CSIG-18. HYPERMETHYLATION OF SLIT-ROBO PATHWAY GENES RESULTS IN INACTIVATION IN GLIOMA PROGRESSION

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi37-vi37
Author(s):  
Amber Kerstetter-Fogle ◽  
Peggy Harris ◽  
Harry Hoffman ◽  
Anthony Sloan ◽  
Theresa Elder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
The Cancer Genome Atlas ◽  
Glioma Stem Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Western Blots

Abstract INTRODUCTION Glioblastomas (GBM) are the most common and malignant primary brain tumors. Their rapid growth and invasion into neuronal parenchyma is devastating, with limited treatment options. Genomic alterations have been extensively studied in gliomas tumors, including aberrations of the Slit-Robo pathway genes. The Slit family of secreted proteins modulates migration of somatic cells during development and mediate their effect by binding to its receptor Roundabout (Robo). Genes in the Slit-Robo pathways have been shown to be inactivated by promoter hypermethylation in a number of human cancers. We hypothesize that Slit-Robo signaling pathways are modulated via promotor methylation, controlling GBM malignancy and regulating the invasive capacity of glioma stem cells (GSCs). METHODS We characterized expression of mRNA and protein via real-time PCR and Western blots in primary GBM tissue. We then assessed whether epigenetic alterations correlate with Slit-Robo expression by conducting in vitro demethylation assays in patient derived GSCs followed by real-time PCR. We conducted invasion assays to elucidate the role of Slit in GSC invasion. RESULTS The Cancer Genome Atlas indicates a negative correlation between Robo2 expression and glioma patient survival (17.1 months versus 37.4 months; p < 1.4E-4). We discovered differential expression of the Slit (1-3) and Robo (2, 3) genes and protein of up to 8-fold between grades 3 and 4 astrocytomas (8.34 versus 4.73 density). Treating patient derived glioma stem cells with 5-aza-2-deoxycytidine results in induction in Slit-Robo gene expression ranging from 5 – 40-fold (p < 0.01). Addition of Slit 2 and 3 in an invasion assay induced migration of GSCs in a differential and concentration dependent manner of 10 - 25% (p < 0.05). CONCLUSIONS Our results suggest that promoter methylation and gene expression of the Slit-Robo genes correlates with protein expression, tumor invasiveness, and prognosis and a potential therapeutic target.

(-)-Epigallocatechin Gallate Promotes MicroRNA 145 Expression against Myocardial Hypoxic Injury through Dab2/Wnt3a/β-catenin

2020 ◽  
Vol 48 (02) ◽  
pp. 341-356
Author(s):  
Chiu-Mei Lin ◽  
Wei-Jen Fang ◽  
Bao-Wei Wang ◽  
Chun-Ming Pan ◽  
Su-Kiat Chua ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Real Time ◽  
Real Time Pcr ◽  
Myocardial Infarct ◽  
Epigallocatechin Gallate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rat Cardiomyocytes

MicroRNA 145 (miR-145) is a critical modulator of cardiovascular diseases. The downregulation of myocardial miR-145 is followed by an increase in disabled-2 (Dab2) expression in cardiomyocytes. (-)-epigallocatechin gallate (EGCG) is a flavonoid that has been evaluated extensively due to its diverse pharmacological properties including anti-inflammatory effects. The aim of this study was to investigate the cardioprotective effects of EGCG under hypoxia-induced stress in vitro and in vivo. The hypoxic insult led to the suppression of miR-145 expression in cultured rat cardiomyocytes in a concentration-dependent manner. Western blotting and real-time PCR were performed. In rat myocardial infarction study, in situ hybridization, and immunofluorescent analyses were adopted. The western blot and real-time PCR data revealed that hypoxic stress with 2.5% O2 suppressed the expression of miR-145 and Wnt3a/[Formula: see text]-catenin in cultured rat cardiomyocytes but augmented Dab2. Treatment with EGCG attenuated Dab2 expression, but increased Wnt3a and [Formula: see text]-catenin in hypoxic cultured cardiomyocytes. Following in vivo myocardial infarction (MI) study, the data revealed the myocardial infarct area reduced by 48.5%, 44.6%, and 48.5% in EGCG (50[Formula: see text]mg/kg) or miR-145 dominant or Dab2 siRNA groups after myocardial infarction for 28 days, respectively. This study demonstrated that EGCG increased miR-145, Wnt3a, and [Formula: see text]-catenin expression but attenuated Dab2 expression. Moreover, EGCG ameliorated myocardial ischemia in vivo. The novel suppressive effect was mediated through the miR-145 and Dab2/Wnt3a/[Formula: see text]-catenin pathways.

