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  • 92
    ATCC bnl cl2
    Coupling of IgG to target neuroblastoma and NSC34 cells and to CD16 on effector cells. FACS histograms presenting a shift in binding of serum pools of ALS patients to human neuroblastoma cells in comparison to binding of healthy control (CON), inflammatory bowel disease and multiple sclerosis serum pools to neuroblastoma cells ( A ); FACS histogram presenting shift in binding of purified IgG from serum pools of ALS patients to neuroblastoma cells relative to binding of purified IgG from healthy control (CON) ( B ); Dose-dependent coupling of purified ALS-IgG to human <t>PANC1,</t> <t>HeLa,</t> and neuroblastoma cells performed as described in A( C ); Mean fluorescent intensity (MFI) calculated relative to control sample containing cells and serum that was free of IgG. Dose-dependent coupling of ALS-IgG to mouse NSC34 cells was performed as described above ( D ); Secretion of IFNγ by enriched human peripheral NK cells in response to interactions with pools of ALS, inflammatory bowel disease patients, patients of multiple sclerosis, and healthy control (CON) sera ( E ); Secretion of IL-2 by BW-CD16 transfectants or BW cells in response to interactions with pools of ALS and healthy control sera ( F ), and in response to interactions with ALS-IgG and ALS IgG-depleted sera ( G ). Comparing the specificity of dose-dependent coupling of PNGase F-treated or untreated IgG of ALS patients and of the IgG of healthy volunteers, to CD16 ( H ). Data represent the mean ± SD of triplicate measurements from independent duplicate experiments. Pools of healthy and patient samples contained a mixture of four individual serum samples with similar glycan amounts represented in peaks 12 and 13. Statistical significance, *** p
    Bnl Cl2, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher ice centra cl 2 centrifuge
    The efficiency of miR-21 mimic or inhibitor transfection in hepatocytes. qRT-PCR analysis confirmed that miR-21 mimic ( A ) increased and miR-21 inhibitor ( B ) reduced miR-21 level in <t>BNL</t> <t>CL.2</t> cells (n=5). ** P
    Ice Centra Cl 2 Centrifuge, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    ATCC mouse hepatocyte line bnl cl2
    AK054386 functions as a ceRNA and interacts with miR-199. Subcellular localization of AK054386 by qRT-PCR of the subcellular fractionations of <t>BNL-CL2</t> cells. The in situ expression of AK054386 was analyzed by FISH using a FITC-AK054386 RNA probe or a FITC-scrambled sequence RNA probe (green), and the nuclear DNA was counterstained with DAPI (blue). (b) The relative levels of AK054386 were analyzed by qRT-PCR after miR-199 overexpression or inhibition. Data are shown as the mean ± S.D. of 6 independent experiments. ANOVA, ∗∗ P
    Mouse Hepatocyte Line Bnl Cl2, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    SCIENION scireader cl2
    AK054386 functions as a ceRNA and interacts with miR-199. Subcellular localization of AK054386 by qRT-PCR of the subcellular fractionations of <t>BNL-CL2</t> cells. The in situ expression of AK054386 was analyzed by FISH using a FITC-AK054386 RNA probe or a FITC-scrambled sequence RNA probe (green), and the nuclear DNA was counterstained with DAPI (blue). (b) The relative levels of AK054386 were analyzed by qRT-PCR after miR-199 overexpression or inhibition. Data are shown as the mean ± S.D. of 6 independent experiments. ANOVA, ∗∗ P
    Scireader Cl2, supplied by SCIENION, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Coupling of IgG to target neuroblastoma and NSC34 cells and to CD16 on effector cells. FACS histograms presenting a shift in binding of serum pools of ALS patients to human neuroblastoma cells in comparison to binding of healthy control (CON), inflammatory bowel disease and multiple sclerosis serum pools to neuroblastoma cells ( A ); FACS histogram presenting shift in binding of purified IgG from serum pools of ALS patients to neuroblastoma cells relative to binding of purified IgG from healthy control (CON) ( B ); Dose-dependent coupling of purified ALS-IgG to human PANC1, HeLa, and neuroblastoma cells performed as described in A( C ); Mean fluorescent intensity (MFI) calculated relative to control sample containing cells and serum that was free of IgG. Dose-dependent coupling of ALS-IgG to mouse NSC34 cells was performed as described above ( D ); Secretion of IFNγ by enriched human peripheral NK cells in response to interactions with pools of ALS, inflammatory bowel disease patients, patients of multiple sclerosis, and healthy control (CON) sera ( E ); Secretion of IL-2 by BW-CD16 transfectants or BW cells in response to interactions with pools of ALS and healthy control sera ( F ), and in response to interactions with ALS-IgG and ALS IgG-depleted sera ( G ). Comparing the specificity of dose-dependent coupling of PNGase F-treated or untreated IgG of ALS patients and of the IgG of healthy volunteers, to CD16 ( H ). Data represent the mean ± SD of triplicate measurements from independent duplicate experiments. Pools of healthy and patient samples contained a mixture of four individual serum samples with similar glycan amounts represented in peaks 12 and 13. Statistical significance, *** p

    Journal: PLoS ONE

    Article Title: Glycans in Sera of Amyotrophic Lateral Sclerosis Patients and Their Role in Killing Neuronal Cells

    doi: 10.1371/journal.pone.0035772

    Figure Lengend Snippet: Coupling of IgG to target neuroblastoma and NSC34 cells and to CD16 on effector cells. FACS histograms presenting a shift in binding of serum pools of ALS patients to human neuroblastoma cells in comparison to binding of healthy control (CON), inflammatory bowel disease and multiple sclerosis serum pools to neuroblastoma cells ( A ); FACS histogram presenting shift in binding of purified IgG from serum pools of ALS patients to neuroblastoma cells relative to binding of purified IgG from healthy control (CON) ( B ); Dose-dependent coupling of purified ALS-IgG to human PANC1, HeLa, and neuroblastoma cells performed as described in A( C ); Mean fluorescent intensity (MFI) calculated relative to control sample containing cells and serum that was free of IgG. Dose-dependent coupling of ALS-IgG to mouse NSC34 cells was performed as described above ( D ); Secretion of IFNγ by enriched human peripheral NK cells in response to interactions with pools of ALS, inflammatory bowel disease patients, patients of multiple sclerosis, and healthy control (CON) sera ( E ); Secretion of IL-2 by BW-CD16 transfectants or BW cells in response to interactions with pools of ALS and healthy control sera ( F ), and in response to interactions with ALS-IgG and ALS IgG-depleted sera ( G ). Comparing the specificity of dose-dependent coupling of PNGase F-treated or untreated IgG of ALS patients and of the IgG of healthy volunteers, to CD16 ( H ). Data represent the mean ± SD of triplicate measurements from independent duplicate experiments. Pools of healthy and patient samples contained a mixture of four individual serum samples with similar glycan amounts represented in peaks 12 and 13. Statistical significance, *** p

