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: Frontiers in Physiology

Article Title: A Single Conserved Amino Acid Residue as a Critical Context-Specific Determinant of the Differential Ability of Mdm2 and MdmX RING Domains to Dimerize

doi: 10.3389/fphys.2019.00390

Figure Lengend Snippet: Mutations mimicking MdmX RING sequence inhibit Mdm2 activity toward MdmX. (A) Schematic representation of human Mdm2 and MdmX proteins. RING domain sequences of selected mammalian Mdm2 and MdmX proteins were aligned using BOXSHADE 3.21 software at http://www.ch.embnet.org/software/BOX_form.html . Zinc coordinating residues are marked with an asterisk (*). Sites individually mutated in this study are marked in red. (B) Mdm2 mutants generated for this study. Selected amino acid residues were replaced with the corresponding MdmX RING residues. Some mutants presented in the table were created at later stages of the project (shown in italics ). (C) The activity of selected mutants was tested in the MdmX degradation assay. U2OS cells were transiently transfected with combinations of plasmid vectors coding for Myc-tagged MdmX, GFP, and wild-type Mdm2 or the RING domain Mdm2 mutants. Lysates of transfected cells were analyzed by Western blotting.

Article Snippet: Human U2OS and HEK293 cell lines were obtained from ECACC and DSMZ, respectively.

Techniques: Sequencing, Activity Assay, Software, Generated, Degradation Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Frontiers in Physiology

Article Title: A Single Conserved Amino Acid Residue as a Critical Context-Specific Determinant of the Differential Ability of Mdm2 and MdmX RING Domains to Dimerize

doi: 10.3389/fphys.2019.00390

Figure Lengend Snippet: Most Mdm2 mutants mimicking MdmX RING sequence retain the ability to heterodimerize with MdmX. (A) MdmX relocalization assay. U2OS cells were transfected with GFP-tagged MdmX expression plasmid together with plasmids coding for wild-type Mdm2 or the Mdm2 RING mutants. MdmX was detected by GFP fluorescence (green) and Mdm2 by immunofluorescence (red). DAPI was used to label cell nuclei (blue). (B) Immunoprecipitations. Plasmid constructs encoding selected Mdm2 mutants were transiently transfected into HEK293 cells together with Myc-tagged MdmX plasmid construct. Cells were treated 24 h post-transfection with the proteasome inhibitor MG132 for 4 h, lysed, and immunoprecipitated with anti-Mdm2 and anti-Myc antibodies. Immunoprecipitates were analyzed by Western blotting. INPUT: Mdm2 and MdmX levels in cell lysates. IP MdmX: Mdm2 and MdmX levels in samples immunoprecipitated using the anti-Myc tag antibody. IP Mdm2: MdmX and Mdm2 levels in samples immunoprecipitated with the anti-Mdm2 antibody.

Article Snippet: Human U2OS and HEK293 cell lines were obtained from ECACC and DSMZ, respectively.

Techniques: Sequencing, Transfection, Expressing, Plasmid Preparation, Fluorescence, Immunofluorescence, Construct, Immunoprecipitation, Western Blot

Journal: Frontiers in Physiology

Article Title: A Single Conserved Amino Acid Residue as a Critical Context-Specific Determinant of the Differential Ability of Mdm2 and MdmX RING Domains to Dimerize

doi: 10.3389/fphys.2019.00390

Figure Lengend Snippet: C449N mutation disrupts Mdm2-MdmX heterodimerization and MdmX degradation. (A) MdmX relocalization assay. U2OS cells were transfected with GFP-tagged MdmX together with wild-type Mdm2 or selected Mdm2 C449 mutants. MdmX was detected by GFP fluorescence (green) and Mdm2 by immunofluorescence (red). DAPI was used to label cell nuclei (blue). (B) Immunoprecipitations. Selected Mdm2 mutants were transiently transfected into HEK293 cells together with Myc-tagged MdmX. Cells were treated 24 h post-transfection with the proteasome inhibitor MG132 for 4 h, lysed, and immunoprecipitated with anti-Mdm2 and anti-Myc antibodies. Immunoprecipitates were analyzed by Western blotting. INPUT: Mdm2 and MdmX levels in cell lysates. IP MdmX: Mdm2 and MdmX levels in samples immunoprecipitated using the anti-Myc tag antibody. IP Mdm2: MdmX and Mdm2 levels in samples immunoprecipitated with the anti-Mdm2 antibody.

