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

Journal: Pharmacogenomics and Personalized Medicine

Article Title: Hsa_circ_0078767 Enhances Osteosarcoma Chemoresistance to Doxorubicin Through the Regulation of the miR-188-3p/GPX4 Axis

doi: 10.2147/PGPM.S473702

Figure Lengend Snippet: DOX-resistant OS tumors and cell lines express higher levels of hsa_circ_0078767. ( A ) qPCR was used to detect hsa_circ_0078767 levels in 65 and 61 non-respond and respond OS tissue samples, respectively. ( B ) Kaplan-Meier curves for assessment of the association between OS patient survival andhsa_circ_0078767 levels. ( C ) hsa_circ_0078767 expression in parental (HOS, U2OS) and DOX-resistant (HOS/DOX, U2OS/DOX) OS cell lines, shown by qPCR. ( D and E ) RNase R was used to treat U2OS/DOX and HOS/DOX cells, after which qPCR was utilized to assess hsa_circ_0078767 and DYNC1H1 expression. ( F ) Hsa_circ_0078767, GAPDH, and U6 were detected from the nuclear and cytoplasmic fractions of U2OS/DOX and HOS/DOX cells. ( G ) hsa_circ_0078767 and linear DYNC1H1 transcript stability levels were analyzed in U2OS/DOX and HOS/DOX cells. *P < 0.05.

Article Snippet: The KHOS and U2OS human OS cell lines from Biovector (Beijing, China) were grown in DMEM (Gibco, MA, USA) supplemented with 10% FBS (Gibco) and penicillin/streptomycin (Invitrogen, MA, USA) in a 5% CO 2 37°C incubator.

Techniques: Expressing

Journal: Pharmacogenomics and Personalized Medicine

Article Title: Hsa_circ_0078767 Enhances Osteosarcoma Chemoresistance to Doxorubicin Through the Regulation of the miR-188-3p/GPX4 Axis

doi: 10.2147/PGPM.S473702

Figure Lengend Snippet: Knocking down hsa_circ_0078767 reduces in vitro OS cell resistance to DOX. ( A and B ) qPCR was employed to gauge knockdown efficiency in U2OS/DOX and HOS/DOX cells following si-NC or si-hsa_circ_0078767 transfection. ( C ) CCK-8 assays were utilized to calculate IC 50 values for DOX-treated OS cells. *P < 0.05.

Article Snippet: The KHOS and U2OS human OS cell lines from Biovector (Beijing, China) were grown in DMEM (Gibco, MA, USA) supplemented with 10% FBS (Gibco) and penicillin/streptomycin (Invitrogen, MA, USA) in a 5% CO 2 37°C incubator.

Techniques: In Vitro, Knockdown, Transfection, CCK-8 Assay

Journal: Pharmacogenomics and Personalized Medicine

Article Title: Hsa_circ_0078767 Enhances Osteosarcoma Chemoresistance to Doxorubicin Through the Regulation of the miR-188-3p/GPX4 Axis

doi: 10.2147/PGPM.S473702

Figure Lengend Snippet: miR-188-3p is targeted directly by hsa_circ_0078767. ( A ) Putative hsa_circ_0078767 and miR-188-3p binding sites. ( B ) miR-188-3p levels in 65 and 61 non-respond and respond OS tissue samples, respectively, shown by qPCR. ( C ) Spearman correlation coefficients between hsa_circ_0078767 and miR-188-3p levels in DOX-resistant OS patient tissue samples. ( D and E ) Dual-luciferase reporter assays for assessment of HOS/DOX and U2OS/DOX cells following hsa_circ_0078767-wt or hsa_circ_0078767-mut co-transfection with the miR-188-3p or miR-NC constructs. ( F and G ) Interaction betweenhsa_circ_0078767 and miR-188-3p shown by RIP. ( H ) Interaction between hsa_circ_0078767 and miR-188-3p shown by RNA pull-down. ( I ) miR-188-3p levels in parental and DOX-resistant OS cells. ( J and K ) qPCR determination of miR-188-3p expression in U2OS/DOX and HOS/DOX cells following si-NC, si-hsa_circ_0078767, pCD-ciR, or hsa_circ_0078767 transfection. *P < 0.05.