    Article Snippet: Cell cultures The human SHSy5y neuroblastoma (CRL2266, ATCC, Manassas, VA), HeLa (CL-2, ATCC) and PANC1 (CRL1469, ATCC) cell lines and the mouse neuroblastoma-spinal cord motoneuron hybrid cell line (NSC 34, a generous gift from Prof. D. Offen,) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS), 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine, in a humidified 5% CO2 atmosphere at 37°C.

    Techniques: FACS, Binding Assay, Purification

    The efficiency of miR-21 mimic or inhibitor transfection in hepatocytes. qRT-PCR analysis confirmed that miR-21 mimic ( A ) increased and miR-21 inhibitor ( B ) reduced miR-21 level in BNL CL.2 cells (n=5). ** P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-21 Contributes to Liver Regeneration by Targeting PTEN

    doi: 10.12659/MSM.896157

    Figure Lengend Snippet: The efficiency of miR-21 mimic or inhibitor transfection in hepatocytes. qRT-PCR analysis confirmed that miR-21 mimic ( A ) increased and miR-21 inhibitor ( B ) reduced miR-21 level in BNL CL.2 cells (n=5). ** P

    Article Snippet: BNL CL.2 cells were transfected with miR-21 mimic (50 nM), inhibitor (100 nM), or their negative controls for 48 h using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.

    Techniques: Transfection, Quantitative RT-PCR

    miR-21 regulation exhibits no effect on the cell size of hepatocytes. Dil (red) fluorescent staining showed that miR-21 mimic or inhibitor did not affect the size of BNL CL.2 cells in vitro . Nuclei were counterstained with DAPI (blue) (n=5). Scale bar=50 μm.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-21 Contributes to Liver Regeneration by Targeting PTEN

    doi: 10.12659/MSM.896157

    Figure Lengend Snippet: miR-21 regulation exhibits no effect on the cell size of hepatocytes. Dil (red) fluorescent staining showed that miR-21 mimic or inhibitor did not affect the size of BNL CL.2 cells in vitro . Nuclei were counterstained with DAPI (blue) (n=5). Scale bar=50 μm.

    Article Snippet: BNL CL.2 cells were transfected with miR-21 mimic (50 nM), inhibitor (100 nM), or their negative controls for 48 h using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.

    Techniques: Staining, In Vitro

    Silencing PTEN reverses the proliferation-suppressing effect of miR-21 inhibitor in hepatocytes. ( A ) qRT-PCR analysis verified that PTEN siRNA (si-01 or si-02) reduced PTEN expression at mRNA level in BNL CL.2 cells (n=5). ( B ) EdU (green) staining demonstrated that the presence of PTEN siRNA (si-01 or si-02) can abolish the reducing effect of miR-21 inhibitor on the proliferation of BNL CL.2 cells. Nuclei were counterstained with DAPI (blue) (n=5). Scale bar=50 μm. ** P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-21 Contributes to Liver Regeneration by Targeting PTEN

    doi: 10.12659/MSM.896157

    Figure Lengend Snippet: Silencing PTEN reverses the proliferation-suppressing effect of miR-21 inhibitor in hepatocytes. ( A ) qRT-PCR analysis verified that PTEN siRNA (si-01 or si-02) reduced PTEN expression at mRNA level in BNL CL.2 cells (n=5). ( B ) EdU (green) staining demonstrated that the presence of PTEN siRNA (si-01 or si-02) can abolish the reducing effect of miR-21 inhibitor on the proliferation of BNL CL.2 cells. Nuclei were counterstained with DAPI (blue) (n=5). Scale bar=50 μm. ** P

    Article Snippet: BNL CL.2 cells were transfected with miR-21 mimic (50 nM), inhibitor (100 nM), or their negative controls for 48 h using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.

    Techniques: Quantitative RT-PCR, Expressing, Staining

    PTEN expression is inversely correlated with miR-21 both in vitro and in vivo . qRT-PCR ( A ) and Western blot ( B ) analysis showed that miR-21 negatively regulated PTEN expression at both mRNA and protein levels in BNL CL.2 cells in vitro (n=5 and n=3, respectively). A reduction of PTEN expression was also found at mRNA ( C ) and protein ( D ) levels in mouse livers at PH-48 h versus those at PH-0 h (n=5 and n=5, respectively). * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-21 Contributes to Liver Regeneration by Targeting PTEN

    doi: 10.12659/MSM.896157

    Figure Lengend Snippet: PTEN expression is inversely correlated with miR-21 both in vitro and in vivo . qRT-PCR ( A ) and Western blot ( B ) analysis showed that miR-21 negatively regulated PTEN expression at both mRNA and protein levels in BNL CL.2 cells in vitro (n=5 and n=3, respectively). A reduction of PTEN expression was also found at mRNA ( C ) and protein ( D ) levels in mouse livers at PH-48 h versus those at PH-0 h (n=5 and n=5, respectively). * P

