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: PLoS Genetics

Article Title: The DNA Helicase Recql4 Is Required for Normal Osteoblast Expansion and Osteosarcoma Formation

doi: 10.1371/journal.pgen.1005160

Figure Lengend Snippet: (A) Kaplan-Meier survival plots of Osx -Cre Recql4 (left) and Osx -Cre p53 fl/fl Recql4 (right) mice. P value calculated by Log-Rank statistical test. (B) H&E stained sections of primary OS tumors from Osx -Cre p53 fl/fl Recql4 animals of indicated genotype. (C) Representative reconstructed μCT images of primary OS tumors from Osx -Cre p53 fl/fl Recql4 fl/+ and Recql4 fl/fl mice. (D) Flow cytometry percentages of tumor cells stained with CD51, Sca1 and PDGFRα. n = 3 for Recql4 +/+ , n = 8 for Recql4 fl/+ , n = 7 for Recql4 fl/fl ; Data presented as mean±SEM. (E) Representative photos of Alizarin Red-stained tumor cells that were subjected to osteogenic differentiation conditions. (F) qPCR profiling of Osx -Cre p53 fl/fl Recql4 +/+ , Recql4 fl/+ and Recql4 fl/fl tumors for the indicated genes. n = 3–4; Data presented as mean±SEM. (G) Assessment of genomic excision of Recql4 in tumor-derived cell lines. 40ng of genomic DNA was used for PCR and subjected to gel electrophoresis.

Article Snippet: The Kusa4b10, long-bone primary osteoblastic cells and mouse OS cell lines (no authentication performed) were cultured in αMEM (Lonza), 10% non heat inactivated FBS (SAFC Biosciences) and 1% Penicillin/Streptamycin/Glutamine (Life Technologies).

Techniques: Staining, Flow Cytometry, Derivative Assay, Nucleic Acid Electrophoresis