cyclin D1 and survivin expression was measured by Western blotting as described in the Materials and Methods , and beta-actin was used as an internal control. (C) Expression of cyclin D1 and survivin is mediated primarily by HCMV in HepG2 cells and PHH. The histogram shows survivin and cyclin D1 expression at day 3 post-infection as quantified using Image J 1.40 software. Results of western-blots are representative of two independent experiments; histogram represents means (± SD) of two independent experiments.

    Article Snippet: Anti-beta-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Infection, Expressing, Western Blot, Software

    Growth curves of HCMV in HepG2 cells and PHH. (A) Growth curves of HCMV in HepG2 cells, PHH and MRC5 cells . HepG2 cells, PHH, and MRC5 cells were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). Inocula were left in place for 2 hours and then removed with three washes of EMEM without serum. Viral titers were determined in the culture supernatants at the indicated times post-infection by real-time PCR. Results are representative of two independent experiments. (B) Viral entry into HepG2 cells, PHH and MRC5 cells . Uninfected cells and cultures infected with HCMV-AD169 at the indicated MOI for 2 hours were treated with trypsin for 10 min and then washed. Samples of extracted DNA were analyzed by PCR using primers specific for the MIEP of HCMV and for beta-globin (internal loading control). The amplification products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Results are representative of two independent experiments. (C) Detection of IE1 pp72 HCMV antigen in infected HepG2 cells and PHH . HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV-AD169 (MOI = 1 and 10). IE1 pp72 HCMV antigen expression was measured at day 3 post-infection by Western blotting as described in the Materials and Methods section. beta-actin was used as control. Results are representative of two independent experiments. (D) Detection of IE1 pp72, but not US28, pp65 antigen and 65-kD structural late antigen in infected HepG2 cells . HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV-AD169 (MOI = 1). IE1 pp72, US28, pp65 and 65-kD structural late HCMV antigen expression was measured up to day 4 post-infection by Western blotting as described in the Materials and Methods section. beta-actin was used as control. Results are representative of two independent experiments. (E) Detection of IE1 pp72 transcript, but not of US28 transcript, in HCMV-infected HepG2 cells . HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV-AD169 (MOI = 1). IE1 pp72 and US28 transcript expression was measured up to day 6 post-infection by RT-PCR assay as described in the Materials and Methods section. beta-globin was used as control. Results represent means (± SD) of two independent experiments. IE: Immediate Early; MIEP: Major immediate-early promoter; MOI: Multiplicity of infection; PHH: Primary human hepatocytes; U: Uninfected; P: positive control (extract of MRC5 cells infected with HCMV-AD169).

    Journal: PLoS ONE

    Article Title: HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes

    doi: 10.1371/journal.pone.0059591

    Figure Lengend Snippet: Growth curves of HCMV in HepG2 cells and PHH. (A) Growth curves of HCMV in HepG2 cells, PHH and MRC5 cells . HepG2 cells, PHH, and MRC5 cells were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). Inocula were left in place for 2 hours and then removed with three washes of EMEM without serum. Viral titers were determined in the culture supernatants at the indicated times post-infection by real-time PCR. Results are representative of two independent experiments. (B) Viral entry into HepG2 cells, PHH and MRC5 cells . Uninfected cells and cultures infected with HCMV-AD169 at the indicated MOI for 2 hours were treated with trypsin for 10 min and then washed. Samples of extracted DNA were analyzed by PCR using primers specific for the MIEP of HCMV and for beta-globin (internal loading control). The amplification products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Results are representative of two independent experiments. (C) Detection of IE1 pp72 HCMV antigen in infected HepG2 cells and PHH . HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV-AD169 (MOI = 1 and 10). IE1 pp72 HCMV antigen expression was measured at day 3 post-infection by Western blotting as described in the Materials and Methods section. beta-actin was used as control. Results are representative of two independent experiments. (D) Detection of IE1 pp72, but not US28, pp65 antigen and 65-kD structural late antigen in infected HepG2 cells . HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV-AD169 (MOI = 1). IE1 pp72, US28, pp65 and 65-kD structural late HCMV antigen expression was measured up to day 4 post-infection by Western blotting as described in the Materials and Methods section. beta-actin was used as control. Results are representative of two independent experiments. (E) Detection of IE1 pp72 transcript, but not of US28 transcript, in HCMV-infected HepG2 cells . HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV-AD169 (MOI = 1). IE1 pp72 and US28 transcript expression was measured up to day 6 post-infection by RT-PCR assay as described in the Materials and Methods section. beta-globin was used as control. Results represent means (± SD) of two independent experiments. IE: Immediate Early; MIEP: Major immediate-early promoter; MOI: Multiplicity of infection; PHH: Primary human hepatocytes; U: Uninfected; P: positive control (extract of MRC5 cells infected with HCMV-AD169).

    Article Snippet: Anti-beta-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Infection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control

    HCMV induces IL-6-mediated activation of the JAK-STAT3 axis in HepG2 cells and PHH. (A) Time course of STAT3 activation in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 0.5). PHH (2×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). STAT3 activation was measured by Western blotting as described in the Materials and Methods section. Unphosphorylated STAT3 and beta-actin were used as controls, and ganciclovir was used at a concentration of 5 microg/ml. (B) Time course of JAK1/JAK2 activation in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 0.5). PHH (2×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). JAK1/JAK2 activation was measured by Western blotting, and beta-actin was used as an internal control. The histogram shows JAK activation at 2 hours post-infection as quantified using Image J 1.40 software. (C) STAT3 activation is mediated by the IL-6-JAK pathway in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV (MOI = 0.5) in the presence or absence of a JAK inhibitor (1 micromol/l), a STAT3 inhibitor (10 micromol/l), a neutralizing anti-IL-6R mAb (10 microg/ml), and a neutralizing anti-EGFR mAb (20 microg/ml). Cells were left uninfected or incubated with the recombinant HCMV glycoprotein gB (10 microg/ml) for 2 hours. STAT3 activation was measured by Western blotting at day 1 post-infection in PHH incubated with JAK inhibitor, STAT3 inhibitor, anti-IL-6R mAb, and in HepG2 cells incubated with JAK and STAT3 inhibitors. STAT3 activation was measured at 2 hours post-infection in HepG2 cells incubated with anti-IL-6R mAb and anti-EGFR mAb. beta-actin was used as an internal control. The histogram shows STAT3 activation as quantified using Image J 1.40 software. (D) STAT3 activation is mediated primarily by HCMV in HepG2 cells and PHH . HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV or UV-inactivated HCMV (AD169, MOI = 1). The activation of STAT3 and JAK2 was measured by western blot at day 3 post-infection. beta-actin was used as a control for equal loading. The histogram shows STAT3 and JAK2 activation as quantified using Image J 1.40 software. Results of western-blots are representative of two independent experiments; histograms represent means (± SD) of two independent experiments. Ab: Antibody; EGFR: Epidermal growth factor receptor; GCV: ganciclovir.

    Journal: PLoS ONE

    Article Title: HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes

    doi: 10.1371/journal.pone.0059591

    Figure Lengend Snippet: HCMV induces IL-6-mediated activation of the JAK-STAT3 axis in HepG2 cells and PHH. (A) Time course of STAT3 activation in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 0.5). PHH (2×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). STAT3 activation was measured by Western blotting as described in the Materials and Methods section. Unphosphorylated STAT3 and beta-actin were used as controls, and ganciclovir was used at a concentration of 5 microg/ml. (B) Time course of JAK1/JAK2 activation in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 0.5). PHH (2×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). JAK1/JAK2 activation was measured by Western blotting, and beta-actin was used as an internal control. The histogram shows JAK activation at 2 hours post-infection as quantified using Image J 1.40 software. (C) STAT3 activation is mediated by the IL-6-JAK pathway in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV (MOI = 0.5) in the presence or absence of a JAK inhibitor (1 micromol/l), a STAT3 inhibitor (10 micromol/l), a neutralizing anti-IL-6R mAb (10 microg/ml), and a neutralizing anti-EGFR mAb (20 microg/ml). Cells were left uninfected or incubated with the recombinant HCMV glycoprotein gB (10 microg/ml) for 2 hours. STAT3 activation was measured by Western blotting at day 1 post-infection in PHH incubated with JAK inhibitor, STAT3 inhibitor, anti-IL-6R mAb, and in HepG2 cells incubated with JAK and STAT3 inhibitors. STAT3 activation was measured at 2 hours post-infection in HepG2 cells incubated with anti-IL-6R mAb and anti-EGFR mAb. beta-actin was used as an internal control. The histogram shows STAT3 activation as quantified using Image J 1.40 software. (D) STAT3 activation is mediated primarily by HCMV in HepG2 cells and PHH . HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV or UV-inactivated HCMV (AD169, MOI = 1). The activation of STAT3 and JAK2 was measured by western blot at day 3 post-infection. beta-actin was used as a control for equal loading. The histogram shows STAT3 and JAK2 activation as quantified using Image J 1.40 software. Results of western-blots are representative of two independent experiments; histograms represent means (± SD) of two independent experiments. Ab: Antibody; EGFR: Epidermal growth factor receptor; GCV: ganciclovir.

