Journal: Molecular and Cellular Biology

Article Title: LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia

doi: 10.1128/MCB.00652-19

Figure Lengend Snippet: Halo-tagged proteins expressed from recombinant lentiviruses have physiological abundance and localization and can be precisely quantified in lysates or live cells. (A) Cell lysates were prepared from untransduced Jurkat cells; Jurkat cells transduced with Halo-LMO2; untransduced K562 cells; K562 cells transduced with Halo-LMO2; and KOPT-K1, LOUCY, and U937 cells. Lysates were subjected to 4% to 15% SDS-PAGE, transferred, and blotted with anti-LDB1, anti-LMO2 monoclonal antibody, anti-Halo, anti-TAL1, and anti-VCP (gel loading control). Molecular weight standards were run on the same gel and are shown at the right. (B) The top panel shows an immunoblot of lysates prepared from U2OS with a Halo knock-in at CTCF (lane 1) or lysates from Jurkat cells transduced with the constructs shown in the grid above (lanes 2 to 14). Empty vector control is shown as EBFPII-Hygro (lane 2). Lanes 2 to 9 show lysates from Jurkat cells lentivirally expressing Halo-CTCF at increasing MOIs. Lanes 10 to 14 show lysates from Jurkat cells lentivirally expressing Halo-SOX2 at increasing MOIs. The top panel shows gradient SDS-PAGE, transfer, and blotting with anti-CTCF antibody. The middle panel shows direct in-gel Halo fluorescence. Live cells prepared as described for panel A were labeled with cell-permeative fluorescent ligand R110, lysed, and subjected to gradient SDS-PAGE. The gel was visualized for green fluorescence as described in Materials and Methods. (C) Live Jurkat cells expressing Halo-tagged proteins were labeled with R110 Halo ligand, washed, and subjected to flow cytometry. Histograms show FITC fluorescence of the various cells in comparison to the labeled U2OS cells, which have a Halo tag knocked into the CTCF gene. (D) Table showing calculated copy numbers of Halo-tagged proteins based on the absolute values derived from the Halo knock-in cell line. Lentiviral Halo CTCF and Halo SOX2 were quantified from cell populations infected at intermediate and low MOIs, respectively. (E) Confocal microscopy of Jurkat cells lentivirally expressing Halo-LMO2 and labeled with Halo ligand, R110, and nuclear stain (Syto 17 Red). The right panel shows a merged image. Voxel quantification showed that 95% of Halo-LMO2 was nuclear.

Article Snippet: Catalog no. S7579 pBluescript SK Stratagene Iscove's modified Dulbecco's medium (IMDM) Gibco Catalog no. 12200-036 RPMI 1640 Gibco Catalog no. 31800-022 Penicillin-streptomycin solution 10× Corning Catalog no. 30-022-CI Geneticin Gibco Catalog no. 10131-027 0.05% trypsin, 0.53 mM EDTA 1× [−]sodium bicarbonate Corning Catalog no. 20116004 Puromycin dihydrochloride Fisher Bioreagents Catalog no. BP2956-100 Pierce protease inhibitor tablets Thermo Scientific Catalog no. A32965 Hygromycin B-PBS (50 mg/ml) Invitrogen Catalog no. 10687010 Anti-FLAG M2 resin Sigma Catalog no. A2220 Protein A/G resin Santa Cruz Polyvinylidene difluoride (PVDF) membrane GE Catalog no. 10600022 SuperSignal PicoWest Plus Thermo/Pierce Catalog no. 1863099 Experimental models: cell lines Human: HEK 293 ATCC Human: Jurkat ATCC Human: K562 ATCC Human: KOPTK1 ATCC Human: LOUCY ATCC Human: U937 ATCC Human: U2OS C32 Halo-CTCF Hansen et al. ( 27 ) U2OS endogenous knock-in cell line where all endogenous copies of CTCF have been N-terminally tagged with FLAG-HaloTag; clone 32 Software and algorithms Flowjo 10.3 analysis software FlowJo, LLC Contact corresponding author for URL Ideas software Amnis Corporation Contact corresponding author for URL ImageLab 5.2.1 BioRad Contact corresponding author for URL Imaris Bitplane Inc. Other CytoFLEX benchtop cytometer Beckman Contact corresponding author for URL Leica TCS SP8 confocal imaging system Leica Contact corresponding author for URL ImageStream Mk II Amnis Contact corresponding author for URL Open in a separate window Reagents and resources Experimental model and subject details. (i) Cell lines.