Article Snippet: The KHOS and U2OS human OS cell lines from Biovector (Beijing, China) were grown in DMEM (Gibco, MA, USA) supplemented with 10% FBS (Gibco) and penicillin/streptomycin (Invitrogen, MA, USA) in a 5% CO 2 37°C incubator.

Techniques: Binding Assay, Luciferase, Cotransfection, Construct, Expressing, Transfection

Journal: Pharmacogenomics and Personalized Medicine

Article Title: Hsa_circ_0078767 Enhances Osteosarcoma Chemoresistance to Doxorubicin Through the Regulation of the miR-188-3p/GPX4 Axis

doi: 10.2147/PGPM.S473702

Figure Lengend Snippet: GPX4 is a miR-188-3p target ( A ) Overlap between predicted miR-188-3p and GPX4 interaction sites. ( B ) GPX4 levels in 65 and 61 non-respond and respond OS tissue samples, respectively, shown by qPCR. ( C ) Spearman correlations of miR-188-3p and GPX4 levels in DOX-resistant OS tissue samples. ( D and E ) Interaction between miR-188-3p and GPX4 shown by dual-luciferase reporter assays. ( F and G ) Interaction betweenmiR-188-3p and GPX4 shown by RIP. ( H ) GPX4 mRNA levels were analyzed in U2OS, U2OS/DOX, HOS, and HOS/DOX cells. ( I and J ) GPX4 levels were detected via qPCR in U2OS/DOX and HOS/DOX cells following miR-NC, miR-188-3p, anti-miR-NC, or anti-miR-188-3p transfection. *P < 0.05.

Article Snippet: The KHOS and U2OS human OS cell lines from Biovector (Beijing, China) were grown in DMEM (Gibco, MA, USA) supplemented with 10% FBS (Gibco) and penicillin/streptomycin (Invitrogen, MA, USA) in a 5% CO 2 37°C incubator.

Techniques: Luciferase, Transfection

Journal: Pharmacogenomics and Personalized Medicine

Article Title: Hsa_circ_0078767 Enhances Osteosarcoma Chemoresistance to Doxorubicin Through the Regulation of the miR-188-3p/GPX4 Axis

doi: 10.2147/PGPM.S473702

Figure Lengend Snippet: Hsa_circ_0078767 interacts with miR-188-3p to derepress the expression of GPX4. ( A ) Associations between hsa_circ_0078767 and GPX4 within DOX-resistant OS tissue samples were examined through Spearman correlation analyses. ( B and C ) GPX4 expression in U2OS/DOX and HOS/DOX cells following si-NC, si-hsa_circ_0078767, si-hsa_circ_0078767+anti-miR-NC, or si-hsa_circ_0078767+anti-miR-188-3p transfection, shown by qPCR and Western immunoblotting. *P<0.05.

Article Snippet: The KHOS and U2OS human OS cell lines from Biovector (Beijing, China) were grown in DMEM (Gibco, MA, USA) supplemented with 10% FBS (Gibco) and penicillin/streptomycin (Invitrogen, MA, USA) in a 5% CO 2 37°C incubator.

Techniques: Expressing, Transfection, Western Blot

Journal: Cancer Science

Article Title: N‐acetylgalactosaminyltransferase GALNT6 is a potential therapeutic target of clear cell renal cell carcinoma progression

doi: 10.1111/cas.16296

Figure Lengend Snippet: In vivo clear cell renal cell carcinoma xenograft model confirms the pro‐tumorigenic and pro‐metastatic roles of overexpression of N‐acetylgalactosaminyltransferase GALNT6. (A) Tumor volumes were measured using calipers every 4 days. *<0.05, and **<0.01. (B) After 28 days, mice were killed and tumors were collected and photographed. Immunohistochemistry of (C) GALNT6 and (D) Ki‐67 in CAKI1 xenografts. Scale bar, 100 μ m. (E) After 28 days, pulmonary metastasis was monitored by bioluminescence imaging. (F) Number of metastatic lung nodules was visually counted. (G) Histological assessment of H&E staining from lung parenchyma within the metastases. Scale bar, 500 μ m. Data are expressed as the mean ± SD.

Article Snippet: Human ccRCC cell lines CAKI1 and OS‐RC‐2 were obtained from iCell Bioscience.