    Article Snippet: BNL CL.2 cells were transfected with miR-21 mimic (50 nM), inhibitor (100 nM), or their negative controls for 48 h using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, In Vitro, In Vivo, Quantitative RT-PCR, Western Blot

    miR-21 accelerates the proliferation and G1/S phase transition of the cell cycle in hepatocytes. CCK-8 cell proliferation assay ( A ) and EdU (green) incorporation assay ( B ) demonstrated that miR-21 overexpression promoted and miR-21 inhibition reduced the proliferation of BNL CL.2 cells in vitro . Nuclei were counterstained with DAPI (blue) (n=10 and n=5, respectively). Scale bar=50 μm. ( C ) Flow cytometry showed that miR-21 mimic induced a G1/S phase transition of the cell cycle in BNL CL.2 cells, and miR-21 inhibitor resulted in G1 phase arrest (n=5). * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-21 Contributes to Liver Regeneration by Targeting PTEN

    doi: 10.12659/MSM.896157

    Figure Lengend Snippet: miR-21 accelerates the proliferation and G1/S phase transition of the cell cycle in hepatocytes. CCK-8 cell proliferation assay ( A ) and EdU (green) incorporation assay ( B ) demonstrated that miR-21 overexpression promoted and miR-21 inhibition reduced the proliferation of BNL CL.2 cells in vitro . Nuclei were counterstained with DAPI (blue) (n=10 and n=5, respectively). Scale bar=50 μm. ( C ) Flow cytometry showed that miR-21 mimic induced a G1/S phase transition of the cell cycle in BNL CL.2 cells, and miR-21 inhibitor resulted in G1 phase arrest (n=5). * P

    Article Snippet: BNL CL.2 cells were transfected with miR-21 mimic (50 nM), inhibitor (100 nM), or their negative controls for 48 h using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.

    Techniques: Sublimation, CCK-8 Assay, Proliferation Assay, Over Expression, Inhibition, In Vitro, Flow Cytometry, Cytometry

    Analysis of CCDC80 and CD14 protein content in SAT and VAT depots from lean and obese patients and CCDC80 protein in human plasma. (A) Western blot analysis of SAT and VAT extracts (20 μg protein). Membranes were hybridized with antibodies against CCDC80, CD14 and FAA. A representative blot is shown. Bands were quantified and the ratio of intensities between CCDC80 or CD14 and control FAA was calculated. (B) Relative CCDC80 protein content. Data are expressed in arbitrary units and are means ± SEM from 5 samples. # P

    Journal: Molecular Medicine

    Article Title: Adipose Tissue and serum CCDC80 in Obesity and its Association with related Metabolic Disease

    doi: 10.2119/molmed.2017.00067

    Figure Lengend Snippet: Analysis of CCDC80 and CD14 protein content in SAT and VAT depots from lean and obese patients and CCDC80 protein in human plasma. (A) Western blot analysis of SAT and VAT extracts (20 μg protein). Membranes were hybridized with antibodies against CCDC80, CD14 and FAA. A representative blot is shown. Bands were quantified and the ratio of intensities between CCDC80 or CD14 and control FAA was calculated. (B) Relative CCDC80 protein content. Data are expressed in arbitrary units and are means ± SEM from 5 samples. # P

    Article Snippet: Probes for 18S rRNA and CCDC80 were purchased from Applied Biosystems.

    Techniques: Western Blot

    CCDC80 protein expression in human SGBS adipocytes during differentiation. (A) Western blot analysis of CCDC80 protein content in SGBS cell extracts of confluent preadipocytes (d 0), differentiating adipocytes (d 1–8) and mature adipocytes (d 14). (B) Confluent preadipocytes incubated with or without 25 nM dexamethasone, 0.5 mM IBMX and 2 μM rosiglitazone for 16 h. In A and B, a representative image is shown. Ratios of intensity of CCDC80 bands compared with intensity of GAPDH are expressed as a percentage of (untreated) confluent preadipocyte values and are means ± standard error of the mean from two experiments performed in triplicate. * P

    Journal: Molecular Medicine

    Article Title: Adipose Tissue and serum CCDC80 in Obesity and its Association with related Metabolic Disease

    doi: 10.2119/molmed.2017.00067

    Figure Lengend Snippet: CCDC80 protein expression in human SGBS adipocytes during differentiation. (A) Western blot analysis of CCDC80 protein content in SGBS cell extracts of confluent preadipocytes (d 0), differentiating adipocytes (d 1–8) and mature adipocytes (d 14). (B) Confluent preadipocytes incubated with or without 25 nM dexamethasone, 0.5 mM IBMX and 2 μM rosiglitazone for 16 h. In A and B, a representative image is shown. Ratios of intensity of CCDC80 bands compared with intensity of GAPDH are expressed as a percentage of (untreated) confluent preadipocyte values and are means ± standard error of the mean from two experiments performed in triplicate. * P

    Article Snippet: Probes for 18S rRNA and CCDC80 were purchased from Applied Biosystems.

    Techniques: Expressing, Western Blot, Incubation

    The increase in PI-positive cells among DNase II-knockdown cells treated with ABT-737 is attenuated by the inhibition of RIP1 or IFN-β but not by a pan-caspase inhibitor. At 72 h after transfection with Dnase2a siRNA or control siRNA, BNL CL.2 cells were treated with 0 or 4 μM ABT-737 for the indicated time. a – c BNL CL.2 cells were pretreated with or without ODN2088 1 h before ABT-737 treatment. Ifnb1 mRNA expression levels after 6 h of ABT-737 treatment ( a ); n = 4 per group. Representative fluorescence micrographs after 3 h of ABT-737 treatment and their quantitative analysis ( b ); n = 6 per group. Scale bar 100 μm. Cell viability after 6 h of ABT-737 treatment, assessed with a WST assay, was expressed relative to the mean of the ABT-737 0 µM group. ( c ); n = 4 per group. BNL CL2 cells were co-transfected with Infb1 siRNA ( d , f ) or Ripk1 siRNA ( e , f ). BNL CL.2 cells were pretreated with Necrostatin-1 30 min before ABT-737 treatment ( e , f ). Cell viability after 6 h of ABT-737 treatment, assessed with a WST assay, was expressed relative to the mean of the ABT-737 0 µM group. ( d , e ); n = 3 - 4 per group. Representative fluorescence micrographs after 3 h of ABT-737 treatment and their quantitative analysis ( f ); n = 6 per group. Scale bar 100 μm. ( g, h ) BNL CL.2 cells were pretreated with Necrostatin-1 and/or ZVAD-FMK 30 min before ABT-737 treatment for 6 h. BNL CL.2 cells were pretreated with Necrostatin-1 and/or ZVAD-FMK 30 min before ABT-737 treatment for 6 h. The cell viability was expressed relative to the mean of the ABT-737 0 µM group ( g ). Live cell imaging via fluorescence microscopy of BNL CL.2 cells stained with PI ( h ); n = 6 per group. Quantitative analysis. In each analysis, the ABT-737 0 µM group was used as a reference ( a–h ). N.S. not significant. Data are shown as the mean + SD. * P

