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PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
For instance, imatinib, as a first‐line therapy, has significantly improved GIST patient survival .
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
However, most patients eventually experience disease progression due to KIT secondary mutations, including activation loop and gatekeeper mutations .
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Although regorafenib, sunitinib and ripretinib have been approved to overcome imatinib‐resistant mutations of KIT, they both show toxicity and poor clinical responses .
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Several new KIT inhibitors against imatinib‐resistant secondary mutations are currently being evaluated in clinical trials, such as pazopanib , anlotinib and dovitinib .
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In addition, many candidate KIT inhibitors are in preclinical development, such as CHMFL‐KIT‐033/8140 and AZD3229 .
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
However, it takes a long time to complete clinical research, and these drugs are temporarily unable to fulfil the urgent clinical demand.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
It has been demonstrated that imatinib resistance in GISTs is caused by the feedback activation of FGF signalling, which results in a rebound in ERK phosphorylation .
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The combination of the pan‐FGFR inhibitors BGJ398 and imatinib repressed ERK rebound and enhanced the antitumour activity of imatinib in GISTs.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In addition, the Memorial Sloan Kettering Cancer Center (MSKCC) conducted a phase I clinical trial with a combination of imatinib and BGJ398 (Trial Registration: NCT02257541) for patients with locally advanced or metastatic GISTs.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Approximately 30% of the evaluable study cohort attained durable stabilization of disease lasting at least 32 weeks.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
However, combination toxicity was an important factor that limited the ability of this study to meet its primary end point .
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Therefore, there remains an urgent need for more agents that can inhibit both the different resistance mutations of KIT and FGFRs, which are usually highly expressed in GISTs and participate signalling crosstalk with KIT, causing imatinib resistance .
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In this study, through high‐throughput screening using a functional isogenic BaF3 cell library, we found that nintedanib, a triple angiokinase inhibitor of FGFR/VEGFR/PDGFR that has been approved for the treatment of IPF and NSCLC, exhibited potent activity against a panel of primary gain‐of‐function (GOF) mutations and secondary drug resistance mutations in KIT kinase.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Interestingly, in addition to better efficacy against KIT wt, nintedanib showed potent inhibitory effects against primary GOF mutations (V559A/D/G and L576P), a few secondary imatinib‐resistant mutations in the ATP binding pocket (such as V654A and T670I) and activation loops (such as D820E/G/Y, D816E/H/V, A829P) and sunitinib‐resistant mutations such as D816H/V and A829P).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Because nintedanib is a multi‐targeted kinase inhibitor, we believe that this antitumour activity is not only contributed by KIT inhibition alone, its inhibition on other kinases, such as VEGFR and PDGFR, may also play a role.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
However, nintedanib could not completely overcome the resistance mediated by MAP kinase activation via FGF signalling, which may be due to the low drug concentration in tumours or the weak inhibition of FGFR by nintedanib.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In summary, nintedanib eliminated the adaptive response after KIT inhibition in imatinib‐sensitive GISTs.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Additionally, nintedanib may be effective in the imatinib‐resistant clones containing acquired mutations in KIT kinase and FGFR feedback activation that have emerged.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The findings described here support a basis for extending the application of nintedanib as a new strategy to improve the treatment of GIST patients with de novo or acquired resistance to imatinib.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The peer review history for this article is available at https://publons.com/publon/10.1002/1878‐0261.13199.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The most commonly occurring sarcoma of the soft tissue is gastrointestinal stromal tumor (GIST).
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Treatment and prevention of the disease necessitate an understanding of the molecular mechanisms involved.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
However, the role of BRD4 in the progression of GIST is still unclear.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
While it is known there are abundant infiltrating tumor-associated macrophages (TAMs) in the tumor microenvironment, the exact role of these cells has yet to be studied.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
This work showed an upregulation of BRD4 in GIST that was associated with GIST prognosis.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Through gain and loss of function studies, it was found that BRD4 promotes GIST growth and angiogenesis in vitro and in vivo.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Mechanistically, BRD4 enhances CCL2 expression by activating the NF-κB signaling pathway.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Furthermore, this CCL2 upregulation causes recruitment of macrophages into the tumor leading to tumor growth.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
A likely mechanism for interactions in the GIST microenvironment has been outlined by this work to show the role and potential use of BRD4 as a treatment target in GIST.Among sarcomas of soft tissue, the most ubiquitous cancer is gastrointestinal stromal tumor (GIST) with gastrointestinal tract localization.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Previous research suggests the role of KIT and PDGFRA oncogenic mutations in interstitial cells of Cajal, which are mesenchymal pacemaker cells, in disease metastasis, proliferation, and tumorigenesis.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
These mutations in GIST are targeted by molecular drugs, such as imatinib.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Despite the use of such specific drugs that alter the treatment scenario, resistance leads to recurrence in a substantial number of patients.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Another challenge is a lack of effective therapy for GIST when the abovementioned genes are not mutated.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
