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: Developmental Cell

Article Title: Mitochondrial dynamics regulate genome stability via control of caspase-dependent DNA damage

doi: 10.1016/j.devcel.2022.03.019

Figure Lengend Snippet:

Article Snippet: Human osteosarcoma, U2OS , ATCC , Cat# 300364/p489_U-2_OS, RRID:CVCL_0042.

Techniques: Virus, Plasmid Preparation, Recombinant, Staining, Knock-In, Knock-Out, Software

Journal: eLife

Article Title: A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms

doi: 10.7554/eLife.93183

Figure Lengend Snippet: ( A ) Cartoon illustration of SHR function. Under basal conditions, SHRs are sequestered in complex with HSP90 and other co-factors. Upon ligand binding, the receptor dissociates from the inactive complex, dimerizes, and binds to DNA. ( B ) mRNA transcript levels in log(FPKM) of AR, ESR1, PR, and NR3C1 for the engineered U2OS cell lines compared to the parental U2OS line as well as three reference breast cancer cell lines. ( C ) Distribution of diffusive states for Halo-AR, Halo-ER, Halo-GR, and Halo-PR in U2OS cells before and after stimulation with an activating ligand. Arrows indicate the mean D free . Shaded error bands represent 1 SD. ( D ) Selectivity of individual SHRs to their cognate ligand compared with other steroids, as determined by D free . Error bars represent SEM. ( E ) Reference gene sets induced after 24 hr of stimulation with 25 nM estradiol. The top 5 induced gene sets for Halo-ER ectopic expression increases were not significantly induced in the parental U2OS line. ( F ) Bar plot showing the −log( q value) from the top 50 most significantly induced gene sets after estradiol stimulation. Gene sets characteristic of ESR1 or the estrogen response are colored green.

Article Snippet: The Homo sapiens cell lines U2OS (ATCC Cat. No. HTB-96; RRID: CVCL_0042 ), MCF7 (ATCC Cat. No. HTB-22; RRID: CVCL_0031 ), T47d (ATCC Cat. No. HTB-133; RRID: CVCL_0553 ), and SK-BR-3 (ATCC Cat. No. HTB-30; RRID: CVCL_0033 ) were grown in Dulbecco's Modified Eagle Medium (DMEM, Cat. No. 1056601, Gibco DMEM, high glucose, GlutaMAX Supplement, Thermo Fisher) supplemented with 10% fetal bovine serum (Cat. No. 16000044, Thermo Fisher) and 1% pen-strep (Cat. No. 15140122, Thermo Fisher) and maintained in a humidified 37°C incubator at 5% CO 2 and subcultivated approximately every 2–3 days.

Techniques: Ligand Binding Assay, Expressing

Journal: eLife

Article Title: A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms

doi: 10.7554/eLife.93183

Figure Lengend Snippet: ( A ) Distribution of diffusive states for Halo-AR, Halo-ER, Halo-GR, and Halo-PR in U2OS cells before and after stimulation with an activating ligand. The area in the shaded region is f bound . Shaded error bands represent SD. ( B ) Selectivity of individual SHRs to their cognate ligand compared with other steroids, as determined by f bound . Error bars represent SEM across three biological replicates. Figure 2—source data 1. Tabular data to generate plots from and related figure supplements.

Article Snippet: The Homo sapiens cell lines U2OS (ATCC Cat. No. HTB-96; RRID: CVCL_0042 ), MCF7 (ATCC Cat. No. HTB-22; RRID: CVCL_0031 ), T47d (ATCC Cat. No. HTB-133; RRID: CVCL_0553 ), and SK-BR-3 (ATCC Cat. No. HTB-30; RRID: CVCL_0033 ) were grown in Dulbecco's Modified Eagle Medium (DMEM, Cat. No. 1056601, Gibco DMEM, high glucose, GlutaMAX Supplement, Thermo Fisher) supplemented with 10% fetal bovine serum (Cat. No. 16000044, Thermo Fisher) and 1% pen-strep (Cat. No. 15140122, Thermo Fisher) and maintained in a humidified 37°C incubator at 5% CO 2 and subcultivated approximately every 2–3 days.

Techniques:

Journal: eLife

Article Title: A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms

doi: 10.7554/eLife.93183

Figure Lengend Snippet: ( A ) Western blot of the ER-expressing breast cancer cell lines MCF7 and T47d compared to ER expression in ER-null lines SK-BR-3 or U2OS. Samples were treated with fulvestrant for 24 hr prior to lysis. Fulvestrant treatment leads to the degradation of ER, even when fused to HaloTag. ( B ) Example of GDC-0927-induced degradation of ER in different cell lines measured by immunofluorescence against ER. Cells were exposed to compound for 24 hr prior to fixation. Image pixel intensities are equivalently scaled. ( C ) Example compound dose titrations showing change in mean nuclear intensity as a function of compound concentration. ( D ) Example compound dose titrations measuring cell proliferation, normalized to DMSO-treated cells, of cells treated with analogs of GDC-0927.

Article Snippet: The Homo sapiens cell lines U2OS (ATCC Cat. No. HTB-96; RRID: CVCL_0042 ), MCF7 (ATCC Cat. No. HTB-22; RRID: CVCL_0031 ), T47d (ATCC Cat. No. HTB-133; RRID: CVCL_0553 ), and SK-BR-3 (ATCC Cat. No. HTB-30; RRID: CVCL_0033 ) were grown in Dulbecco's Modified Eagle Medium (DMEM, Cat. No. 1056601, Gibco DMEM, high glucose, GlutaMAX Supplement, Thermo Fisher) supplemented with 10% fetal bovine serum (Cat. No. 16000044, Thermo Fisher) and 1% pen-strep (Cat. No. 15140122, Thermo Fisher) and maintained in a humidified 37°C incubator at 5% CO 2 and subcultivated approximately every 2–3 days.

Techniques: Western Blot, Expressing, Lysis, Immunofluorescence, Concentration Assay