    Journal: Cell Death and Differentiation

    Article Title: DNase II activated by the mitochondrial apoptotic pathway regulates RIP1-dependent non-apoptotic hepatocyte death via the TLR9/IFN-β signaling pathway

    doi: 10.1038/s41418-018-0131-6

    Figure Lengend Snippet: The increase in PI-positive cells among DNase II-knockdown cells treated with ABT-737 is attenuated by the inhibition of RIP1 or IFN-β but not by a pan-caspase inhibitor. At 72 h after transfection with Dnase2a siRNA or control siRNA, BNL CL.2 cells were treated with 0 or 4 μM ABT-737 for the indicated time. a – c BNL CL.2 cells were pretreated with or without ODN2088 1 h before ABT-737 treatment. Ifnb1 mRNA expression levels after 6 h of ABT-737 treatment ( a ); n = 4 per group. Representative fluorescence micrographs after 3 h of ABT-737 treatment and their quantitative analysis ( b ); n = 6 per group. Scale bar 100 μm. Cell viability after 6 h of ABT-737 treatment, assessed with a WST assay, was expressed relative to the mean of the ABT-737 0 µM group. ( c ); n = 4 per group. BNL CL2 cells were co-transfected with Infb1 siRNA ( d , f ) or Ripk1 siRNA ( e , f ). BNL CL.2 cells were pretreated with Necrostatin-1 30 min before ABT-737 treatment ( e , f ). Cell viability after 6 h of ABT-737 treatment, assessed with a WST assay, was expressed relative to the mean of the ABT-737 0 µM group. ( d , e ); n = 3 - 4 per group. Representative fluorescence micrographs after 3 h of ABT-737 treatment and their quantitative analysis ( f ); n = 6 per group. Scale bar 100 μm. ( g, h ) BNL CL.2 cells were pretreated with Necrostatin-1 and/or ZVAD-FMK 30 min before ABT-737 treatment for 6 h. BNL CL.2 cells were pretreated with Necrostatin-1 and/or ZVAD-FMK 30 min before ABT-737 treatment for 6 h. The cell viability was expressed relative to the mean of the ABT-737 0 µM group ( g ). Live cell imaging via fluorescence microscopy of BNL CL.2 cells stained with PI ( h ); n = 6 per group. Quantitative analysis. In each analysis, the ABT-737 0 µM group was used as a reference ( a–h ). N.S. not significant. Data are shown as the mean + SD. * P

    Article Snippet: CL2 cells were transfected with 5 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol.

    Techniques: Inhibition, Transfection, Expressing, Fluorescence, WST Assay, Live Cell Imaging, Microscopy, Staining

    AK054386 functions as a ceRNA and interacts with miR-199. Subcellular localization of AK054386 by qRT-PCR of the subcellular fractionations of BNL-CL2 cells. The in situ expression of AK054386 was analyzed by FISH using a FITC-AK054386 RNA probe or a FITC-scrambled sequence RNA probe (green), and the nuclear DNA was counterstained with DAPI (blue). (b) The relative levels of AK054386 were analyzed by qRT-PCR after miR-199 overexpression or inhibition. Data are shown as the mean ± S.D. of 6 independent experiments. ANOVA, ∗∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: lncRNA AK054386 Functions as a ceRNA to Sequester miR-199 and Induce Sustained Endoplasmic Reticulum Stress in Hepatic Reperfusion Injury

    doi: 10.1155/2019/8189079

    Figure Lengend Snippet: AK054386 functions as a ceRNA and interacts with miR-199. Subcellular localization of AK054386 by qRT-PCR of the subcellular fractionations of BNL-CL2 cells. The in situ expression of AK054386 was analyzed by FISH using a FITC-AK054386 RNA probe or a FITC-scrambled sequence RNA probe (green), and the nuclear DNA was counterstained with DAPI (blue). (b) The relative levels of AK054386 were analyzed by qRT-PCR after miR-199 overexpression or inhibition. Data are shown as the mean ± S.D. of 6 independent experiments. ANOVA, ∗∗ P

    Article Snippet: The mouse hepatocyte line BNL-CL2 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained using the provided guidelines.

    Techniques: Quantitative RT-PCR, In Situ, Expressing, Fluorescence In Situ Hybridization, Sequencing, Over Expression, Inhibition

    AK054386 affects hepatic injury from ischemia and reperfusion in vivo and in vitro . (a) AK054386 impacts apoptosis in the BNL-CL2 IRI cell model. Apoptosis rates were assayed by flow cytometry. Representative data from three independent experiments are shown. (b) BNL-CL2 cell death levels were assayed by LDH release. Data are shown as the mean ± S.D. of three independent experiments. (c, d) The protein levels of ER-related genes were analyzed by Western blot. (e) The relative RNA expression levels of inflammatory cytokines (IL-6 and TNF- α ) of liver tissues from mouse hepatic IRI models after lentivirus infection. 5 mice were analyzed in each group. The RNA levels were measured by qRT-PCR and normalized to GAPDH. Data are shown as the mean ± S.D. of three independent experiments. ANOVA, ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: lncRNA AK054386 Functions as a ceRNA to Sequester miR-199 and Induce Sustained Endoplasmic Reticulum Stress in Hepatic Reperfusion Injury

    doi: 10.1155/2019/8189079

    Figure Lengend Snippet: AK054386 affects hepatic injury from ischemia and reperfusion in vivo and in vitro . (a) AK054386 impacts apoptosis in the BNL-CL2 IRI cell model. Apoptosis rates were assayed by flow cytometry. Representative data from three independent experiments are shown. (b) BNL-CL2 cell death levels were assayed by LDH release. Data are shown as the mean ± S.D. of three independent experiments. (c, d) The protein levels of ER-related genes were analyzed by Western blot. (e) The relative RNA expression levels of inflammatory cytokines (IL-6 and TNF- α ) of liver tissues from mouse hepatic IRI models after lentivirus infection. 5 mice were analyzed in each group. The RNA levels were measured by qRT-PCR and normalized to GAPDH. Data are shown as the mean ± S.D. of three independent experiments. ANOVA, ∗ P

    Article Snippet: The mouse hepatocyte line BNL-CL2 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained using the provided guidelines.