    Article Snippet: Anti-beta-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Activation Assay, Infection, Western Blot, Concentration Assay, Software, Incubation, Recombinant

    HCMV upregulates p53 and p21 in HepG2 cells and PHH. Time course of p53, p21 and Mdm2 in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB at MOI = 0.5 and 1, respectively. P53, p21 and Mdm2 protein expressions were measured by Western blotting, and beta-actin was used as an internal control. Results are representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes

    doi: 10.1371/journal.pone.0059591

    Figure Lengend Snippet: HCMV upregulates p53 and p21 in HepG2 cells and PHH. Time course of p53, p21 and Mdm2 in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB at MOI = 0.5 and 1, respectively. P53, p21 and Mdm2 protein expressions were measured by Western blotting, and beta-actin was used as an internal control. Results are representative of two independent experiments.

    Article Snippet: Anti-beta-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Infection, Western Blot

    Ikaros overexpression attenuates EBV reactivation by LEN ( A ) KEM-I and DAUDI were treated with either vehicle, various concentrations of LTP (1–5 µM) or MTX (1 µM) for 48 hours, and cell lysates were immunoblotted for Ikaros, BZLF1, BMRF1, and β-actin. ( B ) MUTU-I cells were infected with a control Lentivirus or a Lentivirus inducing expression of Ikaros for 48 hours, and then treated with LEN (0.1–5 µM) for 48 hours. Cell lysates were immunoblotted for Ikaros, BZLF1, BMRF1, and β-Actin as a loading control. ( C ) DAUDI cells were treated with LEN (1 µM), BZB (5 nM), or both for 24 hours, and cell lysates were immunoblotted with the indicated sera.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Lenalidomide, Thalidomide, and Pomalidomide Reactivate the Epstein-Barr Virus Lytic Cycle Through Phosphoinositide 3-kinase Signaling and Ikaros Expression

    doi: 10.1158/1078-0432.CCR-15-2242

    Figure Lengend Snippet: Ikaros overexpression attenuates EBV reactivation by LEN ( A ) KEM-I and DAUDI were treated with either vehicle, various concentrations of LTP (1–5 µM) or MTX (1 µM) for 48 hours, and cell lysates were immunoblotted for Ikaros, BZLF1, BMRF1, and β-actin. ( B ) MUTU-I cells were infected with a control Lentivirus or a Lentivirus inducing expression of Ikaros for 48 hours, and then treated with LEN (0.1–5 µM) for 48 hours. Cell lysates were immunoblotted for Ikaros, BZLF1, BMRF1, and β-Actin as a loading control. ( C ) DAUDI cells were treated with LEN (1 µM), BZB (5 nM), or both for 24 hours, and cell lysates were immunoblotted with the indicated sera.

    Article Snippet: Antibody to BMRF1 was obtained from Millipore (Billerica, MA), anti-β-actin from Sigma-Aldrich, and anti-VCA p18 antibodies were from Thermo-Scientific (Waltham, MA).

    Techniques: Over Expression, Infection, Expressing

    Immunomodulatory agents reactivate lytic EBV infection ( A ) B95.8 and D4 LCL cell lines were treated for 48 hours with vehicle, LTP, or MTX as a positive control, and extracts were immunoblotted with the indicated antibodies. ( B ) Reverse-transcriptase (RT) PCR on D4 LCLs following treatment with LEN for 48 hours with primers for BZLF1, BCRF1, and a loading control using β2M or a 1:10 dilution of the cDNA. ( C ) The EBV + BL cell lines DAUDI, KEM-I and MUTU-I were treated as above. Protein levels of BZLF1, BMRF1 and VCA, along with β-actin as a loading control, were determined. Representative images are shown from 1 of 3 independent experiments.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Lenalidomide, Thalidomide, and Pomalidomide Reactivate the Epstein-Barr Virus Lytic Cycle Through Phosphoinositide 3-kinase Signaling and Ikaros Expression

    doi: 10.1158/1078-0432.CCR-15-2242

    Figure Lengend Snippet: Immunomodulatory agents reactivate lytic EBV infection ( A ) B95.8 and D4 LCL cell lines were treated for 48 hours with vehicle, LTP, or MTX as a positive control, and extracts were immunoblotted with the indicated antibodies. ( B ) Reverse-transcriptase (RT) PCR on D4 LCLs following treatment with LEN for 48 hours with primers for BZLF1, BCRF1, and a loading control using β2M or a 1:10 dilution of the cDNA. ( C ) The EBV + BL cell lines DAUDI, KEM-I and MUTU-I were treated as above. Protein levels of BZLF1, BMRF1 and VCA, along with β-actin as a loading control, were determined. Representative images are shown from 1 of 3 independent experiments.

    Article Snippet: Antibody to BMRF1 was obtained from Millipore (Billerica, MA), anti-β-actin from Sigma-Aldrich, and anti-VCA p18 antibodies were from Thermo-Scientific (Waltham, MA).

    Techniques: Infection, Positive Control, Reverse Transcription Polymerase Chain Reaction

    3- O -Methylated flavonols do not increase caspase-1 activity in THP-1 cells. A , levels of IL-1β secreted into culture media by cells stimulated with Pam3CSK4 and 10 μ m methylated flavonol. B , Western blot analysis of proIL-1β levels in cell extracts after stimulation. β-Actin was used as the loading control. C , caspase-1 activity in cell extracts after stimulation. Fold-change in caspase-1 activity was determined by comparing the level found in stimulated cells with those of non-stimulated cells. Cells treated with 10 m m DTT at 37 °C for 1 h were used as a positive control. Data in A and C are expressed as the mean ± S.D. from three independent experiments. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Regiospecific Methylation of a Dietary Flavonoid Scaffold Selectively Enhances IL-1? Production following Toll-like Receptor 2 Stimulation in THP-1 Monocytes *

    doi: 10.1074/jbc.M113.453514

    Figure Lengend Snippet: 3- O -Methylated flavonols do not increase caspase-1 activity in THP-1 cells. A , levels of IL-1β secreted into culture media by cells stimulated with Pam3CSK4 and 10 μ m methylated flavonol. B , Western blot analysis of proIL-1β levels in cell extracts after stimulation. β-Actin was used as the loading control. C , caspase-1 activity in cell extracts after stimulation. Fold-change in caspase-1 activity was determined by comparing the level found in stimulated cells with those of non-stimulated cells. Cells treated with 10 m m DTT at 37 °C for 1 h were used as a positive control. Data in A and C are expressed as the mean ± S.D. from three independent experiments. *, p

    Article Snippet: The lysates (50 μg) were clarified by centrifugation and separated on 12% SDS gels, transferred to 0.2 μm PVDF membranes and immunoblotted with anti-IL-1β antibody (Ab), anti-β-actin Ab (Sigma-Aldrich), anti-phospho-NF-κB p65(S536) Ab, anti-IκB-α Ab, anti-phospho-STAT1(S727) Ab, or anti-STAT1 Ab (Cell Signaling), followed by goat anti-rabbit or anti-mouse HRP-conjugated Ab (Santa Cruz Biotechnology), and were detected with the ECL Plus kit (GE Healthcare).

    Techniques: Methylation, Activity Assay, Western Blot, Positive Control

    Methylated flavonols do not affect steady state levels of IL-1β mRNA and associated transcriptional regulators within the first 2 h of stimulation of THP-1 cells. A , real-time qPCR analysis of steady-state IL-1β mRNA levels in cells stimulated with Pam3CSK4 alone or costimulated with 10 μ m methylated flavonol for 2 h. B , time course analyses of phospho-NF-κB p65(S536), IκB-α, phospho-STAT1 (S727), and total STAT1 in stimulated cells. Target proteins were detected on Western blots using specific Ab. β-Actin was used as the loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Regiospecific Methylation of a Dietary Flavonoid Scaffold Selectively Enhances IL-1? Production following Toll-like Receptor 2 Stimulation in THP-1 Monocytes *

    doi: 10.1074/jbc.M113.453514

    Figure Lengend Snippet: Methylated flavonols do not affect steady state levels of IL-1β mRNA and associated transcriptional regulators within the first 2 h of stimulation of THP-1 cells. A , real-time qPCR analysis of steady-state IL-1β mRNA levels in cells stimulated with Pam3CSK4 alone or costimulated with 10 μ m methylated flavonol for 2 h. B , time course analyses of phospho-NF-κB p65(S536), IκB-α, phospho-STAT1 (S727), and total STAT1 in stimulated cells. Target proteins were detected on Western blots using specific Ab. β-Actin was used as the loading control.

