Journal: FEBS letters

Article Title: Differential effects of the N-terminal helix of FGF8b on the activity of a small-molecule FGFR inhibitor in cell culture and for the extracellular domain of FGFR3c in solution.

doi: 10.1002/1873-3468.14976

Figure Lengend Snippet: Fig. 1. Activity of SSR against FGF2 and FGF8b. (A–D) Fluorescence intensity analysis of U-2 OS cells immunolabeled for pMAPK (pY202/ 204) (A, B) or pPLCγ1 (pY783) (C, D) after stimulation with FGF2 (A, C) and FGF8b (B, D) and in the presence and absence of 25 μM SSR. The statistical significance was evaluated using the Mann–Whitney U test (*P < 0.05; **P < 0.01; ***P < 0.001; n.s., non-significant). Number of biologically independent replicates is n = 4 for A, B, and D; and 3 for panel C. (E) Representative microscopic images of proliferating bEnd.3 cells (EdU+, green; upper panel) and total bEnd.3 cells (nuclear marker DAPI, gray; bottom panel). Length of the scale bar equals 100 μM. (F) Percentage of proliferating human bEnd.3 cells upon stimulation by FGF2 and FGF8b in the presence and absence of either 50 nM or 100 μM SSR. BSA was used as a negative control. Error bars represent the SEM. Number of biologically independent replicates is n = 3.

Article Snippet: The human osteosarcoma cell line U-2 OS (CLS Cell Lines Service GmbH, Eppelheim, Germany; cat. no. 300364) was grown in growth medium consisting of high glucose DMEM/F-12 (Gibco, Grand Island, NY, USA; cat. no. 21041025) and supplemented with 1% GlutaMAX (Gibco; cat. no. 35050061), 1 unit mL 1 penicillin, 1 μg mL 1 streptomycin (Gibco; cat. no. 10378016), and 10% FBS (Satorius, Goettingen, Germany) at 37 °C and 5% CO2.

Techniques: Activity Assay, Fluorescence, Immunolabeling, MANN-WHITNEY, Marker, Negative Control

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: Nature genetics

Article Title: Gene regulation by convergent promoters.

doi: 10.1038/s41588-024-02025-w

Figure Lengend Snippet: Fig. 6 | Characteristics of convergent promoters. a, Schematics (left) highlight the TSSs that have been used to identify the extended set of convergent promoters and that have been compared for their log2FC (Nutlin-3a compared with DMSO control). Spearman correlation in MCF-7 (left panel), U2OS (middle panel) and RPE-1 cells (right panel). Linear regression with 95% confidence intervals. Spearman correlation with two-tailed significance. b, Summary profile (top panel) and heatmap with individual convergent promoter regions (bottom panels) starting from the host TSS display CpG island signals. The extended set of MCF-7 convergent promoters is length-sorted in descending order. c,d,f,g, ENCODE data from MCF-7 cells57. Summary profiles of H3K4me3 (left panels c and f), H3K27ac (middle panel c, right panel f), H3K4me1 (right panel c) and Pol II (d,g) signals for the extended set of scale-adjusted MCF-7 convergent promoters. e, Violin plots summarize the read counts at GENCODE-annotated TSS-overlapping

Article Snippet: Cell culture, drug treatment and transfection MCF-7 (ATCC, cat. no. HTB-22) and U2OS (DSMZ, cat. no. ACC 785) cells were grown in high glucose Dulbecco’s modified Eagle’s media (DMEM) with pyruvate (ThermoFisher Scientific).

Techniques: Control, Two Tailed Test