scholarly journals Hypermethylation in Calca Promoter Inhibited ASC Osteogenic Differentiation in Rats with Type 2 Diabetic Mellitus

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Lei Wang ◽  
Feng Ding ◽  
Shaojie Shi ◽  
Xingxing Wang ◽  
Sijia Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Methylation Level ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Target Fragment

The abnormal environment of type 2 diabetes mellitus (T2DM) leads to a substantial decrease in osteogenic function of stem cells. However, the gene sequence does not vary before and after disease for the patient. This phenomenon may be related to changes in osteogenesis-related gene expression caused by DNA methylation. In this study, we established T2DM models to extract adipose-derived stem cells (ASCs) for different gene identifications through DNA methylation sequencing. Specific fragments of methylation changes in the target gene (Calca) were identified by IGV analysis. CGRP was applied to compare the effects on ASCs-T2DM morphology via phalloidin staining, proliferation through CCK-8 assay, and osteogenic differentiation with osteogenic staining, qPCR, and repair of calvarial defect. Furthermore, 5-azacytidine (5-az) was used to intervene ASCs-T2DM to verify the relationship between the methylation level of the target fragment and expression of Calca. We found that the DNA methylation level of target fragment of Calca in ASCs-T2DM was higher than that in ASCs-C. CGRP intervention showed that it did not change the morphology of ASCs-T2DM but could improve proliferation within a certain range. Meanwhile, it could significantly enhance the formation of ALP and calcium nodules in ASCs-T2DM, increase the expression of osteogenesis-related genes in vitro, and promote the healing of calvarial defects of T2DM rat in a concentration-dependent manner. 5-az intervention indicated that the reduction of the methylation level in Calca target fragment of ASCs-T2DM indeed escalated the gene expression, which may be related to DNMT1. Taken together, the environment of T2DM could upregulate the methylation level in the promoter region of Calca and then decrease the Calca expression. The coding product of Calca revealed a promoting role for osteogenic differentiation of ASCs-T2DM. This result provides an implication for us to understand the mechanism of the decreased osteogenic ability of ASCs-T2DM and improve its osteogenic capacity.

scholarly journals Cell surface GRP78 regulates BACE2 via lysosome-dependent manner to maintain mesenchymal phenotype of glioma stem cells

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zihang Chen ◽  
Huizhi Wang ◽  
Zongpu Zhang ◽  
Jianye Xu ◽  
Yanhua Qi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Stem Cells ◽  
Cell Surface ◽  
Bioinformatic Analysis ◽  
The Cancer Genome Atlas ◽  
Glioma Stem Cells ◽  
Dependent Manner ◽  
Cell Surface Grp78