Techniques: In Vivo, Over Expression, Immunohistochemistry, Imaging, Staining

Journal: Cancer Science

Article Title: N‐acetylgalactosaminyltransferase GALNT6 is a potential therapeutic target of clear cell renal cell carcinoma progression

doi: 10.1111/cas.16296

Figure Lengend Snippet: Prohibitin 2 (PHB2) is a substrate for GALNT6‐mediated O‐glycosylation in clear cell renal cell carcinoma (ccRCC) cells. (A) Immunofluorescence of vicia villosa agglutinin (VVA) lectins in ccRCC cells. Scale bar, 50 μ m. (B) Venn diagram illustrating the number of specific GALNT6 interactors in CAKI1 cells. IgG was used to excluded nonspecific binders. (C) Co‐immunoprecipitation (IP) of GALNT6 with PHB2 in lysates from ccRCC cells. (D) The IP combined with VVA blot assays was used to assess whether GALNT6 affects O‐GalNAc glycosylation of PHB2 in ccRCC cells. (E) Top panel, schematic diagram of plasmid transfection in CAKI1 cells. Bottom panel, IP combined with VVA blot assays was used to identify the main amino acid sites of PHB2 O‐glycosylation mediated by GALNT6 in CAKI1 cells.

Article Snippet: Human ccRCC cell lines CAKI1 and OS‐RC‐2 were obtained from iCell Bioscience.

Techniques: Glycoproteomics, Immunofluorescence, Immunoprecipitation, Plasmid Preparation, Transfection

Journal: Cancer Science

Article Title: N‐acetylgalactosaminyltransferase GALNT6 is a potential therapeutic target of clear cell renal cell carcinoma progression

doi: 10.1111/cas.16296

Figure Lengend Snippet: Silencing of prohibitin 2 (PHB2) inhibits GALNT6 overexpression‐induced proliferation, migration, and invasion of clear cell renal cell carcinoma cells. (A) MTT assay for viability of CAKI1 cells 48 h after plasmid transfection. (B) Transwell assays for cell migration and invasion capacity in CAKI1 cells 48 h after plasmid transfection. Scale bar, 100 μ m. Data are expressed as the mean ± SD. OD, optical density.

Article Snippet: Human ccRCC cell lines CAKI1 and OS‐RC‐2 were obtained from iCell Bioscience.

Techniques: Over Expression, Migration, MTT Assay, Plasmid Preparation, Transfection

Journal: Cancer Science

Article Title: N‐acetylgalactosaminyltransferase GALNT6 is a potential therapeutic target of clear cell renal cell carcinoma progression

doi: 10.1111/cas.16296

Figure Lengend Snippet: Lens epithelium‐derived growth factor (LEDGF) is responsible for GALNT6‐mediated clear cell renal cell carcinoma progression. (A) LEDGF expression levels in an mRNA expression profile dataset GSE15641. (B) Real time‐quantitative PCR and western blot analyses for transfection efficiencies in CAKI1 cells. (C) MTT assay for proliferation of CAKI1 cells. (D) Transwell assays for cell migration and invasion capacity in CAKI1 cells. Scale bar, 100 μ m. (E) Dual‐luciferase reporter assay for luciferase activity of GALNT6 in CAKI1 cells. (F) MTT assay for viability of CAKI1 cells 48 h after transfection. (G) Transwell assays for cell migration and invasion capacity in CAKI1 cells. Scale bar, 100 μ m. Data are expressed as the mean ± SD.

Article Snippet: Human ccRCC cell lines CAKI1 and OS‐RC‐2 were obtained from iCell Bioscience.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, MTT Assay, Migration, Luciferase, Reporter Assay, Activity Assay

Journal: Cell Death & Disease

Article Title: Rho-GEF Trio regulates osteosarcoma progression and osteogenic differentiation through Rac1 and RhoA

doi: 10.1038/s41419-021-04448-3

Figure Lengend Snippet: A The Trio mRNA levels in different cancer type cell lines from the CCLE database. B The expression levels of Trio in sarcoma and normal control tissues were achieved from the TCGA database. C and D Kaplan–Meier curves show the disease-free survival and the overall survival of patients with sarcoma (TCGA database) in terms of Trio gene expression. E and F Kaplan–Meier curves show the disease-free survival and the overall survival of patients with osteosarcoma (TARGET database) in terms of Trio gene expression. G – I The expression levels of Trio in hFOB1.19, U2OS,143B were measured by Western blot and qRT-PCR. J The expression levels of Trio in osteosarcoma and normal control tissues were detected by IHC. Representative images are shown (magnification at ×200). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01. Scale bars: 100 μm.

