Journal: medRxiv

Article Title: ARF1 prevents aberrant type I IFN induction by regulating STING activation and recycling

doi: 10.1101/2023.04.28.23289152

Figure Lengend Snippet: a , Induction of the ISRE promoter in THP-1-Dual WT (grey) and THP-1-Dual KO-cGAS (pink) cells transduced with lentiviral vectors expressing ARF1 WT or R99C as indicated, quantified by Lucia luciferase (LLuc) activity 72 h post transduction. IFN-β (1000 U/mL, 16 h) and cGAMP (10 µg/ml, 16 h) served as positive controls. Bars represent mean of n=3 ± SEM (biological replicates). Lower panel: Corresponding immunoblots of WCLs stained by anti-FLAG, anti-STING, anti-cGAS and anti-GAPDH. b , Exemplary electron microscopy analysis of HEK293T cells transiently transfected with ARF1 WT or ARF1 R99C as indicated. Mitochondria (m) are highlighted in insets in bottom panels. Electron-dense granules and inflated cristae are highlighted by black and white arrows, respectively. Annotations: cp, cytoplasm; er, endoplasmic reticulum; g , Golgi apparatus; lv, large vesicle; ly, lysosome; m, mitochondria; nc, nucleus. c, Exemplary immunoblots showing fractionation of ARF1 WT, R99C, Q71L and vector transfected HEK293T cells as indicated. WCLs and fraction blots stained by anti-FLAG, anti-TFAM (mitochondria), anti-LAMIN B1 (nucleus) and anti-GAPDH (cytosol). d, qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of ( c ) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using the ΔΔCT method. n = 3 ± SEM. e , qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of primary fibroblasts from healthy donors (n2, f1, I7) or a patient (Patient 1) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using the ΔΔCT method. n = 5 ± SEM. f, qPCR of representative ISG OAS1 in U2OS cells stably expressing STING and depleted of mtDNA by ddC, or untreated (NT), upon transfection with empty vector, ARF1 WT or R99C n=3 ± SEM. g , Exemplary immunoblot of WCLs of HEK293T cells transiently expressing ARF1 WT, R99C or vector. Blots were stained with anti-MFN1, anti-RHOT1, anti-FLAG and anti-GAPDH. Quantification of the band intensities for MFN1 normalized to the band intensities of GAPDH. Bars represent mean of n = 6 ± SEM (biological replicates). h , Exemplary immunoblots showing fractionation of HEK293T cells expressing ARF1 WT, R99C or vector control as well as VCP. WCLs and fraction blots stained by anti-FLAG, anti-HA, anti-TFAM (mitochondria), anti LAMIN B1 (nucleus) and anti-GAPDH (cytosol). i , qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of (h) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using 725 the ΔΔCT method. n = 5 ± SEM.

Article Snippet: U2OS cells (ATCC) were maintained in McCoy medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum.

Techniques: Transduction, Expressing, Luciferase, Activity Assay, Western Blot, Staining, Electron Microscopy, Transfection, Fractionation, Plasmid Preparation, Stable Transfection, Control

Journal: bioRxiv

Article Title: Single Particle Tracking of Genetically Encoded Nanoparticles: Optimizing Expression for Cytoplasmic Diffusion Studies

doi: 10.1101/2024.11.17.623896

Figure Lengend Snippet: A) Characteristic log-log plots of MSD vs. lag time (τ). Slope α indicates diffusion type: Brownian (α=1), subdiffusive (α<1), superdiffusive (α>1). Plateau indicates confinement. B) Log-log plot of ensemble- and time-averaged MSD ( ate ) for particles longer than 10 frames (50 ms), corrected for static error and fitted to a power law with different lag time Δt values of 5, 10, 15, or 20 ms (τ = nΔt), the corresponding alpha values are given in the legend; C) image of U2OS with lower (fluorescent intensity = 16 r.u.) and higher GEM expression (fluorescent intensity = 147 r.u.). Sample size was 60 cells. D) Density probability function of collected D α , E) α,and F) D eff collected after fit with power-law relationship with σ/Δt = 0.5; G) Plot of D eff vs. fluorescence intensity in cells throughout the U2OS colony. The red line indicates the division between cells with “High GEM expression” (30 cells) and “Low GEM expression” (30 cells); H) Corresponding plot of α vs. fluorescence intensity.