Abstract Background Glioma stem cells (GSCs) are considered the initial cells of gliomas, contributing to therapeutic resistance. Patient-derived GSCs well recapitulate the heterogeneity of their parent glioma tissues, which can be classified into different subtypes. Likewise, previous works identified GSCs as two distinct subtypes, mesenchymal (MES) and proneural (PN) subtypes, and with general recognition, the MES subtype is considered a more malignant phenotype characterized by high invasion and radioresistance. Therefore, understanding the mechanisms involved in the MES phenotype is necessary for glioblastoma treatment. Methods Data for bioinformatic analysis were obtained from The Cancer Genome Atlas (TCGA) and The Gene Expression Omnibus (GEO) database. An antibody was used to block cell surface glucose-regulated protein 78 (csGRP78). Apoptosis and cell cycle analyses were performed to evaluate radiation damage. Immunofluorescence staining was applied to assess protein expression and distribution. Mass spectrometry combined with bioinformatic analysis was used to screen downstream molecules. Intracranial GSC-derived xenografts were established for in vivo experiments. Results Total GRP78 expression was associated with MES GSC stemness, and csGRP78 was highly expressed in MES GSCs. Targeting csGRP78 suppressed the self-renewal and radioresistance of MES GSCs in vitro and in vivo, accompanied by downregulation of the STAT3, NF-κB and C/EBPβ pathways. Mass spectrometry revealed the potential downstream β-site APP-cleaving enzyme 2 (BACE2), which was regulated by csGRP78 via lysosomal degradation. Knockdown of BACE2 inactivated NF-κB and C/EBPβ and significantly suppressed the tumorigenesis and radioresistance of MES GSCs in vitro and in vivo. Conclusions Cell surface GRP78 was preferentially expressed in MES GSCs and played a pivotal role in MES phenotype maintenance. Thus, blocking csGRP78 in MES GSCs with a high-specificity antibody might be a promising novel therapeutic strategy.

scholarly journals Modulatory Effects of Osthole on Lipopolysaccharides-Induced Inflammation in Caco-2 Cell Monolayer and Co-Cultures with THP-1 and THP-1-Derived Macrophages

Nutrients ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 123
Author(s):  
Natalia K. Kordulewska ◽  
Justyna Topa ◽  
Małgorzata Tańska ◽  
Anna Cieślińska ◽  
Ewa Fiedorowicz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inflammatory Reaction ◽  
Wide Spectrum ◽  
Cell Monolayer ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tnf Α

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150–450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1β, -6, -8, and TNF-α, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p < 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/μL for IL1R1 and COX-2 (p < 0.01) and 300 ng/μL for NF-κB (p < 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

Oxygen modulates the differentiation of human fetal lung in vitro and its responsiveness to cAMP

1993 ◽  
Vol 264 (5) ◽  
pp. L465-L474 ◽  
Author(s):  
M. J. Acarregui ◽  
J. M. Snyder ◽  
C. R. Mendelson
Keyword(s):  
Gene Expression ◽  
Atmospheric Oxygen ◽  
Protein A ◽  
Morphological Differentiation ◽  
Fetal Lung ◽  
Morphological Development ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Human Fetal Lung

Previously, it was found that lung explants from mid-trimester human abortuses differentiate spontaneously in organ culture in serum-free defined medium in an atmosphere of 95% air-5% CO2. Dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) treatment of human fetal lung in culture increases the rate of morphological differentiation and enhances expression of the surfactant protein A (SP-A) gene. To begin to define the factors responsible for this accelerated in vitro differentiation, we analyzed the effects of atmospheric oxygen on the morphological and biochemical development of human fetal lung in culture and on responsiveness of the cultured tissue to DBcAMP. We found that when lung explants were maintained in an atmosphere containing 1% oxygen they failed to differentiate spontaneously and no induction of SP-A gene expression was apparent. Furthermore, at 1% oxygen, DBcAMP had no effect to stimulate morphological differentiation or SP-A gene expression. When lung tissues that had been maintained for 5 days in 1% oxygen were transferred to an environment containing 20% oxygen, there was rapid morphological development and induction of SP-A gene expression. The effects on morphological development were manifest within 24 h of transfer to the 20% oxygen environment; within 72 h, a marked stimulatory effect of DBcAMP on SP-A gene expression also was observed. Our findings further suggest that the effects of oxygen on the levels of SP-A and SP-A mRNA are concentration dependent. Interestingly, the inductive effects of DBcAMP on SP-A gene expression were apparent only at oxygen concentrations > or = 10%. Morphological differentiation of the cultured human fetal lung tissue also was influenced by oxygen in a concentration-dependent manner. These findings suggest that oxygen plays an important permissive role in the spontaneous differentiation of human fetal lung in vitro.