Article Snippet: Normal human osteoblast cell line hFOB1.19, as well as human OS cell line U2OS, were purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Control, Gene Expression, Western Blot, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Rho-GEF Trio regulates osteosarcoma progression and osteogenic differentiation through Rac1 and RhoA

doi: 10.1038/s41419-021-04448-3

Figure Lengend Snippet: A – C Efficiency of siRNA-mediated Trio knockdown in U2OS and 143B cells was measured by qRT-PCR and Western blot. D SiRNA-mediated Trio knockdown suppressed OS cell growth, as determined by the CCK-8 assay. E Efficiency of shRNA-mediated Trio knockdown in U2OS and 143B cells was detected by GFP. F and G Efficiency of shRNA-mediated Trio knockdown in U2OS and 143B cells was measured by qRT-PCR and Western blot. H and I Trio knockdown suppressed the colony formation of U2OS and 143B cells. J Cell apoptosis was measured by cytometry. Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01. Scale bars: 100 μm.

Article Snippet: Normal human osteoblast cell line hFOB1.19, as well as human OS cell line U2OS, were purchased from GeneChem (Shanghai, China).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, CCK-8 Assay, shRNA, Cytometry

Journal: Cell Death & Disease

Article Title: Rho-GEF Trio regulates osteosarcoma progression and osteogenic differentiation through Rac1 and RhoA

doi: 10.1038/s41419-021-04448-3

Figure Lengend Snippet: A and B Wound-healing assay was used to measure the migration capacity of U2OS and 143B cells. Representative images are shown (magnification at ×100). C and D Transwell migration and Matrigel invasion assays were used to measure the migration and invasion ability of U2OS and 143B cells. Representative images are shown (magnification at ×200). E Cells of the siRNA-Trio group displayed more stress fibers and stable actin structures compared with the control group. Representative images are shown (magnification at ×400). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01. Scale bars: 100 μm.

Article Snippet: Normal human osteoblast cell line hFOB1.19, as well as human OS cell line U2OS, were purchased from GeneChem (Shanghai, China).

Techniques: Wound Healing Assay, Migration, Control

Journal: Cell Death & Disease

Article Title: Rho-GEF Trio regulates osteosarcoma progression and osteogenic differentiation through Rac1 and RhoA

doi: 10.1038/s41419-021-04448-3

Figure Lengend Snippet: A and B EMT markers and Snail were measured by western blotting in U2OS and 143B cells after transfection. C EMT markers and transcription factors (MMP9 and Snail) were measured by qRT-PCR. D U2OS and 143B cells were subjected to immunofluorescence with E-Cadherin and N-Cadherin antibodies. Local enlarged images showed the membrane localization of E-cad. Representative images are shown (magnification at ×200). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01. Scale bars: 100 μm.

Article Snippet: Normal human osteoblast cell line hFOB1.19, as well as human OS cell line U2OS, were purchased from GeneChem (Shanghai, China).

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Immunofluorescence, Membrane

Journal: Cell Death & Disease

Article Title: Rho-GEF Trio regulates osteosarcoma progression and osteogenic differentiation through Rac1 and RhoA

doi: 10.1038/s41419-021-04448-3

Figure Lengend Snippet: A and B osteogenic markers were measured by western blotting in U2OS and 143B cells after transfection. C osteogenic markers and transcription factors were measured by qRT-PCR. D , E U2OS and 143B cells were stained with the ALP kit. Representative images are shown (magnification at ×200). F , G U2OS and 143B cells were stained with ARS solution. Representative images are shown (magnification at 200×). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01. Scale bars: 100 μm.

Article Snippet: Normal human osteoblast cell line hFOB1.19, as well as human OS cell line U2OS, were purchased from GeneChem (Shanghai, China).

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Staining