Journal: medRxiv

Article Title: ARF1 prevents aberrant type I IFN induction by regulating STING activation and recycling

doi: 10.1101/2023.04.28.23289152

Figure Lengend Snippet: a , Induction of the ISRE promoter in THP-1-Dual WT (grey) and THP-1-Dual KO-cGAS (pink) cells transduced with lentiviral vectors expressing ARF1 WT or R99C as indicated, quantified by Lucia luciferase (LLuc) activity 72 h post transduction. IFN-β (1000 U/mL, 16 h) and cGAMP (10 µg/ml, 16 h) served as positive controls. Bars represent mean of n=3 ± SEM (biological replicates). Lower panel: Corresponding immunoblots of WCLs stained by anti-FLAG, anti-STING, anti-cGAS and anti-GAPDH. b , Exemplary electron microscopy analysis of HEK293T cells transiently transfected with ARF1 WT or ARF1 R99C as indicated. Mitochondria (m) are highlighted in insets in bottom panels. Electron-dense granules and inflated cristae are highlighted by black and white arrows, respectively. Annotations: cp, cytoplasm; er, endoplasmic reticulum; g , Golgi apparatus; lv, large vesicle; ly, lysosome; m, mitochondria; nc, nucleus. c, Exemplary immunoblots showing fractionation of ARF1 WT, R99C, Q71L and vector transfected HEK293T cells as indicated. WCLs and fraction blots stained by anti-FLAG, anti-TFAM (mitochondria), anti-LAMIN B1 (nucleus) and anti-GAPDH (cytosol). d, qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of ( c ) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using the ΔΔCT method. n = 3 ± SEM. e , qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of primary fibroblasts from healthy donors (n2, f1, I7) or a patient (Patient 1) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using the ΔΔCT method. n = 5 ± SEM. f, qPCR of representative ISG OAS1 in U2OS cells stably expressing STING and depleted of mtDNA by ddC, or untreated (NT), upon transfection with empty vector, ARF1 WT or R99C n=3 ± SEM. g , Exemplary immunoblot of WCLs of HEK293T cells transiently expressing ARF1 WT, R99C or vector. Blots were stained with anti-MFN1, anti-RHOT1, anti-FLAG and anti-GAPDH. Quantification of the band intensities for MFN1 normalized to the band intensities of GAPDH. Bars represent mean of n = 6 ± SEM (biological replicates). h , Exemplary immunoblots showing fractionation of HEK293T cells expressing ARF1 WT, R99C or vector control as well as VCP. WCLs and fraction blots stained by anti-FLAG, anti-HA, anti-TFAM (mitochondria), anti LAMIN B1 (nucleus) and anti-GAPDH (cytosol). i , qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of (h) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using 725 the ΔΔCT method. n = 5 ± SEM.

Article Snippet: U2OS cells (ATCC) were maintained in McCoy medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum.

Techniques: Transduction, Expressing, Luciferase, Activity Assay, Western Blot, Staining, Electron Microscopy, Transfection, Fractionation, Plasmid Preparation, Stable Transfection, Control

Journal: Oncology Letters

Article Title: miR-150 is downregulated in osteosarcoma and suppresses cell proliferation, migration and invasion by targeting ROCK1

doi: 10.3892/ol.2017.5709

Figure Lengend Snippet: miR-150 is downregulated in OS patient tissues and cell lines. (A) Relative expression levels of miR-150 in 40 OS patient tissue samples and adjacent normal tissue sample counterparts. The relative expression levels of miR-150 were determined using RT-qPCR analysis. (B) Relative expression levels of miR-150, determined using RT-qPCR analysis in U2OS, MG63 and SaOS2 OS cell lines and the normal hFOB1.19 human OS cell line. Student's t -test was used to analyze significant differences.*P<0.05. Data are presented as the mean + standard deviation. OS, osteosarcoma; miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: The SaOS2, U2OS and MG63 human OS cell lines and the hFOB1.19 normal human osteoblast cell line were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: miR-150 is downregulated in osteosarcoma and suppresses cell proliferation, migration and invasion by targeting ROCK1

doi: 10.3892/ol.2017.5709

Figure Lengend Snippet: miR-150 overexpression is inversely correlated with OS cell growth. (A) U2OS, (B) MG63 and (C) SaOS2 OS cell lines were transfected with miR-150 and the numbers of cells were determined at different time points using a fluorescence-based CyQUANT cell proliferation assay kit. In all cases, Student's t -test was used to analyze significant differences, *P<0.05. Data are presented as the mean + standard deviation. OS, osteosarcoma; miR, microRNA; NC, negative control.

Article Snippet: The SaOS2, U2OS and MG63 human OS cell lines and the hFOB1.19 normal human osteoblast cell line were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Over Expression, Transfection, Fluorescence, CyQUANT Assay, Proliferation Assay, Standard Deviation, Negative Control

Journal: Oncology Letters

Article Title: miR-150 is downregulated in osteosarcoma and suppresses cell proliferation, migration and invasion by targeting ROCK1

doi: 10.3892/ol.2017.5709

Figure Lengend Snippet: miR-150 overexpression is inversely correlated with OS cell migration and invasion. Transwell migration and invasion assays of (A) U2OS, (B) MG63 and (C) SaOS2 OS cell lines. The assays were performed following transfection of the cell lines with miR-150. Representative images of migrated and invaded cells on the membrane (magnification, ×100). OS, osteosarcoma; miR, microRNA; NC, negative control.

Article Snippet: The SaOS2, U2OS and MG63 human OS cell lines and the hFOB1.19 normal human osteoblast cell line were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Over Expression, Migration, Transfection, Membrane, Negative Control

Journal: Oncology Letters

Article Title: miR-150 is downregulated in osteosarcoma and suppresses cell proliferation, migration and invasion by targeting ROCK1

doi: 10.3892/ol.2017.5709

Figure Lengend Snippet: miR-150 negatively regulates gene expression of ROCK1 in OS cell lines. (A) Reverse transcription-quantitative polymerase chain reaction analysis of mRNA expression of ROCK1 in U2OS, MG63 and SaOS2 OS cell lines overexpressing miR150. Student's t -test was used to analyze significant differences. *P<0.05. Data are presented as the mean + standard deviation. (B) Western blot analysis of protein expression of ROCK1 in U2OS, MG63 and SaOS2 OS cell lines overexpressing miR150. A corresponding NC was used in the absence of miR-150. β-actin protein was used as an additional control. miR, microRNA; ROCK1, Rho-associated kinase 1; NC, negative control.

Article Snippet: The SaOS2, U2OS and MG63 human OS cell lines and the hFOB1.19 normal human osteoblast cell line were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Western Blot, Control, Negative Control

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