    Techniques: In Vivo, In Vitro, Flow Cytometry, Cytometry, Western Blot, RNA Expression, Infection, Mouse Assay, Quantitative RT-PCR

    Analyses of tumor-specific immune response in the LV-TAA-DC-immunized mice. (A) ELISPOT assays for immune cells releasing IFN-γ. Splenocytes harvested from the DC-injected mice were restimulated with medium alone, 1MEA7R hepatoma cells or BNL.CL2 normal hepatocytes. 1MEA7R or BNL.CL2 cells were cultured with splenocytes at 1:5 ratios for 24 h at 37 °C, and the IFN-γ-secreting cells (spots) were visualized and quantified. The column represents the average of counted spots per well which was normalized against background from three independent experiments with S.D. error bars. Representative spot-containing wells are shown below each corresponding column. (B) FATAL assay for anti-cancer cytotoxicity. The splenocytes were restimulated for 5 days with irradiated 1MEA7R tumor cells. The FATAL assays, as described in Section 2, were performed to evaluate the cytotoxic activity against 1MEA7R and BNL.CL2 cells. The representative of three experiments is shown. (C) In vitro antibody blocking experiments. The pooled splenocytes (1 × 10 6 ) were pre-incubated with blocking antibodies for 1 h at 37 °C as indicated at bottom of the graph, and then stimulated with 1MEA7R (2 × 10 5 ) for 16 h and treated with Brefeldin A. Intracellular IFN-γ was stained and analyzed by FACS. The experiments were repeated three times and presented with S.D. error bars. Student’s t -test was performed with P

    Journal: Vaccine

    Article Title: An effective cancer vaccine modality: Lentiviral modification of dendritic cells expressing multiple cancer-specific antigens

    doi: 10.1016/j.vaccine.2006.02.025

    Figure Lengend Snippet: Analyses of tumor-specific immune response in the LV-TAA-DC-immunized mice. (A) ELISPOT assays for immune cells releasing IFN-γ. Splenocytes harvested from the DC-injected mice were restimulated with medium alone, 1MEA7R hepatoma cells or BNL.CL2 normal hepatocytes. 1MEA7R or BNL.CL2 cells were cultured with splenocytes at 1:5 ratios for 24 h at 37 °C, and the IFN-γ-secreting cells (spots) were visualized and quantified. The column represents the average of counted spots per well which was normalized against background from three independent experiments with S.D. error bars. Representative spot-containing wells are shown below each corresponding column. (B) FATAL assay for anti-cancer cytotoxicity. The splenocytes were restimulated for 5 days with irradiated 1MEA7R tumor cells. The FATAL assays, as described in Section 2, were performed to evaluate the cytotoxic activity against 1MEA7R and BNL.CL2 cells. The representative of three experiments is shown. (C) In vitro antibody blocking experiments. The pooled splenocytes (1 × 10 6 ) were pre-incubated with blocking antibodies for 1 h at 37 °C as indicated at bottom of the graph, and then stimulated with 1MEA7R (2 × 10 5 ) for 16 h and treated with Brefeldin A. Intracellular IFN-γ was stained and analyzed by FACS. The experiments were repeated three times and presented with S.D. error bars. Student’s t -test was performed with P

    Article Snippet: The 1MEA7R mouse liver hepatoma cell line and its normal counterpart BNL.CL2 cell line, derived from Balb/c mice, were obtained from ATCC (Manassas, VA) and maintained in tissue culture at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Gaithersburg, MD) containing 10% heat-inactivated fetal bovine serum and 100 U/ml of penicillin-streptomycin (Gibco BRL).

    Techniques: Mouse Assay, Enzyme-linked Immunospot, Injection, Cell Culture, Irradiation, Activity Assay, In Vitro, Blocking Assay, Incubation, Staining, FACS

    Analysis of in vivo immunogenicity of LV-TAA-DCs. (A) Expression of TAAs in LV-transduced BNL.CL2 cells. Normal hepatocytes (BNL.CL2) were transduced with LV-Sca-2, LV-GP38 or LV-RABP1. The level of transgene expression was determined by RT-PCR using total cellular RNA and the TAA gene-specific primers as depicted. N, negative control (BNL.CL2); P, positive control (1MEA7R); T, transduced BNL.CL2. RT-PCR of GAPDH RNA was included as control. (B) and (C) Analysis of cell-mediated immue response by intracellular cytokine staining. Balb/c mice were injected with the LV-TAA-DCs twice in weekly intervals and the splenocytes (1 × 10 6 ) of five mice were pooled and stimulated for 16 h with 2 × 10 5 BNL.CL2, BNL.CL2 transduced with a LV-TAA or 1MEA7R cells. The intracellular IFN-γ or TNF-α was stained using specific Abs; left panel represents CD8 T cells and IFN-γ staining, and right panel CD4 T cells and TNFa staining. Error bars depict standard deviations (S.D.) of three independent experiments.

    Journal: Vaccine

    Article Title: An effective cancer vaccine modality: Lentiviral modification of dendritic cells expressing multiple cancer-specific antigens

    doi: 10.1016/j.vaccine.2006.02.025

    Figure Lengend Snippet: Analysis of in vivo immunogenicity of LV-TAA-DCs. (A) Expression of TAAs in LV-transduced BNL.CL2 cells. Normal hepatocytes (BNL.CL2) were transduced with LV-Sca-2, LV-GP38 or LV-RABP1. The level of transgene expression was determined by RT-PCR using total cellular RNA and the TAA gene-specific primers as depicted. N, negative control (BNL.CL2); P, positive control (1MEA7R); T, transduced BNL.CL2. RT-PCR of GAPDH RNA was included as control. (B) and (C) Analysis of cell-mediated immue response by intracellular cytokine staining. Balb/c mice were injected with the LV-TAA-DCs twice in weekly intervals and the splenocytes (1 × 10 6 ) of five mice were pooled and stimulated for 16 h with 2 × 10 5 BNL.CL2, BNL.CL2 transduced with a LV-TAA or 1MEA7R cells. The intracellular IFN-γ or TNF-α was stained using specific Abs; left panel represents CD8 T cells and IFN-γ staining, and right panel CD4 T cells and TNFa staining. Error bars depict standard deviations (S.D.) of three independent experiments.

