Journal: Frontiers in Cell and Developmental Biology

Article Title: Hsa_circ_0000652 Aggravates Inflammation by Activation of Macrophages and Enhancement of OX40/OX40L Interaction in Ankylosing Spondylitis

doi: 10.3389/fcell.2021.737599

Figure Lengend Snippet: Hsa_circ_0000652 is upregulated in PBMCs from AS patients and correlated with disease activity. (A–B) Expression of hsa_circ_0000652 in PBMCs from 76 AS patients with different disease activities and 40 healthy controls was analyzed by quantitative real-time PCR (qRT-PCR). (C–E) Correlation analysis for the expression of hsa_circ_0000652 with the Ankylosing Spondylitis Disease Activity Score (ASDAS CRP ), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), and C-reactive protein (CRP) level (mg/L) of AS patients (n = 72). (F) Schematic illustration showing the formation of hsa_circ_0000652 from its host gene IQGAP1 by back-splicing. (G) Agarose gel electrophoresis of PCR amplification by divergent and convergent primers of hsa_circ_0000652 in cDNA and gDNA. (H) Back-splice junction point of hsa_circ_0000652 was identified by Sanger sequencing. (I) qRT-PCR analysis of hsa_circ_0000652 and IQGAP1 and GAPDH with or without RNase R treatment (n = 3). (J–K) FISH assay and nuclear-cytoplasmic separation assay showed that hsa_circ_0000652 was mainly localized in the cytoplasm of the THP-1 cell line. Data in A and B are presented as median ± quartile and data in I and H are presented as mean ± SD. *** p < 0.001.

Article Snippet: The human THP-1 and Jurkat T-cell lines were purchased from iCell Bioscience (Shanghai, China).

Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Agarose Gel Electrophoresis, Amplification, Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hsa_circ_0000652 Aggravates Inflammation by Activation of Macrophages and Enhancement of OX40/OX40L Interaction in Ankylosing Spondylitis

doi: 10.3389/fcell.2021.737599

Figure Lengend Snippet: Hsa_circ_0000652 promotes the activation of macrophages. (A) Quantitative real-time PCR (qRT-PCR) analysis of IL-1b, IL-12, TNF-α, and hsa_circ_0000652 during the activation of THP-1–derived macrophages (n = 3); (B–C) observation of copGFP and qRT-PCR analysis in hsa_circ_0000652 stably expressed THP-1 cell lines; (D–G) 5-ethynyl-2′-deoxyuridine (EdU) assay and cell counting kit-8 (CCK-8) assay after overexpression and knockdown of hsa_circ_0000652; (H–I) Annexin V–PI assay determined the apoptotic cell ratio of THP-1–derived macrophages with overexpression and knockdown of hsa_circ_0000652 (n = 3); (J–L) ELISA of cytokine levels in the culture medium of THP-1–derived macrophages with overexpression and knockdown of hsa_circ_0000652 (n = 3). Data in bar plots and line plots are presented as mean ± SD, and data in scatter plots are supplemented with line of mean. ** p < 0.01; *** P < 0.001. Each experiment was performed at least in triplicate.

Article Snippet: The human THP-1 and Jurkat T-cell lines were purchased from iCell Bioscience (Shanghai, China).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, Stable Transfection, EdU Assay, Cell Counting, CCK-8 Assay, Over Expression, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hsa_circ_0000652 Aggravates Inflammation by Activation of Macrophages and Enhancement of OX40/OX40L Interaction in Ankylosing Spondylitis

doi: 10.3389/fcell.2021.737599

Figure Lengend Snippet: Hsa_circ_0000652 upregulates OX40L expression by sponging hsa-miR-1179. (A–B) Binding sites of hsa_circ_0000652 and hsa-miR-1179 and 3′ UTR of OX40L mRNA and hsa-miR-1179 predicted by circInteractome, TargetScan, and MiRanda platforms. (C) Co-localization analysis of hsa_circ_0000652 and hsa-miR-1179 in THP-1–derived macrophages by fluorescence in situ hybridization (FISH) assay. (D) Quantitative real-time PCR (qRT-PCR) analysis of hsa-miR-1179 in hsa_circ_0000652 stably expressed THP-1 cells and THP-1 transfected with the hsa-miR-1179 mimic or negative control (n = 3). (E) Correlation analysis for hsa_circ_0000652 and OX40L mRNA in PBMCs from AS patients and healthy controls (n = 103). (F) Dual-luciferase assay detected the binding between hsa_circ_0000652 and hsa-miR-1179. (G) qRT-PCR analysis of hsa-miR-1179 in the immunoprecipitates of RNA immunoprecipitation (RIP) assay using probes for hsa_circ_0000652 or the negative control (n = 3). (H) qRT-PCR analysis of hsa_circ_0000652 and OX40L mRNA in the immunoprecipitates of RIP assay using probes for hsa-miR-1179 or the negative control (n = 3). (I) QRT-PCR analysis of OX40L in hsa_circ_0000652 stably expressed THP-1 cells and THP-1 transfected with hsa-miR-1179 mimic or the negative control (n = 3). (J) Flow cytometry analysis of OX40L on hsa_circ_0000652 stably expressed THP-1 cells (n = 3). Data in bar plots are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The human THP-1 and Jurkat T-cell lines were purchased from iCell Bioscience (Shanghai, China).

