Journal:

Article Title: Modulation of Gamma Interferon-Induced Major Histocompatibility Complex Class II Gene Expression by Porphyromonas gingivalis Membrane Vesicles

doi: 10.1128/IAI.70.3.1185-1192.2002

Figure Lengend Snippet: Inhibition of class II HLA-DRα surface expression in IFN-γ-inducible cells by P. gingivalis membrane vesicles. HUVECs, 143B osteosarcoma cells, MRC-5 fibroblasts, and THP-1 monocytic cells were treated with vesicles (OMVs, 30 μg of protein/ml) alone or IFN-γ (250 U/ml) in the absence or presence of vesicles at 37°C for 3 days. Control cells received no treatment. Cells were stained with either phycoerythrin-conjugated mouse anti-HLA-DR MAb or irrelevant isotype-matched MAb. Stained cells were analyzed by flow cytometry. Averages and standard deviations were calculated from three separate experiments.

Article Snippet: Other cell lines used, obtained from ATCC, included human embryonic lung fibroblasts (MRC-5; ATCC CCL 171), 143B human osteosarcoma cells (ATCC CRL 8304), and THP-1 monocytic cells (ATCC TIB 202).

Techniques: Inhibition, Expressing, Membrane, Control, Staining, Flow Cytometry

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: U2OS and PML KO cells were cultured in 400 μM oleate for 24 h before fixation and immunostaining for CCTα (blue) and PML (red) and imaging by confocal microscopy. LDs were visualized with BODIPY 493/503 (green) (bar, 10 μm). Arrows indicate the region selection for RGB line scan plots showing the co-localization of PML and CCTα on the surface of nuclear LDs and the nuclear envelope.

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Cell Culture, Immunostaining, Imaging, Confocal Microscopy, Selection

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) Lysates of U2OS and PML KO cells that were untreated or incubated with oleate (400 μM) for 24 h were immunoblotted with PML-, CCTα-, and actin-specific antibodies. (B) U2OS and PML KO cells were immunostained for CCTα and LMNA/C, and LDs were visualized with BODIPY 493/503 (bar, 20 μm). Arrows in magnified panels (a) and (b) indicate the regions for RGB line scan plots showing the localization of CCTα on the surface of nLDs. (C, D) Quantitation of cytoplasmic (cLDs) and nLDs in cells treated with oleate (400 μM) for 24 h. (E) Quantitation of CCTα-positive nLDs in oleate-treated cells. (F, G) The size distribution of cLDs (panel F) and nLDs (panel G) in oleate-treated cells was determined by cross-sectional area binning. (H) Average cross-sectional area of total and CCTα-positive nLDs was quantified in oleate-treated cells. In panels (C, D, E, F, G, H), results are presented as box and whisker plots showing the mean and 5 th –95 th percentile for analysis of 50–100 cells from three separate experiments. Significance was determined by two-tailed t test compared with matched U2OS cell controls (** P < 0.01; *** P < 0.001).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Incubation, Quantitation Assay, Whisker Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) 3D surface reconstruction of image stacks of oleate-treated (400 μM for 24 h) U2OS cells immunostained for PML and CCTα. LDs and nuclei were visualized with BODIPY 493/503 and DAPI, respectively. A large nLD was identified in association with CCTα and PML (panel a, red frame). The image was rotated and zoomed to produce a side view of the nLD (panel b, red frame). The framed structure in (b) was assessed by removing the DAPI channel (panels c–f) to reveal how the nLD/BODIPY (blue), CCTα (green), and PML (red) are associated. (A, B) Wide-field images of the same nLD in (A) imaged at the level of the yellow line indicated in panels b–f (image rotated ∼90° clockwise). The nLD is highlighted and magnified in the inset (bar, 5 μm; inset scale bar, 1 μm). (B, C) Images of the same field of view in (B) were acquired by NanoJ SRRF. The nLD is highlighted and magnified in the inset (bar, 5 μm; inset bar, 1 μm).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques:

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: The original SRRF image before cropping from is shown for DAPI, PML (AlexaFluor-647), BODIPY 493/503, and CCTα (AlexaFluor-568) fluorescent micrographs in the top row of each image grouping. The middle row in each grouping represents the resolution estimate divided in ∼750 blocks by Fourier ring correlation (FRC) analysis (mean estimated resolution is shown in the inset at the left). The bottom row represents an Error Map based on the resolution scale Pearson (RSP) correlation and the resolution scale error (RSE).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques:

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: U2OS cells were treated with oleate for 24 h before fixation and immunostaining for CCTα (green) and PML (red). nLDs were localized based on BODIPY 493/503 as described in the legend to . (A, B, C) Spinning disk confocal sections and full 3D rendering (SlideBook) are shown for three representative nuclei (panels A, B, C), and eight nLDs are enlarged (objects 1–6) to better show the relationship between PML and CCTα on nLDs. DNA was visualized with DAPI. The scale bar on confocal images is 10 μm (inset is 2 μm), and the grid on the 3D render is 5 μm.