    Article Snippet: The 1MEA7R mouse liver hepatoma cell line and its normal counterpart BNL.CL2 cell line, derived from Balb/c mice, were obtained from ATCC (Manassas, VA) and maintained in tissue culture at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Gaithersburg, MD) containing 10% heat-inactivated fetal bovine serum and 100 U/ml of penicillin-streptomycin (Gibco BRL).

    Techniques: In Vivo, Expressing, Transduction, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Staining, Mouse Assay, Injection

    Direct regulation of CCL2 by FoxM1 in hepatocytes. ( A ) Quantification of gene expression of FoxM1 ( left ) and Ccl2 ( right ) in FoxM1 siRNA-transfected BNL-CL2 cells (n = 3 per group). ( B ) Quantification of CCL2 levels in supernatant of FoxM1 siRNA-transfected BNL-CL2 cells at indicated time points after transfection (n = 3 per group). ( C ) Quantification of gene expression of FoxM1 ( left ) and Ccl2 ( right ) in FoxM1 siRNA-transfected AML12 cells (n = 3 per group). ( D ) Quantification of CCL2 levels in supernatant of FoxM1 siRNA-transfected AML12 cells at indicated time points after transfection (n = 3 per group). ( E ) Schematic illustration of luciferase (Luc) reporter constructs containing −2468/+67 bp murine Ccl2 promoter and its deletion mutants (−1401/+67 bp and −1136/+67 bp) and quantification of transcriptional activities induced by cotransfection of T7-FoxM1 expression vector (T7-FoxM1) compared with CMV-empty vector (Mock) (n = 3 per group). A 6xCDX2 promoter LUC construct was used as a positive control. ( F ) Quantification of chromatin immunoprecipitation assay to show direct binding of FoxM1 protein to murine Ccl2 promoter DNA using 2 independent antibodies against FoxM1 (K19 and C20) compared with immunoglobulin G control (n = 3 per group). Data are expressed as individual values and mean ± standard deviation; ** P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Forkhead Box M1 Transcription Factor Drives Liver Inflammation Linking to Hepatocarcinogenesis in Mice

    doi: 10.1016/j.jcmgh.2019.10.008

    Figure Lengend Snippet: Direct regulation of CCL2 by FoxM1 in hepatocytes. ( A ) Quantification of gene expression of FoxM1 ( left ) and Ccl2 ( right ) in FoxM1 siRNA-transfected BNL-CL2 cells (n = 3 per group). ( B ) Quantification of CCL2 levels in supernatant of FoxM1 siRNA-transfected BNL-CL2 cells at indicated time points after transfection (n = 3 per group). ( C ) Quantification of gene expression of FoxM1 ( left ) and Ccl2 ( right ) in FoxM1 siRNA-transfected AML12 cells (n = 3 per group). ( D ) Quantification of CCL2 levels in supernatant of FoxM1 siRNA-transfected AML12 cells at indicated time points after transfection (n = 3 per group). ( E ) Schematic illustration of luciferase (Luc) reporter constructs containing −2468/+67 bp murine Ccl2 promoter and its deletion mutants (−1401/+67 bp and −1136/+67 bp) and quantification of transcriptional activities induced by cotransfection of T7-FoxM1 expression vector (T7-FoxM1) compared with CMV-empty vector (Mock) (n = 3 per group). A 6xCDX2 promoter LUC construct was used as a positive control. ( F ) Quantification of chromatin immunoprecipitation assay to show direct binding of FoxM1 protein to murine Ccl2 promoter DNA using 2 independent antibodies against FoxM1 (K19 and C20) compared with immunoglobulin G control (n = 3 per group). Data are expressed as individual values and mean ± standard deviation; ** P

    Article Snippet: The murine nontransformed hepatocyte cell lines BNL-CL2 and AML12 were obtained from the American Type Culture Collection (Manassas, VA).

    Techniques: Expressing, Transfection, Luciferase, Construct, Cotransfection, Plasmid Preparation, Positive Control, Chromatin Immunoprecipitation, Binding Assay, Standard Deviation

    Evi1 regulates a cluster of lncRNAs expression as a transcription factor ( A ) The relative expression of four predicted Evi1 target lncRNAs (AK016494, AK015487, AK021106, and AK044545) in mouse BNL CL.2 cells co-transfected with pEGFP-HBx (HBx) and Evi1-specific siRNA (siEvi1-1 or siEvi1-2) by qRT-PCR. ( B ) EMSA shows that Evi1 protein can bind to the promoter of lncRNA-AK015487. The specificity of binding was examined by competition with the unlabeled probes. ( C ) The binding activity of Evi1 at the promoters of AK016494, AK015487, AK021106, and AK044545 in pEGFP-HBx transfected BNL CL.2 cells was evaluated with ChIP assays. Rabbit total IgG was used as a negative control. For PCR assays, non-template control (NTC) was also used to evaluate the primers. ( D ) Dual luciferase assay of BNL CL.2 cells cotransfected with the reporter vectors containing wild-type (pGL3-lncR) or mutated (pGL3-lncRm) predicted Evi1-binding sites of lncRNA AK015487 promoter. A mock vector (pGL3-Basic) was used as a control. * P