Article Snippet: U2OS cells expressing pCMV-pfv-Sapphire-Ires-DsRed (Addgene #116934) or pCMV-pfv-paGFP were sorted by flow cytometry using a Cell Sorter SH800S, while U2OS cells expressing TRE-pfv-Sapphire were selected with 2 μg/mL puromycin.

Techniques: Diffusion-based Assay, Expressing, Fluorescence

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Western blot immunoreactivity of the polyclonal anti-MPV17 antibody Ab 93374 ( left ) and the monoclonal anti- human MPV17 antibodies 6F5 and 5D2 on extracts of human and murine cells of different MPV17 genotypes. Left extracts from human U2OS cells, untransfected or transfected with the MPV17 expression clone SC118652 (origene), were probed with the antibody ab 93374 (abcam). Center untransfected U2OS cells with MPV17 +/+ endogenous genotype probed with the monoclonal antibodies 6F5 and 5D2. Right Murine embryo fibroblast (MEF) cells of MPV17 +/+ or MPV17 -/- genotype probed with the monoclonal antibodies 6F5 and 5D2. Indicated marker sizes were transferred from Ponceau stained marker bands run in parallel. Loading was controlled as indicated by reaction of the filters with an anti-ß-actin antibody. 10 μg of protein were separated on SDS gels and analysed by Western blot according to ( left panel ), and Kinkley et al. respectively

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Western Blot, Transfection, Expressing, Marker, Staining

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Ab93374 (Abcam) recognizes MPV17 specific structures in U2OS cells transiently transfected with an MPV17 expression construct in immunofluorescence analysis. Ab93374 signal ( green ) is largely missing in nontransfected cells. The immunoreactivity does colocalise with peroxisomal (anti-catalase, mouse polyclonal, Abcam ab88650) and mitochondrial (anti-complex IV mitochondrial subunit I, mouse monoclonal, Invitrogen 459600) markers ( red ). Immunofluorescence (IF) method was according to Pircher et al. using a MicroRadiance confocal scanning system (Bio-Rad) in combination with a Zeiss Axiophot microscope. Colocalisation analysis was performed using the Fiji software of Image J and is illustrated by white dots

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Transfection, Expressing, Construct, Immunofluorescence, Microscopy, Software

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: In U2OS cells, 5D2 antibody detects a punctate pattern colocalising with a peroxisomal (PMP70) but not with a mitochondrial (MitoTracker) marker. Top Rabbit anti PMP70 antibody ( decorated green ) was a gift from W. Just, Heidelberg. Secondary antibodies: Alexa Fluor 555 anti-mouse and Alexa Fluor 488 anti-rabbit. Bottom Mitotracker 7510 (Invitrogen) was used as recommended by the supplier. Secondary antibody: Alexa 488 anti-mouse. IF method and microscopy according to Kinkley et al. using a Zeiss LSM confocal microscope. Mathematical colocalisation analysis was performed using the Fiji software of Image J and is illustrated by white dots

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Marker, Microscopy, Software

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: 5D2 colocalisation with a marker of early endosomes (EEA1) and lysosomes (LAMP1) in U2OS cells. Top Partial colocalisation of 5D2 with EEA1 (rabbit anti-human). Bottom colocalisation of 5D2 with LAMP1 in U2OS cells. The IF method, secondary antibodies, and Image J analysis were used as described in the legend of Fig.

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Marker

Journal: BMC Research Notes

Article Title: A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

doi: 10.1186/s13104-016-1939-0

Figure Lengend Snippet: Colocalisation of 5D2 and LAMP1 mediated immunofluorescence in a U2OS single cell. IF and analysis of 5D2 and Colocalisation of 5D2 with LAMP1 in U2OS cells. The IF method, secondary antibodies, and Image J analysis were used as described in the legend of Fig. . In addition a merge of the two immunofluorescence pictures is depicted

Article Snippet: Commercially available anti-human MPV17 antibodies from different sources (Abcam ab93374; Proteintech 10310-1-AP, 60310-1-Ig; Santa Cruz Biotechnology SC109551; Insight Biotechnology ARP73712-P050; BioCat AP8749a-ev-AB) were tested in western blot (Fig. ) and immunofluorescence studies (Fig. ) on human osteosarcoma cells (U2OS) transfected with a human- MPV17 expression construct (Origene SC118652).