    Article Snippet: The lysates (50 μg) were clarified by centrifugation and separated on 12% SDS gels, transferred to 0.2 μm PVDF membranes and immunoblotted with anti-IL-1β antibody (Ab), anti-β-actin Ab (Sigma-Aldrich), anti-phospho-NF-κB p65(S536) Ab, anti-IκB-α Ab, anti-phospho-STAT1(S727) Ab, or anti-STAT1 Ab (Cell Signaling), followed by goat anti-rabbit or anti-mouse HRP-conjugated Ab (Santa Cruz Biotechnology), and were detected with the ECL Plus kit (GE Healthcare).

    Techniques: Methylation, Real-time Polymerase Chain Reaction, Western Blot

    Localization of PSMB8-AS1 in cells and effects of PSMB8-AS1 on epigenetic markers . (A) The levels of PSMB8-AS1, GAPDH and β-actin mRNAs (cytoplasmic RNA positive controls), and U2snRNA (nuclear RNA positive control) in cytoplasmic and nuclear fractions of A549 cells were determined by real-time PCR. Results are represented for each gene as means ± SE from three independent experiments. (B-D) A549 cells stably expressed dCas9-KRAB were infected with lentiviral gRNAs at a MOI of 200 in the presence of 0.1 µg/ml of puromycin for 48 h and infected with PR/8 at a MOI of 0.01 for 48 h. H3K27m3 and H3K27Ac levels in the nuclei were determined by western blotting and normalized to H3. Representative blots and quantitation were presented in B and C, D, respectively. Results are represented as means ± SE from three independent experiments. No significance was detected by one-way ANOVA.

    Journal: RNA Biology

    Article Title: Long non-coding RNA PSMB8-AS1 regulates influenza virus replication

    doi: 10.1080/15476286.2019.1572448

    Figure Lengend Snippet: Localization of PSMB8-AS1 in cells and effects of PSMB8-AS1 on epigenetic markers . (A) The levels of PSMB8-AS1, GAPDH and β-actin mRNAs (cytoplasmic RNA positive controls), and U2snRNA (nuclear RNA positive control) in cytoplasmic and nuclear fractions of A549 cells were determined by real-time PCR. Results are represented for each gene as means ± SE from three independent experiments. (B-D) A549 cells stably expressed dCas9-KRAB were infected with lentiviral gRNAs at a MOI of 200 in the presence of 0.1 µg/ml of puromycin for 48 h and infected with PR/8 at a MOI of 0.01 for 48 h. H3K27m3 and H3K27Ac levels in the nuclei were determined by western blotting and normalized to H3. Representative blots and quantitation were presented in B and C, D, respectively. Results are represented as means ± SE from three independent experiments. No significance was detected by one-way ANOVA.

    Article Snippet: Mouser anti-β-actin antibodies (Catalog No, MA5-15,739-1MG, lot#QK229411) were purchased from Sigma-Aldrich (St Louis, MO, USA).

    Techniques: Positive Control, Real-time Polymerase Chain Reaction, Stable Transfection, Infection, Western Blot, Quantitation Assay

    lncRNA induction by different influenza viruses . A549 cells were infected with influenza A viruses, PR/8 (MOI: 0.02, 0.2 and 2), WSN (MOI 2) and Pdm/OK (MOI 2) for 24 h. lncRNA expression levels were determined by real-time PCR and normalized to β-actin to determine ΔΔct. Results are represented as means ± SE from three independent experiments. * p

    Journal: RNA Biology

    Article Title: Long non-coding RNA PSMB8-AS1 regulates influenza virus replication

    doi: 10.1080/15476286.2019.1572448

    Figure Lengend Snippet: lncRNA induction by different influenza viruses . A549 cells were infected with influenza A viruses, PR/8 (MOI: 0.02, 0.2 and 2), WSN (MOI 2) and Pdm/OK (MOI 2) for 24 h. lncRNA expression levels were determined by real-time PCR and normalized to β-actin to determine ΔΔct. Results are represented as means ± SE from three independent experiments. * p

    Article Snippet: Mouser anti-β-actin antibodies (Catalog No, MA5-15,739-1MG, lot#QK229411) were purchased from Sigma-Aldrich (St Louis, MO, USA).

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction

    Induction of PSMB8-AS1 transcripts by PR/8 . (A) The PSMB8-AS1 transcripts and real-PCR primers and gRNA locations. (B-C) Human alveolar epithelial (A549) cells (B), human embryonic kidney cells (HEK 293) (C) and human lung adenocarcinoma cell line (H441) (D) were infected with PR/8 at a MOI of 0.2 for 24 h and the relative expression of the four PSMB8-AS1 transcripts were determined by real-time PCR. Data was normalized to β-actin and expressed as means ± SE. n = 3 independent experiments. *p

    Journal: RNA Biology

    Article Title: Long non-coding RNA PSMB8-AS1 regulates influenza virus replication

    doi: 10.1080/15476286.2019.1572448

    Figure Lengend Snippet: Induction of PSMB8-AS1 transcripts by PR/8 . (A) The PSMB8-AS1 transcripts and real-PCR primers and gRNA locations. (B-C) Human alveolar epithelial (A549) cells (B), human embryonic kidney cells (HEK 293) (C) and human lung adenocarcinoma cell line (H441) (D) were infected with PR/8 at a MOI of 0.2 for 24 h and the relative expression of the four PSMB8-AS1 transcripts were determined by real-time PCR. Data was normalized to β-actin and expressed as means ± SE. n = 3 independent experiments. *p

    Article Snippet: Mouser anti-β-actin antibodies (Catalog No, MA5-15,739-1MG, lot#QK229411) were purchased from Sigma-Aldrich (St Louis, MO, USA).

    Techniques: Polymerase Chain Reaction, Infection, Expressing, Real-time Polymerase Chain Reaction

    Validation of RNA-seq results with real-time PCR . (a, b) Relative expression of selected lncRNAs and mRNAs performed on the same samples as for RNA-seq using real-time PCR. Data was normalized to β-actin and expressed as means ± SE. n = 3 independent experiments. * p

    Journal: RNA Biology

    Article Title: Long non-coding RNA PSMB8-AS1 regulates influenza virus replication

    doi: 10.1080/15476286.2019.1572448

    Figure Lengend Snippet: Validation of RNA-seq results with real-time PCR . (a, b) Relative expression of selected lncRNAs and mRNAs performed on the same samples as for RNA-seq using real-time PCR. Data was normalized to β-actin and expressed as means ± SE. n = 3 independent experiments. * p

    Article Snippet: Mouser anti-β-actin antibodies (Catalog No, MA5-15,739-1MG, lot#QK229411) were purchased from Sigma-Aldrich (St Louis, MO, USA).

    Techniques: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Expressing

    Repression of PSMB8-AS1 reduces influenza virus replication . A549 cells stably expressed dCas9-KRAB were infected with lentiviral gRNAs at a MOI of 200 in the presence of 0.1 µg/ml of puromycin for 48 h and infected with PR/8 at a MOI of 0.01 for 48 h. (A) PSMB8-AS1 expression level and (B) NP and NS1 mRNA levels as determined by real-time PCR. (C, D) NP, NS1 and PB1 protein levels as determined by western blotting. The protein bands were quantitated using Image Quant software and normalized to β-actin. (E, F) % NP-positive cells as revealed by immunostaining. (G)Virus titer in medium as determined by a plaque assay. (H) Cell viability as determined by a Cell-Titer Blue assay (Promega G8080). *P

    Journal: RNA Biology

    Article Title: Long non-coding RNA PSMB8-AS1 regulates influenza virus replication

    doi: 10.1080/15476286.2019.1572448

    Figure Lengend Snippet: Repression of PSMB8-AS1 reduces influenza virus replication . A549 cells stably expressed dCas9-KRAB were infected with lentiviral gRNAs at a MOI of 200 in the presence of 0.1 µg/ml of puromycin for 48 h and infected with PR/8 at a MOI of 0.01 for 48 h. (A) PSMB8-AS1 expression level and (B) NP and NS1 mRNA levels as determined by real-time PCR. (C, D) NP, NS1 and PB1 protein levels as determined by western blotting. The protein bands were quantitated using Image Quant software and normalized to β-actin. (E, F) % NP-positive cells as revealed by immunostaining. (G)Virus titer in medium as determined by a plaque assay. (H) Cell viability as determined by a Cell-Titer Blue assay (Promega G8080). *P

    Article Snippet: Mouser anti-β-actin antibodies (Catalog No, MA5-15,739-1MG, lot#QK229411) were purchased from Sigma-Aldrich (St Louis, MO, USA).