Effect of Auraptene on angiogenesis in Xenograft model of breast cancer

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mohammad Reza Shiran ◽  
Elham Mahmoudian ◽  
Abolghasem Ajami ◽  
Seyed Mostafa Hosseini ◽  
Ayjamal Khojasteh ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Animal Model ◽  
Protein Expression ◽  
Western Blot ◽  
Xenograft Model ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Abstract Objectives Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. Methods In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1 × 104 HUVEC treated with Auraptene, 20 ng/mL EGF, 20 ng/mL bFGF and 20 ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Results Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80 μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Conclusions Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

2020 ◽  
pp. 15-34
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Tissue Regeneration ◽  
Real Time Pcr ◽  
Molecular Level ◽  
Northern Blotting ◽  
Tissue Constructs ◽  
Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

209 9-cis RETINOIC ACID INHIBITS EXPRESSION OF AKR1B1 TRANSCRIPT IN THE BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 252
Author(s):  
G. K. Deb ◽  
S. R. Dey ◽  
K. S. Huque ◽  
M. Fokruzzaman ◽  
K. L. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Embryo Implantation ◽  
Cumulus Cell ◽  
Prostaglandin F2α ◽  
Receptor Expression ◽  
Bovine Embryo

Quantitative real-time PCR has enabled quality evaluation of oocyte and pre-implantation embryo through monitoring expression of several molecular markers that are involved in metabolic activity, stress response, reprogramming, and other biological events. The aldo-keto reductase family 1 member B1 (AKR1B1) transcript is potentially involved in pregnancy failure through metabolism of progesterone and synthesis of prostaglandin F2α in the bovine uterine endometrium. High expression of the transcript in blastocysts correlates inhibition of embryo implantation and/or embryo resorption. Maturation of immature oocyte in presence of 9-cis retinoic acid (9-cis RA) increases in vitro bovine embryo development rates and embryo quality. These beneficial effects of 9-cis RA are mediated through multiple mechanisms, including FSH/LH receptor expression, polyadenylation, growth factor signalling, oxidative-stress protection, or decreasing oocyte TNFα gene expression and inhibiting cumulus cell apoptosis during maturation. The present study aimed to evaluate the effect of 9-cis RA on expression pattern of AKR1B1 transcript in the oocyte matured in vitro and embryos (8-cell and Day 8 blastocyst) produced from in vitro matured oocytes in presence or absence of 9-cis RA. Bovine cumulus–oocyte complexes, isolated from ovaries collected at the abattoir, were matured in vitro in the presence of zero (control) or 5 nM 9-cis RA in the maturation medium (TCM199 + 10% fetal bovine serum + 1 µg mL–1 β-oestradiol + 10 µg mL–1 follicle stimulating hormone + 0.6 mM cystein and 0.2 mM Na-pyruvate). After maturation, the oocytes were subjected to standardized in vitro embryo production protocol or oocyte samples were collected for gene expression analysis. The expression of AKR1B1 transcript was quantified in zona-free oocytes, 8-cell embryos, and Day 8 blastocysts by real-time PCR using SYBER green. Not less than 4 biological replicates (oocytes: 50 to 60 per replicate and 8-cell embryos/day-8 blastocyst: 3 to 5 per replicate) were done for each group. The expression was normalized against a minimum of 2 out of 4 reference transcripts (18S rRNA, β-actin, glyceraldehyde-3-phosphate dehydrogenase and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) analysed each time with AKR1B1. The best combination of reference genes was automatically calculated by the CFX manager V1.1 program (Bio-Rad) based on M-value. The differences in gene expression levels were tested by Student’s t-test. Results indicated that 9-cis RA decreased expression of AKR1B1 transcript in the oocyte (1.0- v. 2.0-fold; P < 0.05), 8-cell-embryos (1.0- v. 10.1-fold; P < 0.03), and blastocyst (1.0- v. 2.1-fold; P < 0.03) compared with control. In conclusion, the present study indicates that 9-cis RA inhibits AKR1B1 transcript expression in oocytes and pre-implantation embryos.

scholarly journals Platelet Microparticles Accelerate Proliferation and Growth of Mesenchymal Stem Cells through Longevity-Related Genes