Techniques: Expressing, Binding Assay, Derivative Assay, Fluorescence, In Situ Hybridization, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Stable Transfection, Transfection, Negative Control, Luciferase, Immunoprecipitation, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hsa_circ_0000652 Aggravates Inflammation by Activation of Macrophages and Enhancement of OX40/OX40L Interaction in Ankylosing Spondylitis

doi: 10.3389/fcell.2021.737599

Figure Lengend Snippet: Hsa_circ_0000652 regulates inflammation caused by OX40/OX40L interaction between macrophages and CD4 + T cells. (A) Schematic illustration of macrophages and CD4 + T-cell coculture system. (B) QRT-PCR analysis of OX40 and OX40L in CD4 + T cells after being cocultured with hsa_circ_0000652 stably expressed THP-1 cell lines with or without hsa-miR-1179 mimics (n = 3). (C,D) Flow cytometry of OX40 in CD4 + T cells after coculturing with hsa_circ_0000652 stably expressed THP-1 cell lines with or without hsa-miR-1179 mimics (n = 3). (E–G) ELISA analysis of cytokine levels in the supernatant of CD4 + T cells after coculturing with hsa_circ_0000652 stably expressed THP-1–derived macrophages with or without transfection of hsa-miR-1179 (n = 3). Data are presented as mean ± SD. ns: p ≥ 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001. Each experiment was performed at least in triplicate.

Article Snippet: The human THP-1 and Jurkat T-cell lines were purchased from iCell Bioscience (Shanghai, China).

Techniques: Quantitative RT-PCR, Stable Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Transfection

Journal: Cancers

Article Title: AMPKα1 Regulates Lung and Breast Cancer Progression by Regulating TLR4-Mediated TRAF6-BECN1 Signaling Axis

doi: 10.3390/cancers12113289

Figure Lengend Snippet: AMPKα1 -knockdown THP-1 cells show impaired autophagy flux induced by TLR4 stimulation. ( A ) AMPKα1 KD THP-1 cells were generated as described in Materials and Methods. Knockdown efficacy of AMPKαl was confirmed by western blotting with anti-AMPKαl antibody. ( B ) Control (Ctrl) and AMPKα1 KD THP-1 cells were treated with or without LPS for 2 h. Cell lysates were immunoblotted with antibodies specific for LC3-I/-II, GAPDH, phosphorylated TAK1 (pho-TAK1), or TAK1. ( C ) HEK293T cells were transfected with mock, AMPKαl WT, or AMPKα1 (D159A) DN as indicated. Cells were treated with or without LPS. Cell lysates were immunoblotted with antibodies specific for LC3-I/-II, AMPKαl, or GAPDH. ( D , E ) Ctrl and AMPKα1 KD THP-1 cells were treated with or without LPS for 6 h. Expression levels of autophagy-related Atg genes (13 different genes, ( D )) and autophagosome synthesis-related genes (21 different genes, ( E )) were compared between Ctrl THP-1 cells treated with LPS and Ctrl THP-1 cells without LPS treatment (lane 1), between AMPKα1 KD THP-1 cells treated with LPS and AMPKα1 KD THP-1 cells without LPS treatment (lane 2), or between AMPKα1 KD THP-1 cells treated with LPS and Ctrl THP-1 cells treated with LPS (lane 3). ATG7, Autophagy Related 7; GABARAP, Gamma-aminobutyric acid receptor-associated protein; ATG12, Autophagy Related 12; ATG4A, Autophagy Related 4A Cysteine Peptidase; MAP1B, Microtubule-associated protein 1B; ULK1, Unc-51 Like Autophagy Activating Kinase 1; ATG16L1, Autophagy Related 16 Like 1; ATG9A, Autophagy-related protein 9A; ATG4D, Autophagy Related 4D Cysteine Peptidase; C12orf44, Autophagy-related protein 101; MAP1A, Microtubule Associated Protein 1A; ATG2A, Autophagy Related 2A; PI4K2B, Phosphatidylinositol 4-kinase type 2-beta; IP6K1, Inositol Hexakisphosphate Kinase 1; PI4K2A, Phosphatidylinositol 4-Kinase Type 2 Alpha; INPP1, Inositol Polyphosphate-1-Phosphatase; MTMR3, Myotubularin-related protein 3; PI4KB, Phosphatidylinositol 4-Kinase Beta; PIP5K1A, Phosphatidylinositol-4-Phosphate 5-Kinase Type 1 Alpha; PIP5K1C, Phosphatidylinositol-4-Phosphate 5-Kinase Type 1 Gamma; MTMR6, Myotubularin Related Protein 6; PIK3CD, Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Delta; PI4KA, Phosphatidylinositol 4-kinase alpha; CDS1, CDP-Diacylglycerol Synthase 1; PIKFYVE, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing; PIK3C3, Phosphatidylinositol 3-Kinase Catalytic Subunit Type 3; MTMR1, Myotubularin Related Protein 1; ITPKA, Inositol-Trisphosphate 3-Kinase A. Uncropped western blot images are available in .

Article Snippet: To generate control (Ctrl) THP-1 and AMPKα1 KD THP-1 cells, lentivirus containing small hairpin RNA (shRNA) targeting human AMPKαl (Santa Cruz Biotechnology, sc-29673-V, Santa Cruz, CA, USA) and control shRNA lentivirus (Santa Cruz Biotechnology, Santa Cruz, sc-108080, Santa Cruz, CA, USA) were used to infect THP-1 cells.

Techniques: Generated, Western Blot, Transfection, Expressing