Techniques: Recombinant, Transduction, SDS Page, Control, Molecular Weight, Western Blot, Knock-In, Construct, Plasmid Preparation, Expressing, Fluorescence, Labeling, Flow Cytometry, Comparison, Derivative Assay, Infection, Confocal Microscopy, Staining

Journal: Molecular and Cellular Biology

Article Title: LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia

doi: 10.1128/MCB.00652-19

Figure Lengend Snippet: Reagents and resources

Article Snippet: Catalog no. S7579 pBluescript SK Stratagene Iscove's modified Dulbecco's medium (IMDM) Gibco Catalog no. 12200-036 RPMI 1640 Gibco Catalog no. 31800-022 Penicillin-streptomycin solution 10× Corning Catalog no. 30-022-CI Geneticin Gibco Catalog no. 10131-027 0.05% trypsin, 0.53 mM EDTA 1× [−]sodium bicarbonate Corning Catalog no. 20116004 Puromycin dihydrochloride Fisher Bioreagents Catalog no. BP2956-100 Pierce protease inhibitor tablets Thermo Scientific Catalog no. A32965 Hygromycin B-PBS (50 mg/ml) Invitrogen Catalog no. 10687010 Anti-FLAG M2 resin Sigma Catalog no. A2220 Protein A/G resin Santa Cruz Polyvinylidene difluoride (PVDF) membrane GE Catalog no. 10600022 SuperSignal PicoWest Plus Thermo/Pierce Catalog no. 1863099 Experimental models: cell lines Human: HEK 293 ATCC Human: Jurkat ATCC Human: K562 ATCC Human: KOPTK1 ATCC Human: LOUCY ATCC Human: U937 ATCC Human: U2OS C32 Halo-CTCF Hansen et al. ( 27 ) U2OS endogenous knock-in cell line where all endogenous copies of CTCF have been N-terminally tagged with FLAG-HaloTag; clone 32 Software and algorithms Flowjo 10.3 analysis software FlowJo, LLC Contact corresponding author for URL Ideas software Amnis Corporation Contact corresponding author for URL ImageLab 5.2.1 BioRad Contact corresponding author for URL Imaris Bitplane Inc. Other CytoFLEX benchtop cytometer Beckman Contact corresponding author for URL Leica TCS SP8 confocal imaging system Leica Contact corresponding author for URL ImageStream Mk II Amnis Contact corresponding author for URL Open in a separate window Reagents and resources Experimental model and subject details. (i) Cell lines.

Techniques: Recombinant, Staining, Modification, Protease Inhibitor, Membrane, Knock-In, Software, Cytometry, Imaging

Journal: Bone Research

Article Title: Chemotherapeutic drug-triggered AEP-cleaved G3BP1 orchestrates stress granules/nucleoli/mitochondria in osteosarcoma

doi: 10.1038/s41413-025-00453-w

Figure Lengend Snippet: Chemotherapeutic drugs induced SG assembly and triggered AEP to specifically cleave G3BP1 at N258/N309. a Representative immunofluorescences (IF) images of SG assembly in U2OS, 143B, U87-MG and A549 cells exposed to cisplatin (5 and 50 μmol/L) or vehicle for 6 h. Scale Bar = 10 μm. b Quantification of the counts of SGs per cell ( n = 50) and SG + cell ratio ( n = 6) in cells of ( a ). c WB analysis of G3BP1 and AEP in U2OS and 143B with NC or AEP-knockdown (KD) exposed to different chemotherapeutic drugs for 6 h. The arrows point out the truncated fragments of G3BP1 cleaved by AEP. d In vitro cleavage experiment of AEP and G3BP1 (WT and point mutants) purified recombinant proteins. Data are expressed as mean ± SD. *** P < 0.001, **** P < 0. 0001. Comparisons were conducted using one-way ANOVA

Article Snippet: Stable cell lines of U2OS, 143B, and U87-MG were seeded in 96-well cell culture plates and treated with cisplatin (50 μmol/L) or appropriate vehicle solution for 6 h. OCR Fluorometric Assay Kit (Cat# E-BC-F068, Elabscience, Wuhan, China) was used according to manufacturer’s protocol.