2021 ◽  
Vol 24 (8) ◽  
pp. 607-614
Author(s):  
Maryam Samareh Salavati Pour ◽  
Fatemeh Hoseinpour Kasgari ◽  
Alireza Farsinejad ◽  
Ahmad Fatemi ◽  
Gholamhossein Hassanshahi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Treated Group ◽  
Population Doubling ◽  
Control Group ◽  
Population Doubling Time ◽  
Longevity Genes

Background: Due to their self-renewal and differentiation ability, the mesenchymal stem cells (MSCs) have been studied extensively. However, the MSCs lifespan is restricted; they undergo several divisions in vitro that cause several alternations in cellular features and relatively lessens their application. Thus, this study was aimed to assess the effect of platelet-derived microparticles (PMPs), a valuable source of proteins, microRNAs (miRNAs), and growth factors, on the expression of hTERT, c-MYC, p16, p53, and p21 as the most important aging and cell longevity genes alongside with population doubling time (PDT) of PMP-treated cells in comparison to a control group. Methods: Umbilical cord MSCs (UC-MSCs) were used in this study, whereby they reached a confluency of 30%. MSCs were treated by PMPs (50 µg/mL), and then, PDT was determined for both groups. Quantitative expression of hTERT, c-MYC, p16, p53, and p21 was examined through quantitative real-time PCR at various intervals (i.e. after five and thirty days as well as freezing-thawing process). Results: Our results demonstrated that the treated group had a shorter PDT in comparison to the control group (P<0.050). The real-Time PCR data also indicated that PMPs were able to remarkably up-regulate hTERT and c-MYC genes expression while down-regulating the expression of p16, p21, and p53 genes (P<0.050), especially following five days of treatment. Conclusion: According to these data, it appears that PMPs are a safe and effective candidate for prolonging the lifespan of UC-MSCs; however, further investigations are needed to corroborate this finding.

scholarly journals Novel Roles of Chloroquine and Hydroxychloroquine in Graves’ Orbitopathy Therapy by Targeting Orbital Fibroblasts

2020 ◽  
Vol 105 (6) ◽  
pp. 1906-1917 ◽  
Author(s):  
Yan Guo ◽  
Hai Li ◽  
Xueying Chen ◽  
Huasheng Yang ◽  
Hongyu Guan ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Fluorescent Protein ◽  
Inhibitory Effect ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Graves Orbitopathy ◽  
Orbital Fibroblasts

Abstract Context Graves’ orbitopathy (GO) causes infiltrative exophthalmos by inducing excessive proliferation, adipogenesis, and glycosaminoglycan production in orbital fibroblasts (OFs). Interference with OF autophagy is a potential therapy for proptosis. Objectives Here, we aimed to evaluate the effects of chloroquine (CQ) and hydroxychloroquine (HCQ), the autophagy inhibitors commonly used in clinical practice, on OFs. Design/Setting/Participants OFs isolated from patients with GO (GO-OFs) or control individuals (non-GO-OFs) were cultured in proliferation medium (PM) or subjected to differentiation medium. OFs were treated with CQ or HCQ (0, 0.5, 2, and 10 μM), and subsequently examined in vitro. Main Outcome Measures CCK-8, EdU incorporation, and flow cytometry assays were used to assess cellular viability. Adipogenesis was assessed with Western blot analysis, real-time polymerase chain reaction (PCR) , and Oil Red O staining. Hyaluronan production was determined by real-time PCR and enzyme-linked immunosorbent assay. Autophagy flux was detected through red fluorescent protein (RFP)-green fluorescent protein (GFP)-LC3 fluorescence staining and Western blot analyses. Results CQ/HCQ halted proliferation and adipogenesis in GO-OFs in a concentration-dependent manner through blockage of autophagy, phenotypes that were not detected in non-GO-OFs. The inhibitory effect of CQ/HCQ on hyaluronan secretion of GO-OFs was also concentration dependent, mediated by downregulation of hyaluronan synthase 2 rather than hyaluronidases. Moreover, CQ (10 μM) induced GO-OF apoptosis without aggravating oxidative stress. Conclusions The antimalarials CQ/HCQ affect proliferation, adipogenesis, and hyaluronan generation in GO-OFs by inhibiting autophagy, providing evidence that they can be used to treat GO as autophagy inhibitors.

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