Techniques: Immunofluorescence

Journal: Virology

Article Title: Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency.

doi: 10.1016/j.virol.2006.03.047

Figure Lengend Snippet: Fig. 2. ICN activates RTA promoter in a dose-dependent manner. (A) Scheme to show the Notch expression constructs used for luciferase assay. Delta E is encoded by a cDNA consisting of codons 1–22 fused to codons 1673–2555 and is predicted to result in the synthesis of a mature polypeptide with an amino terminus lying 61 amino acids external to the transmembrane domain. Delta EA has an additional deletion of ankyrin repeats. ICN is encoded by a cDNA consisting of the first two codons of NOTCH1 fused to codon 1770, which lies 24 amino acids internal to the transmembrane domain (Aster et al., 1997). In the diagram, proteolytic cleavage by furin at site S1 produces two subunits, ECN and NTM, which remain non-covalently associated at the cell surface. Sites S2 and S3 identify the sites of proteolytic cleavage mediated by metalloproteases and presenilins induced upon activation by ligand. Cleavage at S3 yields activated form of Notch ICN (Schroeter et al., 1998). (B) Luciferase assay: 1 million of U2OS cells were transfected with 0.5-μg RTA promoter reporter plasmid along with increasing amount of full-length Notch, Delta E, Delta EA or ICN expression vector (0 ng, 10 ng, 20 ng, 50 ng, 100 ng, 200 ng) by lipofectamine 2000. Twenty four hours post-transfection, cells were harvested and lysed for reporter assay.

Article Snippet: A human osteosarcoma cell line U2OS was obtained from the American Type Culture Collection.

Techniques: Expressing, Construct, Luciferase, Activation Assay, Transfection, Plasmid Preparation, Reporter Assay

Journal: Virology

Article Title: Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency.

doi: 10.1016/j.virol.2006.03.047

Figure Lengend Snippet: Fig. 5. Transcriptional activity of ICN on RTA promoters. The reporter plasmid pRpluc contains a 3-kb sequence upstream of the translational initiation site of the RTA gene that drives the expression of firefly luciferase. A series of truncation promoters named as pRplucΔ2570, pRplucΔ1490 and pRplucΔ1327 were made for deletion of RBP–Jκ binding sites (bottom panel). A fixed amount (0.5 μg) of the reporter plasmids was transfected or co-transfected into U2OS cell with 50 ng of pflu-ICN. The promoter activity was expressed as the fold activation relative to the reporters alone control. The means and standard deviations from three independent transfections are shown.

Article Snippet: A human osteosarcoma cell line U2OS was obtained from the American Type Culture Collection.

Techniques: Activity Assay, Plasmid Preparation, Sequencing, Expressing, Luciferase, Binding Assay, Transfection, Activation Assay, Control

Journal: Virology

Article Title: Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency.

doi: 10.1016/j.virol.2006.03.047

Figure Lengend Snippet: Fig. 7. (A) LANA represses ICN transactivation on RTA promoter. 1 million of U2OS cells were transfected with 0.5 μg RTA promoter reporter plasmid and 50 ng ICN expression vector along with increasing amount of LANA expression vector (0 ng, 10 ng, 20 ng, 50 ng, 100 ng, 200 ng) by lipofectamine 2000. Twenty four hours post-transfection, cells were harvested and lysed for reporter assay. (B) For each transfection, 15 million BCBL1 cells were transfected with mock, pA3M empty vector, and ICN with or without LANA. Twenty four hours post-transfection, total RNAwas collected from the cells. A total of 5 μg of RNA was used with the Superscript First Strand Synthesis system to construct cDNA. Real-time PCR was performed using the DyNAmo SYBR Green qPCR kit with β-actin as the standard. The PCR data are expressed as the Ct values for RTA transcription. Each sample was tested in triplicate for the calculation of the mean and standard deviation. The relative transcript abundance (Log molecules) based on the Ct values for RTA. (C) Twenty million of Vero cells stably infected with Bac36 were mock-transfected or transfected with empty vector pA3M, ICN, or ICN plus LANA. Four days post-transfection, supernatant from each transfection was harvested and concentrated for infection of 293 cells. Twenty four hours post-infection, cells were harvested for FACS analysis to detect GFP expression marking the virus existence.

Article Snippet: A human osteosarcoma cell line U2OS was obtained from the American Type Culture Collection.

Techniques: Transfection, Plasmid Preparation, Expressing, Reporter Assay, Construct, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation, Stable Transfection, Infection, Virus