Journal: Physiological reports

Article Title: Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

doi: 10.14814/phy2.12355

Figure Lengend Snippet: Figure 1. Characterization of IPEC-J2 cell monolayer. (A) Light microscope image of confluent cell monolayer grown on a tissue culture flask, scale bar equals 20 lm. (B) Confocal micrograph showing a top view of IPEC-J2 monolayer grown on transwell filter for 9 days, the cell borders can be distinguished by immunofluorescent staining of tight junction protein ZO-1, scale bar equals 20 lm. (C) Top view of well- differentiated IPEC-J2 cell with microvilli observed by scanning electron microscopy, figure contains one cell from cell monolayer grown on transwell filter for 9 days, scale bar equals 5 lm. (D) Progression in transepithelial electrical resistance (TEER) values of cells grown on transwell filter for 9 days. Data are given as means (SEM) of 20 separate experiments. They were assigned to experimental treatment on day 9, respectively.

Article Snippet: Images were acquired using laser scanning confocal microscopy (Nikon C-1 with Plan ApoVC 609/1.40 oil objective; Nikon EZ-C1 software; Nikon, Amsterdam, the Netherlands).

Techniques: Light Microscopy, Staining, Electron Microscopy

Journal: Physiological reports

Article Title: Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

doi: 10.14814/phy2.12355

Figure Lengend Snippet: Figure 7. Cellular distribution of the tight junction protein ZO-1 in IPEC-J2 cells. Cell monolayers grown on transwell filters were left untreated (control) or treated with bacterial cells of ETEC (multiplicity of infection, MOI 10) alone for 3 h or pretreated with Lactobacillus spp. (MOI 100) for 6 h and then challenged by ETEC for 3 h. Monolayers stained for the tight junction protein ZO-1 (green) and nuclei stained with DAPI (blue) were detected by laser scanning confocal microscopy. The images were representatives from three separate experiments. Yellow arrows show the broken lining of ZO-1 expressions. Red circles highlight areas of cell disassociation. Scale bar equals 50 lm for all images.

Article Snippet: Images were acquired using laser scanning confocal microscopy (Nikon C-1 with Plan ApoVC 609/1.40 oil objective; Nikon EZ-C1 software; Nikon, Amsterdam, the Netherlands).

Techniques: Control, Infection, Staining, Confocal Microscopy

Journal: Biochemical Pharmacology

Article Title: HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

doi: 10.1016/j.bcp.2016.03.010

Figure Lengend Snippet: HDAC 3-selective inhibitor RGFP966 attenuates LPS/IFNγ-induced NF-κB p65 activity but affects neither the NF-κB p65 acetylation nor the histone acetylation status in RAW 264.7 macrophages. RAW-Blue cells were subjected to RGFP966 and SAHA for 20 h and stimulated with LPS/IFNγ for the last 4 h of the experiment. In the absence of an inflammatory stimulus, treatment of RAW-Blue cells with HDAC inhibitors did not affect the secretion of embryonic alkaline phosphatase (SEAP; data not shown), whereas treatment of RAW-Blue cells with RGFP966 followed by a pro-inflammatory stimulus significantly reduced the secretion of SEAP (A). Data are presented as mean values ± SD of 3–4 independent experiments. *** p < 0.001, compared to vehicle-treated group. The effect of HDAC 3 inhibition on total NF-κB p65 acetylation was assessed by (B) immunoblotting and quantified by densitometric analysis (C). RAW 264.7 macrophages were incubated with HDAC inhibitors for 20 h and subsequently lysed. For the detection of NF-κB p65 acetylation, lysates were incubated and immunoprecipitated with 5 μg of mouse monoclonal NF-κB p65-specific antibody as described. Data are presented as mean values ± SD expressed as fold change compared to control (vehicle-treated) group of 4 independent experiments. Histones were separated on SDS–PAGE (D), subsequently histones H3 and H4 were excised from the gel. Gel pieces were treated with acetic anhydride d6, followed by trypsin digestion. Resulting peptides were subjected to LC–MS/MS analysis (E). RGFP966 treatment did not affect the acetylation status of histone H3 (res. peptide 18–26: KQLATKAAR) and histone H4 (res. peptide 4–17: GKGGKGLGKGGAKR), whereas SAHA increased acetylation status of both peptides. Data are presented as mean values ± SD of 3–5 independent experiments. No significant differences were observed between untreated and vehicle treated cells (data not shown).