Article Snippet: Human U2OS and HEK293 cell lines were obtained from ECACC and DSMZ, respectively.

Techniques: Mutagenesis, Transfection, Fluorescence, Immunofluorescence, Immunoprecipitation, Western Blot

Journal: Frontiers in Physiology

Article Title: A Single Conserved Amino Acid Residue as a Critical Context-Specific Determinant of the Differential Ability of Mdm2 and MdmX RING Domains to Dimerize

doi: 10.3389/fphys.2019.00390

Figure Lengend Snippet: Replacing N448 of MdmX RING with cysteine to mimic Mdm2 C449 promotes MdmX homodimerization in vivo . (A) Schematic representation of the Mdm2/MdmX chimeric protein (Mdm2/X). (B) MdmX relocalization assay. U2OS cells were transfected with GFP-tagged MdmX plasmid construct together with plasmids coding for Mdm2/X or Mdm2/X:448C in which the critical asparagine residue was replaced with cysteine. MdmX was detected by GFP fluorescence (green) and Mdm2/X by immunofluorescence (red). DAPI was used to label cell nuclei (blue). (C) MdmX relocalization assay. U2OS cells were transfected with plasmid construct encoding Myc-MdmX, in which the critical asparagine residue was replaced with cysteine (MdmX:448C), together with plasmids coding for Mdm2/X or Mdm2/X:448C. MdmX (red) and Mdm2/X (green) were detected by immunofluorescence. DAPI was used to label cell nuclei (blue).

Article Snippet: Human U2OS and HEK293 cell lines were obtained from ECACC and DSMZ, respectively.

Techniques: In Vivo, Transfection, Plasmid Preparation, Construct, Residue, Fluorescence, Immunofluorescence

Journal: Frontiers in Physiology

Article Title: A Single Conserved Amino Acid Residue as a Critical Context-Specific Determinant of the Differential Ability of Mdm2 and MdmX RING Domains to Dimerize

doi: 10.3389/fphys.2019.00390

Figure Lengend Snippet: Mutations of Mdm2/E2 contact residues can have a different impact on Mdm2 activity toward different targets. (A) Structure of the MdmX-Mdm2-UbcH5b complex (PDB: 5MNJ) ( Nomura et al., 2017 ). Mdm2 (blue) binds MdmX (red) and UbcH5b (yellow) via opposite contact surfaces. (B) The Mdm2 contact surface view with the E436, V439, I440, Q442, and R479 contact residues mediating Mdm2-UbcH5b interaction highlighted in yellow. ( C ) p53 degradation assay. U2OS cells were transfected with plasmids coding for GFP, FLAG-tagged p53, and wild-type Mdm2 or selected Mdm2 mutants. Cells were lysed 30 h post-transfection and proteins were resolved by SDS-PAGE and analyzed by Western blotting.

Article Snippet: Human U2OS and HEK293 cell lines were obtained from ECACC and DSMZ, respectively.

Techniques: Activity Assay, Degradation Assay, Transfection, SDS Page, Western Blot

Journal: Frontiers in Physiology

Article Title: A Single Conserved Amino Acid Residue as a Critical Context-Specific Determinant of the Differential Ability of Mdm2 and MdmX RING Domains to Dimerize

doi: 10.3389/fphys.2019.00390

Figure Lengend Snippet: Mutations of the conserved C449 residue can have a different impact on Mdm2 activity toward different targets. (A) The Mdm2 dimerization interface (2VJF) (blue) with highlighted central C449 residue (red stick). (B) The Mdm2-Mdm2 homodimer (2HDP) with highlighted central C449-C449 contact (sticks and dots). (C) In the simulated MdmX-MdmX homodimer (red), the central N448 residues (sticks and dots) are clashing at the dimer interface. (D) Cell-based p53 ubiquitylation assay. U2OS cells were transfected with plasmids encoding HA-tagged ubiquitin, FLAG-tagged p53, and wild-type Mdm2 or selected Mdm2 mutants. Cells were treated with a proteasome inhibitor 24 h post-transfection, lysed under denaturing conditions to disrupt non-covalent protein–protein interactions, lysates were diluted, and p53 was immunoprecipitated. Samples were resolved by SDS-PAGE and Western blotting was used to determine the levels of p53 and Mdm2 in cell lysates and HA-ubiquitin linked to p53 in immunoprecipitates. (E) p53 degradation assay. U2OS cells were transfected with GFP, FLAG-tagged p53, and wild-type Mdm2 or selected Mdm2 mutants. Cells were lysed 30 h post-transfection and proteins were resolved by SDS-PAGE and analyzed by Western blotting.

