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

Journal: Neoplasia (New York, N.Y.)

Article Title: CAND1 Promotes PLK4-Mediated Centriole Overduplication and Is Frequently Disrupted in Prostate Cancer 1

doi:

Figure Lengend Snippet: CAND1 stabilizes PLK4 and synergizes with PLK4 to induce centriole overduplication. (A) Immunoblot analysis of U-2 OS/centrin-GFP cells for CUL1 and CUL1 deletion mutants. Immunoblot for actin is shown for protein loading. (B) Quantification of the percentage of U-2 OS/centrin-GFP cells with centriole overduplication (more than four per cell and at least one maternal centriole with more than one 1 daughter) following transient transfection with empty vector (control), CUL1, or CUL1 mutants. Mean and standard errors of three independent experiments are shown. Asterisks indicate statistically significant differences (P < .05, Student's t test for independent samples, two-tailed). (C) Cell cycle distribution after transient transfection of U-2 OS/centrin-GFP cells as assessed by flow cytometry. (D, E) Immunoblot analysis of U-2 OS/centrin-GFP cells for PLK4 (anti-myc) or CUL1 mutants (anti-HA) 48 hours after transfection with PLK4-myc, CUL1-ΔN53-HA or CUL1-CΔ22-HA transfection (0-hour CHX), or after 6-hour CHX block. GAPDH is shown to demonstrate protein loading. (F) Fluorescence microscopic analysis of U-2 OS/centrin-GFP cells following transfection with either empty vector (control) or PLK4. Note the concurrent formation of multiple daughter centrioles at single maternal centrioles (“centriole flower”), which is highly characteristic for PLK4 overexpression (right panel). Scale bar, 5 µm. (G) Quantification of the percentage of U-2 OS/centrin-GFP cells with centriole overduplication after transfection with PLK4 alone (open bars, µg of PLK4 plasmid DNA transfected as indicated) or in combination with CAND1 (black bars). Mean and standard errors of three independent experiments are shown. Asterisks indicate statistically significant differences (P < .005, Student's t test for independent samples, two-tailed). (H) Immunoblot analysis of U-2 OS/centrin-GFP cells for CAND1 and PLK4 (anti-myc tag), CUL1 or actin after transient transfection of cells with empty myc tag vector (control), CAND1-myc, and/or PLK4-myc and treatment of cells with CHX (60 µg/ml) for the indicated time intervals. Note the increased protein expression of PLK4 in cells cotransfected with CAND1 after 6-hour CHX in comparison to the decreased protein expression when PLK4 is expressed individually.

Article Snippet: Cell Culture and Transfections U-2 OS/centrin-green fluorescent protein (GFP) cells (centrin-GFP construct kindly provided by Michel Bornens, Institut Curie, Paris, France) [ 22 ] were cultured and transiently transfected as previously described [ 12 ].

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Two Tailed Test, Flow Cytometry, Blocking Assay, Fluorescence, Over Expression, Expressing, Comparison

Journal: iScience

Article Title: YAP condensates are highly organized hubs

doi: 10.1016/j.isci.2024.109927

Figure Lengend Snippet:

Article Snippet: The YAP–HaloTag CRISPR knock-in U-2 OS cells were plated into eight-well LabTek chambered coverglass dishes (life technologies, 155409PK) for drug treatment and imaging the following day.

Techniques: Virus, Recombinant, Modification, Protease Inhibitor, Transfection, Reverse Transcription, SYBR Green Assay, Hybridization, Single Particle, CRISPR, Knock-In, Negative Control, Plasmid Preparation, Software