Article Snippet: NF-κB p65 was detected with rabbit monoclonal NF-κB p65 antibody (2 h incubation at 4 °C, 1:250; # 8242, Cell Signaling), followed by 1 h incubation with goat anti-rabbit AlexaFluor 488 (10 μg/ml; A-11008, Molecular Probes, Leiden, The Netherlands).

Techniques: Activity Assay, Inhibition, Western Blot, Incubation, Immunoprecipitation, Control, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: Biochemical Pharmacology

Article Title: HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

doi: 10.1016/j.bcp.2016.03.010

Figure Lengend Snippet: RGFP966 does not affect the nuclear translocation of NF-kB p65 in RAW 264.7 macrophages. After 20 h incubation with RGFP966 followed by 1 h LPS/IFNγ stimulation, RAW 264.7 macrophages were prepared for immunofluorescence microscopy (A). Green signal represents NF-kB p65 protein, while the blue signal visualizes the Hoechst-stained nuclei. RGFP966 did not affect the nuclear translocation of NF-kB p65 compared to (vehicle-treated) control group. The presented data set shows representative images of 3 independent experiments, original magnification 400×. All images were taken under identical instrumental conditions. In addition, the effect of RGFP966 on NF-kB p65 translocation in LPS/IFNγ-stimulated RAW 264.7 macrophages was analyzed by immunoblotting cell fractions (B) and quantified by densitometric analysis (C). PARP-1 and β-actin were used as internal controls. Data are presented as mean values ± SD expressed as fold change compared to control (LPS/IFNγ-treated) group of 4 independent experiments. RAW 264.7 macrophages were stimulated with 10 ng/ml LPS/IFNγ for 15, 30 and 60 min and subsequently lysed. In parallel, to confirm proper LPS/IFNγ-stimulation and to determine the levels of IκBα in the presence of RGFP966, IκBα expression was assessed by Western blotting (D). β-Actin was used as loading control. The presented data set shows a representative blot of 3 independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: NF-κB p65 was detected with rabbit monoclonal NF-κB p65 antibody (2 h incubation at 4 °C, 1:250; # 8242, Cell Signaling), followed by 1 h incubation with goat anti-rabbit AlexaFluor 488 (10 μg/ml; A-11008, Molecular Probes, Leiden, The Netherlands).

Techniques: Translocation Assay, Incubation, Immunofluorescence, Microscopy, Staining, Control, Western Blot, Expressing

Journal: Biochemical Pharmacology

Article Title: HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

doi: 10.1016/j.bcp.2016.03.010

Figure Lengend Snippet: HDAC 3-selective inhibitor RGFP966 reduces the protein expression of HDAC 1 and HDAC 2 in RAW 264.7 macrophages. RAW 264.7 macrophages were incubated with RGFP966, stimulated for 4 h with LPS/IFNγ and subsequently harvested. Gene expression of NF-kB p65, IκBα, HDAC 1, HDAC 2 and HDAC 3 was analyzed by real-time qPCR (A). Data are presented as mean values ± SD; n = 3–4. In parallel, the effect of RGFP966 on total NF-kB p65 and HDAC 1–3 protein expression in LPS/IFNγ-stimulated RAW 264.7 macrophages was analyzed by immunoblotting (B) and quantified by densitometric analysis (C). Protein levels were normalized against β-actin and control (LPS/IFNγ-treated) cells were set at 100%. Data are presented as mean values ± SD of 4 independent experiments and a representative blot is shown in B. * p < 0.05; ** p < 0.01 compared to vehicle.

Article Snippet: NF-κB p65 was detected with rabbit monoclonal NF-κB p65 antibody (2 h incubation at 4 °C, 1:250; # 8242, Cell Signaling), followed by 1 h incubation with goat anti-rabbit AlexaFluor 488 (10 μg/ml; A-11008, Molecular Probes, Leiden, The Netherlands).