    Journal: Oncotarget

    Article Title: EVI1 promotes cell proliferation in HBx-induced hepatocarcinogenesis as a critical transcription factor regulating lncRNAs

    doi: 10.18632/oncotarget.7993

    Figure Lengend Snippet: Evi1 regulates a cluster of lncRNAs expression as a transcription factor ( A ) The relative expression of four predicted Evi1 target lncRNAs (AK016494, AK015487, AK021106, and AK044545) in mouse BNL CL.2 cells co-transfected with pEGFP-HBx (HBx) and Evi1-specific siRNA (siEvi1-1 or siEvi1-2) by qRT-PCR. ( B ) EMSA shows that Evi1 protein can bind to the promoter of lncRNA-AK015487. The specificity of binding was examined by competition with the unlabeled probes. ( C ) The binding activity of Evi1 at the promoters of AK016494, AK015487, AK021106, and AK044545 in pEGFP-HBx transfected BNL CL.2 cells was evaluated with ChIP assays. Rabbit total IgG was used as a negative control. For PCR assays, non-template control (NTC) was also used to evaluate the primers. ( D ) Dual luciferase assay of BNL CL.2 cells cotransfected with the reporter vectors containing wild-type (pGL3-lncR) or mutated (pGL3-lncRm) predicted Evi1-binding sites of lncRNA AK015487 promoter. A mock vector (pGL3-Basic) was used as a control. * P

    Article Snippet: Cell culture and transfection The liver cell lines HepG2, HepG2.2.15, Hep3B, SMMC-7721 and BNL CL.2, Hepa1-6 were obtained from the American Type Culture Collection.

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Luciferase, Plasmid Preparation

    Enforced HBx expression up-regulates EVI1 in both mouse and human liver cells ( A ) Mouse Evi1 mRNA expression after the transfection of pEGFP-HBx or the control pEGFP plasmid in mouse BNL CL.2 and Hepa1-6 cells. ( B ) Human EVI1 mRNA expression after the transfection of pEGFP-HBx or the control pEGFP plasmid in human HepG2 and SMMC-7721 cells. ( C ) Western blot analysis of human EVI1 protein expression in HepG2 and SMMC-7721 cells transfected with pEGFP-HBx or respective controls, and the no treatment control cells (mock). ( D ) Human EVI1 mRNA levels after the transfection of HBx-specific siRNA (siHBx-1 or siHBx-2) or HBs-specific siRNA or HBc-specific siRNA or control siRNA (siNC) in Hep3B and HepG2.2.15 cells. Data are shown as means and standard deviations from at least three independent experiments. * P

    Journal: Oncotarget

    Article Title: EVI1 promotes cell proliferation in HBx-induced hepatocarcinogenesis as a critical transcription factor regulating lncRNAs

    doi: 10.18632/oncotarget.7993

    Figure Lengend Snippet: Enforced HBx expression up-regulates EVI1 in both mouse and human liver cells ( A ) Mouse Evi1 mRNA expression after the transfection of pEGFP-HBx or the control pEGFP plasmid in mouse BNL CL.2 and Hepa1-6 cells. ( B ) Human EVI1 mRNA expression after the transfection of pEGFP-HBx or the control pEGFP plasmid in human HepG2 and SMMC-7721 cells. ( C ) Western blot analysis of human EVI1 protein expression in HepG2 and SMMC-7721 cells transfected with pEGFP-HBx or respective controls, and the no treatment control cells (mock). ( D ) Human EVI1 mRNA levels after the transfection of HBx-specific siRNA (siHBx-1 or siHBx-2) or HBs-specific siRNA or HBc-specific siRNA or control siRNA (siNC) in Hep3B and HepG2.2.15 cells. Data are shown as means and standard deviations from at least three independent experiments. * P

    Article Snippet: Cell culture and transfection The liver cell lines HepG2, HepG2.2.15, Hep3B, SMMC-7721 and BNL CL.2, Hepa1-6 were obtained from the American Type Culture Collection.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

    Intracellular bacterial replication is severely compromised when genes encoding iglC and pdpA are deleted. 24 h and 48 h gentamicin protection assays were performed on liver BNL CL.2 cells infected with wild-type F. novicida , deletion mutants (Δ iglC and Δ pdpA ), and their respective complement strains (Δ iglC :: iglC and Δ pdpA :: pdpA ). Samples were then treated with gentamicin starting from 22 h post-inoculation until the experimental endpoint. After host cells were lysed, the released bacteria were diluted and plated for CFU enumeration. Error bars, S.D. (n = 4).

    Journal: PLoS ONE

    Article Title: IglC and PdpA Are Important for Promoting Francisella Invasion and Intracellular Growth in Epithelial Cells

    doi: 10.1371/journal.pone.0104881

    Figure Lengend Snippet: Intracellular bacterial replication is severely compromised when genes encoding iglC and pdpA are deleted. 24 h and 48 h gentamicin protection assays were performed on liver BNL CL.2 cells infected with wild-type F. novicida , deletion mutants (Δ iglC and Δ pdpA ), and their respective complement strains (Δ iglC :: iglC and Δ pdpA :: pdpA ). Samples were then treated with gentamicin starting from 22 h post-inoculation until the experimental endpoint. After host cells were lysed, the released bacteria were diluted and plated for CFU enumeration. Error bars, S.D. (n = 4).

    Article Snippet: We chose human lung A549 cells over BNL CL.2 cells for this assay primarily because of the low infections rates BNL CL.2 cells show at 4 h when infected with F. tularensis LVS.

    Techniques: Infection

    Proportion of F. novicida associated with LAMP1 during murine hepatocyte infections. (A) wild-type F. novicida , deletion mutants (Δ pdpA , Δ iglC ) and complements strains (Δ pdpA :: pdpA , Δ iglC :: iglC ) invaded BNL CL.2 cells for 3 h, after which extracellular bacteria were washed off and then killed with gentamicin (100 µg mL −1 ). Subsequently, samples were exposed to low gentamicin concentration until the experimental endpoint was reached. Image stacks were assembled and used to determine the frequency of LAMP1-associated bacteria. For time-points 4, 8 and 12 h, between 30 and 50 intracellular bacteria were counted. For the 24 h time-point, more than 50 intracellular bacteria were counted. Error bars, S.E.M. (n = 3).