    Techniques: Stable Transfection, Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software, Immunostaining, Plaque Assay

    Western blot analysis of caspase 3 in sorted HCT-8 cells at various times after C. parvum infection. (A) Sorted C. parvum -infected HCT-8 cells. Lanes: 1, negative control, noninfected HCT-8 cells; 2, positive control, Jurkat cells treated with 1 μg of staurosporine/ml for 3 h; 3, sorted C. parvum -infected HCT-8 cells at 2 h.p.i.; 4, sorted C. parvum -infected HCT-8 cells at 6 h.p.i.; 5, sorted C. parvum -infected HCT-8 cells at 24 h.p.i.; 6, sorted C. parvum -infected HCT-8 cells at 48 h.p.i. (B) Sorted noninfected cells. Lanes: 1, negative control, noninfected HCT-8 cells; 2, positive control, Jurkat cells treated with 1 μg of staurosporine/ml for 3 h; 3, sorted noninfected HCT-8 cells at 2 h.p.i.; 4, sorted noninfected HCT-8 cells at 6 h.p.i.; 5, sorted noninfected HCT-8 cells at 24 h.p.i.; 6, sorted noninfected HCT-8 cells at 48 h.p.i. Molecular masses in kilodaltons are shown on the left. The β-actin housekeeping protein was used as a loading control.

    Journal: Infection and Immunity

    Article Title: Cryptosporidium parvum at Different Developmental Stages Modulates Host Cell Apoptosis In Vitro

    doi: 10.1128/IAI.72.10.6061-6067.2004

    Figure Lengend Snippet: Western blot analysis of caspase 3 in sorted HCT-8 cells at various times after C. parvum infection. (A) Sorted C. parvum -infected HCT-8 cells. Lanes: 1, negative control, noninfected HCT-8 cells; 2, positive control, Jurkat cells treated with 1 μg of staurosporine/ml for 3 h; 3, sorted C. parvum -infected HCT-8 cells at 2 h.p.i.; 4, sorted C. parvum -infected HCT-8 cells at 6 h.p.i.; 5, sorted C. parvum -infected HCT-8 cells at 24 h.p.i.; 6, sorted C. parvum -infected HCT-8 cells at 48 h.p.i. (B) Sorted noninfected cells. Lanes: 1, negative control, noninfected HCT-8 cells; 2, positive control, Jurkat cells treated with 1 μg of staurosporine/ml for 3 h; 3, sorted noninfected HCT-8 cells at 2 h.p.i.; 4, sorted noninfected HCT-8 cells at 6 h.p.i.; 5, sorted noninfected HCT-8 cells at 24 h.p.i.; 6, sorted noninfected HCT-8 cells at 48 h.p.i. Molecular masses in kilodaltons are shown on the left. The β-actin housekeeping protein was used as a loading control.

    Article Snippet: The nonspecific binding of antibodies was blocked by incubating the membranes with 2% FCS in TNT (Tris-HCl [pH 8.0] with 0.05% Tween 20) at room temperature for 1 h. After blocking, the membranes were processed for immunoblotting with a 1:200-diluted anti-Fas monoclonal antibody (Sigma), a 1:1,000-diluted polyclonal anti-caspase 3 antibody (Sigma), a 1:1,000-diluted anti-Bcl-2 monoclonal antibody (Sigma), or a 1:5,000-diluted anti-β-actin monoclonal antibody (Oncogene Research Products, San Diego, Calif.) at room temperature for 1 h. After immunoblotting, the membranes were rinsed three times for 10 min each in TNT and incubated with peroxidase-conjugated secondary antibodies (either anti-rabbit or anti-mouse) at a 1:3,000 final dilution in PBS for 1 h. Fas, caspase 3, and Bcl-2 were detected by using a chemiluminescence system followed by xerography on CL-X Posure film (Pierce, Boston, Mass.).

    Techniques: Western Blot, Infection, Negative Control, Positive Control

    RT-PCR analysis of gene expression of FasL in sorted C. parvum -infected HCT-8 cells at different times postinfection. Lanes: 1, HCT-8 noninfected cells with 100 U of gamma interferon/ml; 2, HCT-8 noninfected control cells; 3, C. parvum -infected HCT-8 cells at 2 h.p.i.; 4, C. parvum -infected HCT-8 cells at 24 h.p.i.; 5, C. parvum -infected HCT-8 cells at 48 h.p.i.; 6, C. parvum -infected HCT-8 cells at 72 h.p.i. Lower panel, RT-PCR of gene expression of β-actin.

    Journal: Infection and Immunity

    Article Title: Cryptosporidium parvum at Different Developmental Stages Modulates Host Cell Apoptosis In Vitro

    doi: 10.1128/IAI.72.10.6061-6067.2004

    Figure Lengend Snippet: RT-PCR analysis of gene expression of FasL in sorted C. parvum -infected HCT-8 cells at different times postinfection. Lanes: 1, HCT-8 noninfected cells with 100 U of gamma interferon/ml; 2, HCT-8 noninfected control cells; 3, C. parvum -infected HCT-8 cells at 2 h.p.i.; 4, C. parvum -infected HCT-8 cells at 24 h.p.i.; 5, C. parvum -infected HCT-8 cells at 48 h.p.i.; 6, C. parvum -infected HCT-8 cells at 72 h.p.i. Lower panel, RT-PCR of gene expression of β-actin.

    Article Snippet: The nonspecific binding of antibodies was blocked by incubating the membranes with 2% FCS in TNT (Tris-HCl [pH 8.0] with 0.05% Tween 20) at room temperature for 1 h. After blocking, the membranes were processed for immunoblotting with a 1:200-diluted anti-Fas monoclonal antibody (Sigma), a 1:1,000-diluted polyclonal anti-caspase 3 antibody (Sigma), a 1:1,000-diluted anti-Bcl-2 monoclonal antibody (Sigma), or a 1:5,000-diluted anti-β-actin monoclonal antibody (Oncogene Research Products, San Diego, Calif.) at room temperature for 1 h. After immunoblotting, the membranes were rinsed three times for 10 min each in TNT and incubated with peroxidase-conjugated secondary antibodies (either anti-rabbit or anti-mouse) at a 1:3,000 final dilution in PBS for 1 h. Fas, caspase 3, and Bcl-2 were detected by using a chemiluminescence system followed by xerography on CL-X Posure film (Pierce, Boston, Mass.).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Infection

    The conditioned medium from the P. aeruginosa PAO1 wild type and QS mutants regulates PEL cell growth and viral gene expression. (A to C) BCBL-1 (A), BCP-1 (B), or BL-41 (C) cells were incubated with filtered conditioned medium from overnight P. aeruginosa PAO1 wt or QS mutant ( lasI , rhlI , pqsC ) cultures (diluted 1:100, 1:50, and 1:25) for 48 h. The cell proliferation status was examined using WST-1 cell proliferation assays (Roche). (D and E) BCBL-1 (D) and BCP-1 (E) cells were incubated with filtered conditioned medium from overnight P. aeruginosa PAO1 wt or QS mutants cultures (diluted 1:25) for 48 h, and then qRT-PCR was used to quantify viral transcripts representing either latent or lytic genes. Data were normalized to those for vehicle-treated cells, and β-actin was used as a loading control. (F and G) BCBL-1 cells were incubated with the filtered conditioned medium described above for 4 days, and then protein expression was measured using immunoblots. Released virions were isolated, purified from the supernatant, and used to infect fresh HUVEC. After 24 h postinfection, Lana transcripts were quantified using qRT-PCR. Error bars represent the SD from 3 independent experiments. *, P

    Journal: Journal of Virology

    Article Title: Regulation of Virus-Associated Lymphoma Growth and Gene Expression by Bacterial Quorum-Sensing Molecules

    doi: 10.1128/JVI.00478-18

    Figure Lengend Snippet: The conditioned medium from the P. aeruginosa PAO1 wild type and QS mutants regulates PEL cell growth and viral gene expression. (A to C) BCBL-1 (A), BCP-1 (B), or BL-41 (C) cells were incubated with filtered conditioned medium from overnight P. aeruginosa PAO1 wt or QS mutant ( lasI , rhlI , pqsC ) cultures (diluted 1:100, 1:50, and 1:25) for 48 h. The cell proliferation status was examined using WST-1 cell proliferation assays (Roche). (D and E) BCBL-1 (D) and BCP-1 (E) cells were incubated with filtered conditioned medium from overnight P. aeruginosa PAO1 wt or QS mutants cultures (diluted 1:25) for 48 h, and then qRT-PCR was used to quantify viral transcripts representing either latent or lytic genes. Data were normalized to those for vehicle-treated cells, and β-actin was used as a loading control. (F and G) BCBL-1 cells were incubated with the filtered conditioned medium described above for 4 days, and then protein expression was measured using immunoblots. Released virions were isolated, purified from the supernatant, and used to infect fresh HUVEC. After 24 h postinfection, Lana transcripts were quantified using qRT-PCR. Error bars represent the SD from 3 independent experiments. *, P

    Article Snippet: For loading controls, lysates were also incubated with antibodies detecting β-actin (Sigma).