Techniques: Expressing, Incubation, Gene Expression, Western Blot, Control

Journal: Biochemical Pharmacology

Article Title: HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

doi: 10.1016/j.bcp.2016.03.010

Figure Lengend Snippet: RGFP966 does not affect the localization of HDAC 3 and HDAC 3 – NF-κB p65 interaction in LPS/IFNγ-stimulated RAW 264.7 macrophages. After 20 h incubation with RGFP966 followed by 1 h LPS/IFNγ stimulation, RAW 264.7 macrophages were prepared for immunofluorescence microscopy (A). The green signal represents NF-kB p65 protein, while the blue signal visualizes the Hoechst-stained nuclei. RGFP966 did not affect the nuclear localization of HDACs 1–3 compared to (vehicle-treated) control group. The presented data set shows representative images of 4 independent experiments. Images of the cells were taken using confocal laser scanning microscopy and all images were taken with identical instrumental conditions, original magnification 630×. In addition, the effect of RGFP966 on HDAC 1–3 localization in LPS/IFNγ-stimulated RAW 264.7 macrophages was analyzed by immunoblotting cell fractions (data not shown) and quantified by densitometric analysis (B). PARP-1 and β-actin were used as internal controls. Control (vehicle-treated) cells were set at 100%. Data are presented as mean values ± SD of 3–4 independent experiments. The HDAC 3 – NF-κB p65 interaction in LPS/IFNγ-stimulated RGFP966-treated RAW 264.7 macrophages was investigated by immunoprecipitation of NF-κB p65 followed by immunoblotting for HDAC 3, which was quantified by densitometric analysis (C). Protein levels were normalized against NF-κB p65. Data are presented as mean values ± SD of 3 independent experiments and a representative blot is shown. * p < 0.05 compared to vehicle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: NF-κB p65 was detected with rabbit monoclonal NF-κB p65 antibody (2 h incubation at 4 °C, 1:250; # 8242, Cell Signaling), followed by 1 h incubation with goat anti-rabbit AlexaFluor 488 (10 μg/ml; A-11008, Molecular Probes, Leiden, The Netherlands).

Techniques: Incubation, Immunofluorescence, Microscopy, Staining, Control, Confocal Laser Scanning Microscopy, Western Blot, Immunoprecipitation

Journal: Biochemical Pharmacology

Article Title: HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

doi: 10.1016/j.bcp.2016.03.010

Figure Lengend Snippet: RGFP966 does not affect the localization of HDAC 3 and HDAC 3 – NF-κB p65 interaction in LPS/IFNγ-stimulated RAW 264.7 macrophages. After 20 h incubation with RGFP966 followed by 1 h LPS/IFNγ stimulation, RAW 264.7 macrophages were prepared for immunofluorescence microscopy (A). The green signal represents NF-kB p65 protein, while the blue signal visualizes the Hoechst-stained nuclei. RGFP966 did not affect the nuclear localization of HDACs 1–3 compared to (vehicle-treated) control group. The presented data set shows representative images of 4 independent experiments. Images of the cells were taken using confocal laser scanning microscopy and all images were taken with identical instrumental conditions, original magnification 630×. In addition, the effect of RGFP966 on HDAC 1–3 localization in LPS/IFNγ-stimulated RAW 264.7 macrophages was analyzed by immunoblotting cell fractions (data not shown) and quantified by densitometric analysis (B). PARP-1 and β-actin were used as internal controls. Control (vehicle-treated) cells were set at 100%. Data are presented as mean values ± SD of 3–4 independent experiments. The HDAC 3 – NF-κB p65 interaction in LPS/IFNγ-stimulated RGFP966-treated RAW 264.7 macrophages was investigated by immunoprecipitation of NF-κB p65 followed by immunoblotting for HDAC 3, which was quantified by densitometric analysis (C). Protein levels were normalized against NF-κB p65. Data are presented as mean values ± SD of 3 independent experiments and a representative blot is shown. * p < 0.05 compared to vehicle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Subsequently, lysates were incubated with 50 μl of Protein A/G PLUS-Agarose Immunoprecipitation Reagent (sc-2003; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at constant rotation for 6 h at 4 °C.

Techniques: Incubation, Immunofluorescence, Microscopy, Staining, Control, Confocal Laser Scanning Microscopy, Western Blot, Immunoprecipitation