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Immunostaining

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) U2OS and PML KO cells transiently expressing a nuclear nGFP-DAG sensor were exposed to 400 μM oleate for 24 h, fixed, and immunostained with a CCTα antibody and incubated with LipidTox Red to visualize LDs (bar, 10 μm). Arrows in the merged image indicate the region selected for an RGB line scan plot showing the association of CCTα and DAG-GFP. (B) Quantification of nLDs in oleate-treated U2OS and PML KO cells containing neither nGFP-DAG or CCTα (CCTα/DAG-GFP-), nGFP-DAG (DAG+), CCTα (CCT+), or both (CCT/DAG+). A Venn diagram shows the percent distribution of nGFP-DAG and CCTα-positive nLDs. (C) In oleate-treated U2OS and PML KO cells, the cross-sectional area of nLDs containing nGFP-DAG and/or CCTα was measured. (B, C) Results are presented as box and whisker plots showing the mean and 5 th –95 th percentile for analysis of 50–100 cells from three separate experiments. Significance was determined by a two-tailed t test compared with matched U2OS controls (** P < 0.01; *** P < 0.001).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Expressing, Incubation, Whisker Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) U2OS cells transiently expressing nuclear localized GFP-C1(2)δ (nGFP-DAG) were exposed to oleate (400 μM) for 24 h and were fixed and immunostained with a PML antibody. LDs were visualized with LipidTox Red (bar, 10 μm). Arrows in the merged image indicate the region selected of an RGB line scan plot showing the localization of DAG-GFP with PML and nLDs. (B) Quantitation of nLDs in oleate-treated U2OS cells containing neither nGFP-DAG or PML (DAG/PML−), nGFP-DAG (DAG+), PML (PML+), or both (DAG/PML+). A Venn diagram shows the percent distribution of nGFP-DAG and PML-positive nLDs. (C) The average cross-sectional area of nLDs in oleate-treated U2OS cells containing nGFP-DAG and/or PML as described above. (D) Quantification of DAG-negative (−) and DAG-negative (+) nLDs in U2OS and PML KO cells. (B, C, D) Results are presented as box and whisker plots showing the mean and 5 th –95 th percentile from analysis of 50–100 cells in three separate experiments. Significance was determined by one-way ANOVA and Tukey’s multiple comparison to total nLD area (panel C) or two-tailed t test compared with U2OS controls (panel D) (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Expressing, Quantitation Assay, Whisker Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) U2OS and PML KO cells transiently expressing Lipin1α-V5 were immunostained with a V5 monoclonal antibody, and nuclei were visualized with DAPI (bar, 10 μm). (B) U2OS cells transiently expressing Lipin1α-V5 were treated with 400 μM oleate for 24 h and immunostained with a V5 monoclonal antibody. LDs were visualized with BODIPY 493/503 (bar, 10 μm). Panels (a) and (b) show magnified regions for Lipin1α and merge images. Arrows show the region in panel (b) selected for an RGB line scan plot (panel c) showing Lipin1α on the surface of nLDs. (C) Quantification of nLDs in oleate-treated U2OS cells containing neither nGFP-DAG nor Lipin1α (Lipin/PML−), nGFP-DAG (DAG+), Lipin1α (Lipin+), or both (Lipin/DAG+). A Venn diagram shows the percent distribution of nGFP-DAG and Lipin1α-positive nLDs. (D) The cross-sectional area of nLDs containing differing compositions of nGFP-DAG, and Lipin1α was quantified in oleate-treated U2OS cells. (E) PML KO cells were treated with oleate and immunostained as described above (bar, 10 μm). (F) Quantification of Lipin-negative (−) and Lipin-positive (+) nLDs in U2OS and PML KO cells. (C, D, F) Results are presented as box and whisker plots showing the mean and 5 th –95 th percentile for analysis of 50–100 cells from three separate experiments. (C, D, F) Significance was determined by one-way ANOVA and Tukey’s multiple comparison (panels C and D) or two-tailed t test compared with control U2OS cells (panel F) (*** P < 0.001).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Expressing, Whisker Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) U2OS cells transiently expressing Lipin1β-V5 were untreated or incubated with 400 μM oleate for 24 h before fixation and immunostaining with a V5 monoclonal antibody (red) and imaged by confocal microscopy. Selected regions from Lipin1β (a) and merge (b) images of oleate-treated cells show the association of Lipin1β with nLD stained with BODIPY 493/503 (green) (bar, 10 μm). Arrows in panel (b) indicate the region selected for RGB line scan plots (panel c) showing Lipin1β is on the surface of nLD. ( B ) Transiently expressed Lipin1β in oleate-treated PML KO cells (as described in panel A) was not detected on the surface of nLDs (bar, 10 μm).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Expressing, Incubation, Immunostaining, Confocal Microscopy, Staining