Article Snippet: Human U2OS and HEK293 cell lines were obtained from ECACC and DSMZ, respectively.

Techniques: Residue, Activity Assay, Ubiquitin Assay, Transfection, Ubiquitin Proteomics, Protein-Protein interactions, Immunoprecipitation, SDS Page, Western Blot, Degradation Assay

Journal: Molecular cancer research : MCR

Article Title: The Microtubule Network and Cell Death are Regulated by a miR-34a/Stathmin 1/βIII-tubulin Axis

doi: 10.1158/1541-7786.MCR-16-0372

Figure Lengend Snippet: (A) Top – Schematic of the alignment between the miR-34a seed sequence and the target recognition sequence of STMN1 mRNA 3′UTR, predicted by microRNA.org. Bottom - Expression of miR-34a and STMN1 mRNA in OS cell lines (n=5) and xenografts (n=9) was measured by RT-qPCR and expressed relative to normal osteoblasts. Values are mean of three measurements. (B) The correlation between miR-34a expression in OS cells and xenografts and STMN1 mRNA was analyzed by Pearson’s correlation coefficient. (C) SaOS and 143B cells were transfected with non-targeting miR-C (control) oligonucleotides or miR-34a precursors and STMN1 mRNA was measured by RT-qPCR. Results are represented as fold-change relative to miR-C. Graph depicts mean ± SE of one representation of three independent experiments. * denotes p<0.05 (D) STMN1 protein expression in untreated, miR-C and miR-34a transfected SaOS, 143B, HOS and U2OS cells was measured by immunoblot. GAPDH was loading control. Representative results of three analyses are shown.

Article Snippet: Cell culture Human OS cell lines SaOS (p53-null), 143B, HOS, U2OS (wild-type p53), MG-63 (mutant p53) and human osteoblasts (CRL-1132) were purchased from ATCC (Manassas, VA).

Techniques: Sequencing, Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot

Journal: Molecular cancer research : MCR

Article Title: The Microtubule Network and Cell Death are Regulated by a miR-34a/Stathmin 1/βIII-tubulin Axis

doi: 10.1158/1541-7786.MCR-16-0372

Figure Lengend Snippet: (A) SaOS, 143B, HOS and U2OS were transfected with either miR-34a or with siRNA targeting Sp1 (bottom panels) and STMN1 and βIII-tubulin protein expression were measured by immunoblot. GAPDH was loading control. Representative results of three analyses are shown. (B) 143B cells were transfected with miR-34a or an STMN1 expression plasmid. Immunoblot for STMN1 protein (left) is indicated. Cell cycle distribution analysis for untreated, miR-34a-expressing or STMN1 overexpressing 143B cells was assessed by PI staining and flow cytometry analysis. Arrow indicates apoptotic cells. (C) Immunoblot analysis of cell cycle markers (cyclin D1 and p27) and apoptotic marker (cleaved PARP) in miR-34a-expressing and STMN1 overexpressing OS cells. GAPDH was loading control. Representative results of three immunoblot analyses are shown. (D) Confocal microscopy images for 143B cells transfected with miR-34a for 48 hours and immunostained for LC3 I/II (red) and βIII-tubulin (green). Cells were counterstained with Hoechst (blue) to visualize nuclei. Scale bars are 25 μm.

Article Snippet: Cell culture Human OS cell lines SaOS (p53-null), 143B, HOS, U2OS (wild-type p53), MG-63 (mutant p53) and human osteoblasts (CRL-1132) were purchased from ATCC (Manassas, VA).

Techniques: Transfection, Expressing, Western Blot, Control, Plasmid Preparation, Staining, Flow Cytometry, Marker, Confocal Microscopy

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Reciprocal Modulation of Antiretroviral Drug and Steroid Receptor Function In Vitro

doi: 10.1128/aac.01890-19

Figure Lengend Snippet: Figure 6: Dose-dependent effects of TDF on ligand-independent PR-B activation. U2OS 973

Article Snippet: U2OS human osteosarcoma cells 139 on N ovem ber 8, 2019 at U C S F LIB R A R Y http://aac.asm .org/ D ow nloaded from 7 were used for the luciferase reporter assays as they are deficient in endogenous steroid 140 receptors (American Type Culture Collection (ATCC), USA).

Techniques: Activation Assay