    Techniques: Expressing, Incubation, Mutagenesis, Quantitative RT-PCR, Western Blot, Isolation, Purification

    P. aeruginosa QS signaling molecules regulate KSHV gene expression from PEL cells. (A to C) BCBL-1 cells were incubated with the indicated concentrations of OdDHL (A), BHL (B), or PQS (C) for 48 h, and then quantitative real-time PCR (qRT-PCR) was used to quantify viral transcripts representing either latent ( Lana ) or lytic ( Rta , vGpcr , K8.1 , and Orf57 ) genes. Data were normalized to those for vehicle-treated cells, and β-actin was used as a loading control. (D) Protein expression was measured using immunoblots. (E) Released virions were isolated, purified from the supernatant of BCBL-1 cells that had been treated with OdDHL or valproic acid (VA; as the positive control) for 4 days, and then used to infect fresh HUVEC. After 24 h postinfection, Lana transcripts were quantified using qRT-PCR. Error bars represent the SD from 3 independent experiments. *, P

    Journal: Journal of Virology

    Article Title: Regulation of Virus-Associated Lymphoma Growth and Gene Expression by Bacterial Quorum-Sensing Molecules

    doi: 10.1128/JVI.00478-18

    Figure Lengend Snippet: P. aeruginosa QS signaling molecules regulate KSHV gene expression from PEL cells. (A to C) BCBL-1 cells were incubated with the indicated concentrations of OdDHL (A), BHL (B), or PQS (C) for 48 h, and then quantitative real-time PCR (qRT-PCR) was used to quantify viral transcripts representing either latent ( Lana ) or lytic ( Rta , vGpcr , K8.1 , and Orf57 ) genes. Data were normalized to those for vehicle-treated cells, and β-actin was used as a loading control. (D) Protein expression was measured using immunoblots. (E) Released virions were isolated, purified from the supernatant of BCBL-1 cells that had been treated with OdDHL or valproic acid (VA; as the positive control) for 4 days, and then used to infect fresh HUVEC. After 24 h postinfection, Lana transcripts were quantified using qRT-PCR. Error bars represent the SD from 3 independent experiments. *, P

    Article Snippet: For loading controls, lysates were also incubated with antibodies detecting β-actin (Sigma).

    Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Isolation, Purification, Positive Control

    Polymicrobial infection activates BAD pro-apoptotic activity in septic shock. Bad −/− mice and their WT littermates ( a , b , e ), or Tnf -R1 −/− mice and their WT littermates ( c – e ) were subjected to high-grade CLP surgery or Sham procedure, as described in Fig. 5a . a – d Total liver tissue extracts were fractionated to determine BAD translocation from the cytosol [(containing cytoskeleton, Cytosol(+C)] to mitochondria (see “Materials and methods” for details) ( a , c and Supplementary information, Figure S9n , S9p ), or BAD release from the cytoskeleton to the cytosol [containing mitochondria; Cytosol(+M)] (see “Materials and methods” for details) ( b , d and Supplementary information, Figure S9o , S9q ). The levels of BAD and the percentage of IKK-phosphorylated BAD in total BAD were determined and quantitated, as described in Fig. 1f (See “Materials and methods” for details). e Phosphorylation of IKKβ, IκBα, phospho-BAD( S26), as well as expression levels of IKKβ, IκBα, BAD, TNF-R1, and β-actin in liver were analyzed by immunoblotting with corresponding antibodies. All the results represent three individual experiments with similar results

    Journal: Cell Research

    Article Title: The BH3-only protein BAD mediates TNFα cytotoxicity despite concurrent activation of IKK and NF-κB in septic shock

    doi: 10.1038/s41422-018-0041-7

    Figure Lengend Snippet: Polymicrobial infection activates BAD pro-apoptotic activity in septic shock. Bad −/− mice and their WT littermates ( a , b , e ), or Tnf -R1 −/− mice and their WT littermates ( c – e ) were subjected to high-grade CLP surgery or Sham procedure, as described in Fig. 5a . a – d Total liver tissue extracts were fractionated to determine BAD translocation from the cytosol [(containing cytoskeleton, Cytosol(+C)] to mitochondria (see “Materials and methods” for details) ( a , c and Supplementary information, Figure S9n , S9p ), or BAD release from the cytoskeleton to the cytosol [containing mitochondria; Cytosol(+M)] (see “Materials and methods” for details) ( b , d and Supplementary information, Figure S9o , S9q ). The levels of BAD and the percentage of IKK-phosphorylated BAD in total BAD were determined and quantitated, as described in Fig. 1f (See “Materials and methods” for details). e Phosphorylation of IKKβ, IκBα, phospho-BAD( S26), as well as expression levels of IKKβ, IκBα, BAD, TNF-R1, and β-actin in liver were analyzed by immunoblotting with corresponding antibodies. All the results represent three individual experiments with similar results

    Article Snippet: Antibody against β-actin was from Sigma.

    Techniques: Infection, Activity Assay, Mouse Assay, Translocation Assay, Expressing

    Cytotoxic dose TNFα is sufficient to induce apoptosis in a BAD-dependent manner despite concurrent activation of IKK and NF-κB. a , b Various primary wild-type (WT) and Bad −/− cells were treated without or with non-cytotoxic (5 ng/ml) or cytotoxic (80 ng/ml) dose TNFα for 12 h, as indicated. Apoptotic cells were detected by Annexin V/Propidium iodide (PI) staining and analyzed by flow cytometric analysis ( a ) or determined by caspase-3 activity assay ( b ). Hep hepatocyte, Thy thymocyte, Mac macrophage, SPC splenocyte, FB fibroblast. c , d WT and Bad −/− embryonic fibroblasts were treated without or with various doses of different fractions (F, fraction; F18, fraction number 18 and etc.) of the TNFα preparation (R D) (TNFα hereinafter for the simplicity unless otherwise specified) for 12 h. Apoptotic cells were determined as described in ( a ). e WT and Bad −/− embryonic fibroblasts were treated without or with various doses of TNFα from three different commercial sources (R D, GeneScript and PeproTech; Supplementary information, Table S4 ) for 24 h. Apoptotic cells were determined as described in a . 80 ng/ml TNFα from R D, 500 ng/ml TNFα from GeneScript, 1000 ng/ml TNFα from PeproTech have similar biological activity (Supplementary information, Table S4 ). f , g Cytotoxic but not non-cytotoxic dose TNFα from the pooled TNFα preparation ( f ) or cytotoxic dose TNFα from different fractions of the TNFα preparation ( g ) induced BAD mitochondrial translocation in primary WT hepatocytes ( f ) and WT fibroblasts ( g ), as determined by the cytosol [containing cytoskeleton, Cytosol (+C)] and mitochondria fractionation (see “Materials and methods” for details), followed by immunoblotting with corresponding antibodies. β-Actin and COX-2 were used as the marker of the cytosol and mitochondria, respectively. The percentage of IKK-phosphorylated BAD in total cytoplasmic BAD protein was determined (Supplementary information, Figure S9a - S9b ), as described in “Materials and methods”. h Cytotoxic and non-cytotoxic dose TNFα induced comparable activation of IKK and NF-κB in primary hepatocytes. Phosphorylation of IKKβ, IκBα and BAD, as well as their protein levels were analyzed by immunoblotting with corresponding antibodies. i Primary WT and Bad −/− hepatocytes were treated without or with non-cytotoxic or cytotoxic dose TNFα for various durations, as indicated. Total RNA was extracted for quantitative real-time PCR analysis with different primers specifically for cIAP2, IκBα, and IL-6

    Journal: Cell Research

    Article Title: The BH3-only protein BAD mediates TNFα cytotoxicity despite concurrent activation of IKK and NF-κB in septic shock

    doi: 10.1038/s41422-018-0041-7

    Figure Lengend Snippet: Cytotoxic dose TNFα is sufficient to induce apoptosis in a BAD-dependent manner despite concurrent activation of IKK and NF-κB. a , b Various primary wild-type (WT) and Bad −/− cells were treated without or with non-cytotoxic (5 ng/ml) or cytotoxic (80 ng/ml) dose TNFα for 12 h, as indicated. Apoptotic cells were detected by Annexin V/Propidium iodide (PI) staining and analyzed by flow cytometric analysis ( a ) or determined by caspase-3 activity assay ( b ). Hep hepatocyte, Thy thymocyte, Mac macrophage, SPC splenocyte, FB fibroblast. c , d WT and Bad −/− embryonic fibroblasts were treated without or with various doses of different fractions (F, fraction; F18, fraction number 18 and etc.) of the TNFα preparation (R D) (TNFα hereinafter for the simplicity unless otherwise specified) for 12 h. Apoptotic cells were determined as described in ( a ). e WT and Bad −/− embryonic fibroblasts were treated without or with various doses of TNFα from three different commercial sources (R D, GeneScript and PeproTech; Supplementary information, Table S4 ) for 24 h. Apoptotic cells were determined as described in a . 80 ng/ml TNFα from R D, 500 ng/ml TNFα from GeneScript, 1000 ng/ml TNFα from PeproTech have similar biological activity (Supplementary information, Table S4 ). f , g Cytotoxic but not non-cytotoxic dose TNFα from the pooled TNFα preparation ( f ) or cytotoxic dose TNFα from different fractions of the TNFα preparation ( g ) induced BAD mitochondrial translocation in primary WT hepatocytes ( f ) and WT fibroblasts ( g ), as determined by the cytosol [containing cytoskeleton, Cytosol (+C)] and mitochondria fractionation (see “Materials and methods” for details), followed by immunoblotting with corresponding antibodies. β-Actin and COX-2 were used as the marker of the cytosol and mitochondria, respectively. The percentage of IKK-phosphorylated BAD in total cytoplasmic BAD protein was determined (Supplementary information, Figure S9a - S9b ), as described in “Materials and methods”. h Cytotoxic and non-cytotoxic dose TNFα induced comparable activation of IKK and NF-κB in primary hepatocytes. Phosphorylation of IKKβ, IκBα and BAD, as well as their protein levels were analyzed by immunoblotting with corresponding antibodies. i Primary WT and Bad −/− hepatocytes were treated without or with non-cytotoxic or cytotoxic dose TNFα for various durations, as indicated. Total RNA was extracted for quantitative real-time PCR analysis with different primers specifically for cIAP2, IκBα, and IL-6

    Article Snippet: Antibody against β-actin was from Sigma.