    Journal: PLoS ONE

    Article Title: IglC and PdpA Are Important for Promoting Francisella Invasion and Intracellular Growth in Epithelial Cells

    doi: 10.1371/journal.pone.0104881

    Figure Lengend Snippet: Proportion of F. novicida associated with LAMP1 during murine hepatocyte infections. (A) wild-type F. novicida , deletion mutants (Δ pdpA , Δ iglC ) and complements strains (Δ pdpA :: pdpA , Δ iglC :: iglC ) invaded BNL CL.2 cells for 3 h, after which extracellular bacteria were washed off and then killed with gentamicin (100 µg mL −1 ). Subsequently, samples were exposed to low gentamicin concentration until the experimental endpoint was reached. Image stacks were assembled and used to determine the frequency of LAMP1-associated bacteria. For time-points 4, 8 and 12 h, between 30 and 50 intracellular bacteria were counted. For the 24 h time-point, more than 50 intracellular bacteria were counted. Error bars, S.E.M. (n = 3).

    Article Snippet: We chose human lung A549 cells over BNL CL.2 cells for this assay primarily because of the low infections rates BNL CL.2 cells show at 4 h when infected with F. tularensis LVS.

    Techniques: Concentration Assay

    Deletion of genes encoding IglC and PdpA perturb F. novicida invasion. Murine BNL CL.2 hepatocytes were infected with wild-type F. novicida , deletion mutants (Δ iglC and Δ pdpA ) as well as complement strains (Δ iglC :: iglC and Δ pdpA :: pdpA ) for 3 h. Subsequently, samples were washed and treated with gentamicin for 1 h. At 4 h PI, lysates were plated onto agar-containing media and bacterial colonies were enumerated the following day. Error bars, S.E.M. (n = 4).

    Journal: PLoS ONE

    Article Title: IglC and PdpA Are Important for Promoting Francisella Invasion and Intracellular Growth in Epithelial Cells

    doi: 10.1371/journal.pone.0104881

    Figure Lengend Snippet: Deletion of genes encoding IglC and PdpA perturb F. novicida invasion. Murine BNL CL.2 hepatocytes were infected with wild-type F. novicida , deletion mutants (Δ iglC and Δ pdpA ) as well as complement strains (Δ iglC :: iglC and Δ pdpA :: pdpA ) for 3 h. Subsequently, samples were washed and treated with gentamicin for 1 h. At 4 h PI, lysates were plated onto agar-containing media and bacterial colonies were enumerated the following day. Error bars, S.E.M. (n = 4).

    Article Snippet: We chose human lung A549 cells over BNL CL.2 cells for this assay primarily because of the low infections rates BNL CL.2 cells show at 4 h when infected with F. tularensis LVS.

    Techniques: Infection

    IglC and PdpA are essential for robust F. novicida growth within hepatocytes. Phase and fluorescence microscopic images were taken of BNL CL.2 cells infected with wild-type F. novicida , deletion mutants (Δ iglC and Δ pdpA ), and complement strains (Δ iglC :: iglC and Δ pdpA :: pdpA ) for 48 h. At 22 h post-inoculation, the samples were washed with PBS and replaced with media containing gentamicin to prohibit further bacterial invasion. F. novicida (green) and DNA (blue, DAPI) were stained in the fixed samples. Each image represents a ‘maximum intensity’ Z-projection comprising a stack through the cell body. Images taken by fluorescence and phase microscopy were merged together to illustrate the cell borders. Scale bar = 10 µm.

    Journal: PLoS ONE

    Article Title: IglC and PdpA Are Important for Promoting Francisella Invasion and Intracellular Growth in Epithelial Cells

    doi: 10.1371/journal.pone.0104881

    Figure Lengend Snippet: IglC and PdpA are essential for robust F. novicida growth within hepatocytes. Phase and fluorescence microscopic images were taken of BNL CL.2 cells infected with wild-type F. novicida , deletion mutants (Δ iglC and Δ pdpA ), and complement strains (Δ iglC :: iglC and Δ pdpA :: pdpA ) for 48 h. At 22 h post-inoculation, the samples were washed with PBS and replaced with media containing gentamicin to prohibit further bacterial invasion. F. novicida (green) and DNA (blue, DAPI) were stained in the fixed samples. Each image represents a ‘maximum intensity’ Z-projection comprising a stack through the cell body. Images taken by fluorescence and phase microscopy were merged together to illustrate the cell borders. Scale bar = 10 µm.

    Article Snippet: We chose human lung A549 cells over BNL CL.2 cells for this assay primarily because of the low infections rates BNL CL.2 cells show at 4 h when infected with F. tularensis LVS.

    Techniques: Fluorescence, Infection, Staining, Microscopy

    Intracellular growth kinetics of F. novicida mutants during hepatocyte infections. BNL CL.2 cells were infected with wild-type F. novicida , deletion mutants (Δ iglC and Δ pdpA ) and their respective complements. Bacteria were allowed to invade for 3 h after which extracellular bacteria were rapidly washed with PBS and killed with 100 µg mL −1 of gentamicin for 1 h. Low concentrations of gentamicin (10 µg mL −1 ) remained in the media (to inhibit extracellular bacteria) until experimental endpoint. Intracellular bacteria were then released by lysing host cells, diluted with TSBC, and plated for bacterial enumeration. Error bars, S.E.M. (n = 3).

    Journal: PLoS ONE

    Article Title: IglC and PdpA Are Important for Promoting Francisella Invasion and Intracellular Growth in Epithelial Cells

    doi: 10.1371/journal.pone.0104881

    Figure Lengend Snippet: Intracellular growth kinetics of F. novicida mutants during hepatocyte infections. BNL CL.2 cells were infected with wild-type F. novicida , deletion mutants (Δ iglC and Δ pdpA ) and their respective complements. Bacteria were allowed to invade for 3 h after which extracellular bacteria were rapidly washed with PBS and killed with 100 µg mL −1 of gentamicin for 1 h. Low concentrations of gentamicin (10 µg mL −1 ) remained in the media (to inhibit extracellular bacteria) until experimental endpoint. Intracellular bacteria were then released by lysing host cells, diluted with TSBC, and plated for bacterial enumeration. Error bars, S.E.M. (n = 3).

    Article Snippet: We chose human lung A549 cells over BNL CL.2 cells for this assay primarily because of the low infections rates BNL CL.2 cells show at 4 h when infected with F. tularensis LVS.

    Techniques: Infection