Techniques: Knockdown, In Vitro, Purification, Recombinant

Journal: Bone Research

Article Title: Chemotherapeutic drug-triggered AEP-cleaved G3BP1 orchestrates stress granules/nucleoli/mitochondria in osteosarcoma

doi: 10.1038/s41413-025-00453-w

Figure Lengend Snippet: tG3BP1-Ns competitively bind to full-length G3BP1 and negatively modulate SG. a Representative images of SGs in U2OS cells with or without AEP-KD exposed to cisplatin (50 μmol/L), doxorubicin (50 μmol/L) for 6 h. Scale bar = 10 μm. b Quantification of the SG counts per cell ( n = 50) and SG + cell ratio ( n = 6) in cells of ( a ). c Representative images of G3BP1-FL colocalized with tG3BP1-Ns or tG3BP1-Cs in Hela cells. Scale bar = 5 μm. d Co-IP and WB assays of mCherry-tagged tG3BP1-Ns or Cs cotransfected with flag-tagged full-length G3BP1 in HEK293T. e Representative images of SGs in tG3BP1-Ns overexpressed U2OS cells exposed to cisplatin (5 μmol/L), doxorubicin (5 μmol/L) for 6 h. Scale bar = 10 μm. f Quantification of SG counts per cell ( n = 50) and SG + cell ratio ( n = 6) of ( e ). Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1. ns no significance. One-way ANOVA

Article Snippet: Stable cell lines of U2OS, 143B, and U87-MG were seeded in 96-well cell culture plates and treated with cisplatin (50 μmol/L) or appropriate vehicle solution for 6 h. OCR Fluorometric Assay Kit (Cat# E-BC-F068, Elabscience, Wuhan, China) was used according to manufacturer’s protocol.

Techniques: Co-Immunoprecipitation Assay

Journal: Bone Research

Article Title: Chemotherapeutic drug-triggered AEP-cleaved G3BP1 orchestrates stress granules/nucleoli/mitochondria in osteosarcoma

doi: 10.1038/s41413-025-00453-w

Figure Lengend Snippet: tG3BP1-Cs translocate into the nucleolus and sequester mRNAs of ribosomal proteins in the nucleolus to inhibit cellular translation. a Representative images of the sub-nucleolar localization of tG3BP1-Cs and sub-nucleolar markers in Hela cells. Scale bar = 5 μm. b Representative images of FISH and IF assays present the nucleolar colocalization of tG3BP1-Cs with FAM-conjugated probes of ribosomal mRNAs, RPS4X, RPL11, and RP27A. c SUnSET experiments analyzed the protein synthesis in U2OS, 143B, and U87-MG cells treated with cisplatin (50 μmol/L) or vehicle for 6 h. d Quantification of protein synthesis of the aforementioned cell lines exposed to cisplatin (50 μmol/L) or vehicle for 6 h were detected with the Click-iT HPG system ( n = 3). Data are expressed as mean ± SD. ** P < 0.01, **** P < 0.000 1. ns no significance. One-way ANOVA

Article Snippet: Stable cell lines of U2OS, 143B, and U87-MG were seeded in 96-well cell culture plates and treated with cisplatin (50 μmol/L) or appropriate vehicle solution for 6 h. OCR Fluorometric Assay Kit (Cat# E-BC-F068, Elabscience, Wuhan, China) was used according to manufacturer’s protocol.

Techniques:

Journal: Bone Research

Article Title: Chemotherapeutic drug-triggered AEP-cleaved G3BP1 orchestrates stress granules/nucleoli/mitochondria in osteosarcoma

doi: 10.1038/s41413-025-00453-w

Figure Lengend Snippet: tG3BP1-Cs bind to mitochondrial mRNA targets and suppress their translation to alleviate mitochondrial stress. a Representative images of the colocalization of tG3BP1-Cs with the mitochondrial marker TOMM20 in Hela cells. Scale bar = 10 μm. b RNP-IP analysis of the mRNA target encoding ribosomal proteins and oxidative phosphorylation binding to tG3BP1-Cs ( n = 3) in tG3BP1-Cs overexpressed U2OS cells. c Ribosome profiling-qPCR analysis demonstrated that tG3BP1 overexpression in U2OS cells significantly downregulates mitochondrial genes translation. d WB analysis of mitochondrial genes expression in cell lines exposed to cisplatin (50 μmol/L) or vehicle for 6 h. e Cisplatin-induced mitochondrial damage was detected by JC-1 probe staining in cells of ( d ). Data are expressed as mean ± SD. *** P < 0.001, **** P < 0.000 1. One-way ANOVA

Article Snippet: Stable cell lines of U2OS, 143B, and U87-MG were seeded in 96-well cell culture plates and treated with cisplatin (50 μmol/L) or appropriate vehicle solution for 6 h. OCR Fluorometric Assay Kit (Cat# E-BC-F068, Elabscience, Wuhan, China) was used according to manufacturer’s protocol.

Techniques: Marker, Phospho-proteomics, Binding Assay, Over Expression, Expressing, Staining