    Techniques: Activation Assay, Staining, Flow Cytometry, Caspase-3 Activity Assay, Activity Assay, Translocation Assay, Fractionation, Marker, Real-time Polymerase Chain Reaction

    Cytotoxic dose TNFα induces substantial BAD release from the cytoskeleton to the cytosol by promoting massive depolymerization of actin stress fibers. a Direct interaction between BAD and F-actin in actin stress fibers at the cytoskeleton was detected by super-resolution microscopy based on Ground State Depletion program. Actin stress fibers were stained with phalloidin - Green (Green color). BAD (Red color) was detected by immunofluorescence staining with anti-BAD antibody. Scale bar, 20 nm. b , c Cytotoxic dose TNFα significantly reduced the interaction of BAD with F-actin, as revealed by proximity ligation assay. Scale bar, 5 μm ( b ), and induced BAD release from the cytoskeleton to the cytosol, as determined by the cytosol [containing mitochondria, Cytosol(+M)] and cytoskeleton fractionation (see “Materials and methods” for details) (Supplementary information, Figure S9g ) ( c ). d Cytotoxic dose TNFα induced massive depolymerization of actin stress fibers, as detected by double immunofluorescence staining. BAD, Red color; Actin, Green color. Scale bar, 1 μm. e WT fibroblasts were stimulated without or with cytotoxic or non-cytotoxic dose TNFα for various durations, as indicated. Cytosol [containing mitochondria, Cytosol(+M)] and cytoskeleton fractions were separated by ultracentrifugation. G-actin and F-actin were detected by immunoblotting with anti-β-actin antibody. Cells treated with Cytochalasin D (CyD; 1 μg/ml; 1 h) were used as positive control. ∞, infinity. GAPDH was used as cytosol marker. The results were quantitated by the ImageJ program. f – i WT and Bad −/− fibroblasts were pre-treated with DMSO or Jasplakinolide (30 nM) ( f , g ), Cytochalasin D (1 μg/ml) ( h , i ) for 1 h, followed by stimulation without or with cytotoxic or non-cytotoxic dose TNFα for various durations, as indicated. Phosphorylated BAD and BAD mitochondrial translocation were determined and quantitated as described in Fig. 1f ( f , h , Supplementary information, Figure S9h - S9i ). Apoptotic cells were determined as described in Fig. 1a . Data are means ± s.d. ** P

    Journal: Cell Research

    Article Title: The BH3-only protein BAD mediates TNFα cytotoxicity despite concurrent activation of IKK and NF-κB in septic shock

    doi: 10.1038/s41422-018-0041-7

    Figure Lengend Snippet: Cytotoxic dose TNFα induces substantial BAD release from the cytoskeleton to the cytosol by promoting massive depolymerization of actin stress fibers. a Direct interaction between BAD and F-actin in actin stress fibers at the cytoskeleton was detected by super-resolution microscopy based on Ground State Depletion program. Actin stress fibers were stained with phalloidin - Green (Green color). BAD (Red color) was detected by immunofluorescence staining with anti-BAD antibody. Scale bar, 20 nm. b , c Cytotoxic dose TNFα significantly reduced the interaction of BAD with F-actin, as revealed by proximity ligation assay. Scale bar, 5 μm ( b ), and induced BAD release from the cytoskeleton to the cytosol, as determined by the cytosol [containing mitochondria, Cytosol(+M)] and cytoskeleton fractionation (see “Materials and methods” for details) (Supplementary information, Figure S9g ) ( c ). d Cytotoxic dose TNFα induced massive depolymerization of actin stress fibers, as detected by double immunofluorescence staining. BAD, Red color; Actin, Green color. Scale bar, 1 μm. e WT fibroblasts were stimulated without or with cytotoxic or non-cytotoxic dose TNFα for various durations, as indicated. Cytosol [containing mitochondria, Cytosol(+M)] and cytoskeleton fractions were separated by ultracentrifugation. G-actin and F-actin were detected by immunoblotting with anti-β-actin antibody. Cells treated with Cytochalasin D (CyD; 1 μg/ml; 1 h) were used as positive control. ∞, infinity. GAPDH was used as cytosol marker. The results were quantitated by the ImageJ program. f – i WT and Bad −/− fibroblasts were pre-treated with DMSO or Jasplakinolide (30 nM) ( f , g ), Cytochalasin D (1 μg/ml) ( h , i ) for 1 h, followed by stimulation without or with cytotoxic or non-cytotoxic dose TNFα for various durations, as indicated. Phosphorylated BAD and BAD mitochondrial translocation were determined and quantitated as described in Fig. 1f ( f , h , Supplementary information, Figure S9h - S9i ). Apoptotic cells were determined as described in Fig. 1a . Data are means ± s.d. ** P

    Article Snippet: Antibody against β-actin was from Sigma.

    Techniques: Microscopy, Staining, Immunofluorescence, Proximity Ligation Assay, Fractionation, Double Immunofluorescence Staining, Positive Control, Marker, Translocation Assay

    Apoptosis induction and telomerase loss in HepG2 cells are independent from ROS production. a) Effect of MTBITC on ROS production in adherent growing cells. Cells were exposed to MTBITC for 1 to 6 h, washed thoroughly with PBS and subsequently exposed to 100 µM spin probe CMH in Krebs-Hepes buffer. ROS were then quantified using ESR spectrometry. Positive control: 100 µM menadione. The pictures display the middle peak of ESR spectra of CMH spin probe labelled samples. Bars are mean ± SD, n = 3. b) Representative immunoblot for HNE Lys-adducts after exposure to MTBITC or DMSO for different time points. Total lysate of HepG2 cells was subjected to immunoblotting using an anti-HNE Lys antibody. Exposure of cells to HNE for 80 min. was used as positive control. The blot was reprobed with anti-β-actin antibody to ensure equal protein loading. c+d) Effect of NAC pre-treatment. HepG2 cells were pre-treated with NAC for 1 h, washed with PBS and subsequently exposed to 25 µM MTBITC or 0.1% DMSO for another 24 h. Cells were then prepared for (c) TRAP analysis or (d) flow cytometric analysis of the mitochondrial membrane potential (MMP) as parameter for apoptosis; *p≤0.05. M: 50 bp DNA marker; 1) 0.1% DMSO 2) 25 µM MTBITC 3) 5 mM NAC +0.1% DMSO,4) 5 mM NAC +25 µM MTBITC 5) cell lytic buffer 6) destilled water 7) 0.1% DMSO, heat treated. e) Effect of 25 µM MTBITC on mtDNA damage after 1 or 24 h exposure of HepG2 cells. Agarose gel electrophoresis of amplified mtDNA multiplex PCR products is shown in representative image details. Each lane contains amplification products obtained with mtDNA from one single cell using specific PCR primers. As positive control for mtDNA damage, cells were exposed to 10 min UV irradiation. f ) Representative pictures of beta-galactosidase staining of HCC cells after exposure to MTBITC for 72 h, captured by light microscopy. beta-galactosidase positive cells are reflected by dark color of the cells. SC: solvent control = 0.1% DMSO. Scale bar = 200 µm, all panels have the same magnification.

    Journal: PLoS ONE

    Article Title: The MAPK Pathway Signals Telomerase Modulation in Response to Isothiocyanate-Induced DNA Damage of Human Liver Cancer Cells

    doi: 10.1371/journal.pone.0053240

    Figure Lengend Snippet: Apoptosis induction and telomerase loss in HepG2 cells are independent from ROS production. a) Effect of MTBITC on ROS production in adherent growing cells. Cells were exposed to MTBITC for 1 to 6 h, washed thoroughly with PBS and subsequently exposed to 100 µM spin probe CMH in Krebs-Hepes buffer. ROS were then quantified using ESR spectrometry. Positive control: 100 µM menadione. The pictures display the middle peak of ESR spectra of CMH spin probe labelled samples. Bars are mean ± SD, n = 3. b) Representative immunoblot for HNE Lys-adducts after exposure to MTBITC or DMSO for different time points. Total lysate of HepG2 cells was subjected to immunoblotting using an anti-HNE Lys antibody. Exposure of cells to HNE for 80 min. was used as positive control. The blot was reprobed with anti-β-actin antibody to ensure equal protein loading. c+d) Effect of NAC pre-treatment. HepG2 cells were pre-treated with NAC for 1 h, washed with PBS and subsequently exposed to 25 µM MTBITC or 0.1% DMSO for another 24 h. Cells were then prepared for (c) TRAP analysis or (d) flow cytometric analysis of the mitochondrial membrane potential (MMP) as parameter for apoptosis; *p≤0.05. M: 50 bp DNA marker; 1) 0.1% DMSO 2) 25 µM MTBITC 3) 5 mM NAC +0.1% DMSO,4) 5 mM NAC +25 µM MTBITC 5) cell lytic buffer 6) destilled water 7) 0.1% DMSO, heat treated. e) Effect of 25 µM MTBITC on mtDNA damage after 1 or 24 h exposure of HepG2 cells. Agarose gel electrophoresis of amplified mtDNA multiplex PCR products is shown in representative image details. Each lane contains amplification products obtained with mtDNA from one single cell using specific PCR primers. As positive control for mtDNA damage, cells were exposed to 10 min UV irradiation. f ) Representative pictures of beta-galactosidase staining of HCC cells after exposure to MTBITC for 72 h, captured by light microscopy. beta-galactosidase positive cells are reflected by dark color of the cells. SC: solvent control = 0.1% DMSO. Scale bar = 200 µm, all panels have the same magnification.

    Article Snippet: The following primary antibodies were used for immunoblotting: anti-p-ERK1/2 (Thr202/Tyr201, 1∶2000), anti-p-JNK (Thr 183/Tyr185, 1∶2000, clone G9), anti-p-c-Jun Ser73 and anti-p-p38 (Thr180/Tyr182, 1∶500) from Cell Signalling, anti- 4-Hydroxynonenal (1∶250; clone 198960) from R & D Systems EUROPE (Abingdon, England) and anti-beta-actin (1∶10000, clone AC-74) from Sigma Aldrich.

    Techniques: Electron Paramagnetic Resonance, Positive Control, Flow Cytometry, Marker, Agarose Gel Electrophoresis, Amplification, Multiplex Assay, Polymerase Chain Reaction, Irradiation, Staining, Light Microscopy

    Reduced number of GnRH neurons in the forebrain of Slit2 –/– mice. ( A ) Schematic of the mouse head area displayed in B,C. ( B , C ) Sagittal sections of E12.5 mouse heads were immunolabelled for GnRH to reveal neurons migrating in the NC and FB. The FB boundary is delineated by a dotted line. Arrows (B,C) indicate areas displayed at higher magnification in insets. Arrow in C also points to abnormal accumulation of cells in the NC of Slit2 –/– mice as compared with wild type (Δ in B). ( D ) Counts of GnRH neurons in the FB of E12.5 wild-type and Slit2 –/– mice revealed a significant reduction in the mutant. ( E-G ′) Schematic (E) and sagittal sections (F,G) of E14.5 mouse heads immunolabelled for GnRH to reveal neurons migrating in the NC and FB. Arrows indicate areas displayed at high magnification in F′,G′. ( H-J ) Schematic (H) and representative images of sagittal sections (I,J) showing migrating GnRH neurons in the basal FB of E14.5 wild-type and Slit2 –/– mice. There is a reduction in cells in the FB of the mutant (Δ in J). Arrowheads indicate examples of migrating GnRH neurons. ( K ) Counts of GnRH neurons in the FB of E14.5 wild-type and Slit2 –/– mice revealed a statistically significant reduction in the mutant. ( L-N , P-R ) Schematic drawings (L,P) and coronal sections (M,N,Q,R) of E18.5 mouse brains immunolabelled for GnRH to reveal neurons projecting to the MPOA (M,N) and extending their axons to the ME (Q,R). Arrowheads indicate examples of migrating GnRH neurons (M,N) or GnRH + fibres projecting to the ME (Q,R). The paucity of such projections in the Slit2 –/– mice is indicated by Δ. ( O ) Counts of GnRH neurons in the brain of E18.5 wild-type and Slit2 –/– mice revealed a statistically significant reduction in the mutant. ( S ) Optical density measurement of GnRH neuron fibres projecting to the ME of E18.5 wild-type and Slit2 –/– mice revealed a statistically significant reduction in the mutant as compared with wild-type littermates. Mean ± s.e.m. * P

    Journal: Development (Cambridge, England)

    Article Title: Slit2 and Robo3 modulate the migration of GnRH-secreting neurons

    doi: 10.1242/dev.079418

    Figure Lengend Snippet: Reduced number of GnRH neurons in the forebrain of Slit2 –/– mice. ( A ) Schematic of the mouse head area displayed in B,C. ( B , C ) Sagittal sections of E12.5 mouse heads were immunolabelled for GnRH to reveal neurons migrating in the NC and FB. The FB boundary is delineated by a dotted line. Arrows (B,C) indicate areas displayed at higher magnification in insets. Arrow in C also points to abnormal accumulation of cells in the NC of Slit2 –/– mice as compared with wild type (Δ in B). ( D ) Counts of GnRH neurons in the FB of E12.5 wild-type and Slit2 –/– mice revealed a significant reduction in the mutant. ( E-G ′) Schematic (E) and sagittal sections (F,G) of E14.5 mouse heads immunolabelled for GnRH to reveal neurons migrating in the NC and FB. Arrows indicate areas displayed at high magnification in F′,G′. ( H-J ) Schematic (H) and representative images of sagittal sections (I,J) showing migrating GnRH neurons in the basal FB of E14.5 wild-type and Slit2 –/– mice. There is a reduction in cells in the FB of the mutant (Δ in J). Arrowheads indicate examples of migrating GnRH neurons. ( K ) Counts of GnRH neurons in the FB of E14.5 wild-type and Slit2 –/– mice revealed a statistically significant reduction in the mutant. ( L-N , P-R ) Schematic drawings (L,P) and coronal sections (M,N,Q,R) of E18.5 mouse brains immunolabelled for GnRH to reveal neurons projecting to the MPOA (M,N) and extending their axons to the ME (Q,R). Arrowheads indicate examples of migrating GnRH neurons (M,N) or GnRH + fibres projecting to the ME (Q,R). The paucity of such projections in the Slit2 –/– mice is indicated by Δ. ( O ) Counts of GnRH neurons in the brain of E18.5 wild-type and Slit2 –/– mice revealed a statistically significant reduction in the mutant. ( S ) Optical density measurement of GnRH neuron fibres projecting to the ME of E18.5 wild-type and Slit2 –/– mice revealed a statistically significant reduction in the mutant as compared with wild-type littermates. Mean ± s.e.m. * P

    Article Snippet: A similar result was obtained following analysis of mice at E12.5 (wild type, 1042±25.41; Slit1–/– , 1023±26.39; Slit2–/– , 1001±9.866; P > 0.05, one-way ANOVA).

    Techniques: Mouse Assay, Mutagenesis

    Slit and Robo in the GnRH neuronal system. ( A-F ) Expression of Slit2 and Slit1 in GnRH neurons. Sagittal sections from E12.5 Slit2 +/– (A-C) and Slit1 +/– (D-F) mouse heads were immunostained for GnRH (A,D) and GFP (B,E); colocalisation between GnRH (red) and Slit2/Slit1 (green) is indicated with arrowheads in the corresponding higher magnification images (C,F). The boundary between NC and FB is indicated by a dotted line. ( G ) RT-PCR on FACS-sorted E14.5 GFP-GnRH neurons revealed expression of Slit2 (210 bp), Robo1 (338 bp) and Robo2 (823 bp). Gapdh , positive control (188 bp). ( H ) Coronal section taken from E14.5 mouse head shows that Slit1 is expressed in GnRH neurons emerging from the VNO (yellow; arrowheads). ( I , J ) Coronal sections through the FB of E16.5 wild-type and Robo1 –/– Robo2 –/– littermates were immunolabelled to visualise GnRH neurons in the MPOA. ( K ) Counts of labelled cells did not show statistically significant differences between the wild-type and Robo1 –/– Robo2 –/– littermates. Mean ± s.e.m. VNO, vomeronasal organ. Scale bars: 150 μm in A,B,D,E; 50 μm in C,F,H; 100 μm in I,J.

    Journal: Development (Cambridge, England)

    Article Title: Slit2 and Robo3 modulate the migration of GnRH-secreting neurons

    doi: 10.1242/dev.079418

    Figure Lengend Snippet: Slit and Robo in the GnRH neuronal system. ( A-F ) Expression of Slit2 and Slit1 in GnRH neurons. Sagittal sections from E12.5 Slit2 +/– (A-C) and Slit1 +/– (D-F) mouse heads were immunostained for GnRH (A,D) and GFP (B,E); colocalisation between GnRH (red) and Slit2/Slit1 (green) is indicated with arrowheads in the corresponding higher magnification images (C,F). The boundary between NC and FB is indicated by a dotted line. ( G ) RT-PCR on FACS-sorted E14.5 GFP-GnRH neurons revealed expression of Slit2 (210 bp), Robo1 (338 bp) and Robo2 (823 bp). Gapdh , positive control (188 bp). ( H ) Coronal section taken from E14.5 mouse head shows that Slit1 is expressed in GnRH neurons emerging from the VNO (yellow; arrowheads). ( I , J ) Coronal sections through the FB of E16.5 wild-type and Robo1 –/– Robo2 –/– littermates were immunolabelled to visualise GnRH neurons in the MPOA. ( K ) Counts of labelled cells did not show statistically significant differences between the wild-type and Robo1 –/– Robo2 –/– littermates. Mean ± s.e.m. VNO, vomeronasal organ. Scale bars: 150 μm in A,B,D,E; 50 μm in C,F,H; 100 μm in I,J.

    Article Snippet: A similar result was obtained following analysis of mice at E12.5 (wild type, 1042±25.41; Slit1–/– , 1023±26.39; Slit2–/– , 1001±9.866; P > 0.05, one-way ANOVA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, FACS, Positive Control

    SCF FBXW7 interacts and targets ZEB2 for degradation in a GSK-3β phosphorylation-dependent manner. a Left, 2DE and MALDI-MS-based identification of novel Fbxw7-associated proteins using crypts (upper panel) isolated from 3-week fbxw 7 fl/fl and fbxw 7 ΔG mice. Yellow circles in the lower panel denote potential Fbxw7-associated proteins. a Right, WB analysis (upper panels), and RT-PCR analysis (lower panels) of fbxw 7 fl/fl vs. fbxw 7 ΔG derived crypts and intestinal proteins and mRNA expression for ZEB2 and β-actin control. Experiments were performed on at least three independent occasions. b Left, schematic representation of the modified yeast two-hybrid reverse Ras Recruitment Screening (rRRS) system identifying proteins interacting with Fbxw7 in a GSK-3β phosphorylation-dependent manner. GSK-3β under the control of the methionine-regulated MET 3 promoter induces phosphorylation of encoded myristoylated proteins through a cDNA library plus positive control expressing FLAG-β-catenin (B—Middle) which only rescued the growth of cdc25–2 mutant yeast by Fbxw7-associated protein(s), if they interact with RasV12-FBXW7ΔF (i.e. human FBXW7α isoform mutant lacking F-box domain; therefore, interaction with Skp1 is lost and degradation of SCF Fbxw7 substrates will not occur in yeast) used as a bait at the restrictive temperature 37 °C, in a methionine-dependent manner. In the FBXW7ΔF mutant, both the N-terminal F-box and Dim-domains are deleted to avoid any interactions with SKP1 and other FBXW7 isoform-associated proteins. Thus, cdc25–2 mutant yeasts can grow only at 37 °C, when a phosphorylation-dependent interaction between a protein target and RasV12-FBXW7ΔF takes place. The FBXW7ΔF(bait)-dependent growth of these clones was further analysed on galactose-containing medium at 37 °C (B—Right). Red circles show the GSK-3β-phosphorylation-dependent interactor, including the Zeb2-clone, green circles show the phosphorylation/non-phosphorylation-dependent interactor and blue circles show the revertant clones (B—Right). c Left, subcellular localisation of GFP-fused human ZEB2 in the absence (top; nuclear) and presence (bottom; nuclear spots indicative of protein degradation) of GSK-3β in HCT116 CRC cells. ( c— Middle and c— Right) WB analysis of total ZEB2 protein level following the inhibition of GSK-3β (e.g. WS119 or LiCl treatment, and siRNA against GSK-3β) and of UPS pathways (MG132) in SW620 CRC cells. d Direct binding and ubiquitin-dependent degradation of ZEB2 by FBXW7. Co-immunoprecipitation (IP) of ZEB2 upon pull-down of FBXW7 in HEK-293T cells (Left); co-IP of FBXW7 upon pull-down of ZEB2 using the TNT-coupled reticulocyte lysate (Middle), and ubiquitination assays with HA-tagged ubiquitin- (HA-Ub) expressing construct in HEK-293T cells (Right). The asterisk indicates a nonspecific band(s). e Co-IP of endogenous ZEB2 upon pull-down of FBXW7 in HCT116 cells with FBXW 7 deletion. f ZEB2 pulse-chase stability assays with 15 µg/ml cycloheximide (CHX) in HCT116 cells with or without FBXW 7 deletion

    Journal: Oncogenesis

    Article Title: An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance

    doi: 10.1038/s41389-019-0125-3

    Figure Lengend Snippet: SCF FBXW7 interacts and targets ZEB2 for degradation in a GSK-3β phosphorylation-dependent manner. a Left, 2DE and MALDI-MS-based identification of novel Fbxw7-associated proteins using crypts (upper panel) isolated from 3-week fbxw 7 fl/fl and fbxw 7 ΔG mice. Yellow circles in the lower panel denote potential Fbxw7-associated proteins. a Right, WB analysis (upper panels), and RT-PCR analysis (lower panels) of fbxw 7 fl/fl vs. fbxw 7 ΔG derived crypts and intestinal proteins and mRNA expression for ZEB2 and β-actin control. Experiments were performed on at least three independent occasions. b Left, schematic representation of the modified yeast two-hybrid reverse Ras Recruitment Screening (rRRS) system identifying proteins interacting with Fbxw7 in a GSK-3β phosphorylation-dependent manner. GSK-3β under the control of the methionine-regulated MET 3 promoter induces phosphorylation of encoded myristoylated proteins through a cDNA library plus positive control expressing FLAG-β-catenin (B—Middle) which only rescued the growth of cdc25–2 mutant yeast by Fbxw7-associated protein(s), if they interact with RasV12-FBXW7ΔF (i.e. human FBXW7α isoform mutant lacking F-box domain; therefore, interaction with Skp1 is lost and degradation of SCF Fbxw7 substrates will not occur in yeast) used as a bait at the restrictive temperature 37 °C, in a methionine-dependent manner. In the FBXW7ΔF mutant, both the N-terminal F-box and Dim-domains are deleted to avoid any interactions with SKP1 and other FBXW7 isoform-associated proteins. Thus, cdc25–2 mutant yeasts can grow only at 37 °C, when a phosphorylation-dependent interaction between a protein target and RasV12-FBXW7ΔF takes place. The FBXW7ΔF(bait)-dependent growth of these clones was further analysed on galactose-containing medium at 37 °C (B—Right). Red circles show the GSK-3β-phosphorylation-dependent interactor, including the Zeb2-clone, green circles show the phosphorylation/non-phosphorylation-dependent interactor and blue circles show the revertant clones (B—Right). c Left, subcellular localisation of GFP-fused human ZEB2 in the absence (top; nuclear) and presence (bottom; nuclear spots indicative of protein degradation) of GSK-3β in HCT116 CRC cells. ( c— Middle and c— Right) WB analysis of total ZEB2 protein level following the inhibition of GSK-3β (e.g. WS119 or LiCl treatment, and siRNA against GSK-3β) and of UPS pathways (MG132) in SW620 CRC cells. d Direct binding and ubiquitin-dependent degradation of ZEB2 by FBXW7. Co-immunoprecipitation (IP) of ZEB2 upon pull-down of FBXW7 in HEK-293T cells (Left); co-IP of FBXW7 upon pull-down of ZEB2 using the TNT-coupled reticulocyte lysate (Middle), and ubiquitination assays with HA-tagged ubiquitin- (HA-Ub) expressing construct in HEK-293T cells (Right). The asterisk indicates a nonspecific band(s). e Co-IP of endogenous ZEB2 upon pull-down of FBXW7 in HCT116 cells with FBXW 7 deletion. f ZEB2 pulse-chase stability assays with 15 µg/ml cycloheximide (CHX) in HCT116 cells with or without FBXW 7 deletion

    Article Snippet: We then conducted a similar cDNA array, where fbxw 7ΔG organoids cocultured with IMFfl/fl and IMFΔG fibroblasts, respectively, at day 1 (EpΔG IMFfl/fl -derived organoids vs. EpΔG IMF∆G -derived organoids).

    Techniques: Two-Dimensional Gel Electrophoresis, Mass Spectrometry, Isolation, Mouse Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing, Modification, cDNA Library Assay, Positive Control, Mutagenesis, Clone Assay, Inhibition, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Construct, Pulse Chase