These shortcoming call for the analysis of the disease mechanisms to consider new therapeutic approaches.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
There are various cells that are not malignant in the tumor microenvironment, such as the most aboundant tumor-associated macrophages (TAMs), which are ubiquitous hematopoietic cells with a migratory nature.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
There are many clinical and epidemiological studies that have highlighted the poor cancer prognosis related to GIST and the number of TAMs.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The progression of cancer involves discrete signals of the microenvironment that influence different cells, such as macrophages.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The tumor microenvironment that influences macrophage orientation and differentiation is influenced by the profile of chemokines at the tumor site.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
An example is the upregulation and role of chemokine (C–C motif) ligand 2 (CCL2) in many cancers, including GIST.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Bromodomain proteins include the mammalian bromodomain and extraterminal domain (BET) family inclusive of BRD2, 3, and 4.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Research has focused on the functioning of BRD2 and 4 in elongation during transcription and regulation of the cell cycle while their possible involvement in inflammation is yet to be uncovered.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
BRD4 has recently been shown to regulate RNA polymerase II elongation and the expression of genes involved in NF-κB-associated inflammation via activation of P-TEFb complex by CDK9.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
In the current study, we identified BRD4, which was markedly upregulated and showed a significant association with pathology and survival in GIST patients.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The expression of CCL2 was increased by BRD4 overexpression via the NF-κB signaling pathway, which led to TAM recruitment, ultimately contributing to tumor growth.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
These findings suggest that BRD4 is involved in GIST via TAMs.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Jonathan Fletcher (DanaFarber Cancer Institute, Boston, MA) kindly provided the GIST882 cell line (from a patient with a K642E: KIT exon 13 homozygous missense mutation).
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Biowit Technologies (Shenzhen, China) was the source of the procured GIST-T1 cell line (from a patient with a V560Y579del: 57 nucleotide inframe mutation in KIT exon 11).
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin were used for culturing at 5% CO2 at 37 °C.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Patients (with written informed consent) who went through surgery at the Second Hospital of Jilin University in the period from February 2012 to March 2015 were the source of the 20 GIST samples used in this study; samples were fixed in formalin and embedded in paraffin.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The samples were subjected to snap freezing in liquid nitrogen and stored at −80 °C untill assayed.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
HE staining was used by two pathologists to pathologically confirm the samples.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The Committees for Ethical Review of Research Involving Human Subjects at the Second Hospital of Jilin University issued an approval for ethical consent.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Sectioning of the samples fixed in formalin and embedded in paraffin into 4-μm sections was performed.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Individually, BRD4, CD31, and CD68 antibody staining was performed with selected slides that were then analyzed by two experienced pathologists.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
After normalization to the staining of the nucleus and the cytoplasm, the staining intensity scores were based on the following scoring system: 0 = no staining; 1 = weak staining; 2 = moderate staining; and 3 = strong staining.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The combination of these scores with the positive cell percentage yielded the final immunohistochemistry (IHC) score.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Quantitative real-time PCR (qRT-PCR) was performed as previously described.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Briefly, TRIzol Reagent (Invitrogen) was used to extract total RNA, followed by reverse transcription with SuperScript II Reverse Transcriptase (Invitrogen) in accordance with the protocols of the manufacturer.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
An ABI 7300 real-time PCR system (Applied Biosystems) was used for the analysis with primers specific for BRD4 and GAPDH.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The 2 method was employed for assessing the changes in the expression of BRD4, with the control being GAPDH mRNA levels.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Western blotting was performed as previously described.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
RIPA buffer (Pierce, Rockford, IL, USA) plus a protease inhibitor cocktail (Roche, Pleasanton, CA, USA) was utilized for lysing cells.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was utilized for resolving proteins, followed by the transfer of proteins to polyvinylidene difluoride membranes.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Overnight incubation with primary antibodies was performed at 4 °C, followed by exposure to the corresponding secondary antibodies conjugated to horseradish peroxidase (HRP).
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Then, blots were incubated with the corresponding HRP-conjugated secondary antibodies.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
ECL regents (Thermo Scientific) were used to observe the resulting bands.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The antibodies used in this study were as follows: BRD4 (ab128874), β-actin (ab8226), MMP9 (ab38898), LOX (ab174316), VEGFA (ab52917), p65 (ab16502), p-p65 (ab86299), Lamin A/C (ab108595), and CCL2 (ab9851; Abcam).
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Cells were seeded into 96-well plates, followed by an exchange of the media to one with CCK8 (100 μl for 10 μl of the latter) for 1 h after overnight culture.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The optical density (OD) was measured using absorbance at 450 nm at various time points: 24, 48, and 72 h. This protocol was in accordance with that used in a previous study.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
To summarize, six-well plates were used for overnight culture of cells, and cells were subsequently incubated with 10 μg/ml BrdU for 20 min.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
This was followed by a double phosphate-buffered saline (PBS) wash and overnight fixation utilizing 70% ethanol at −20 °C.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
This was followed by a 45 min denaturation in 2 N HCL and FITC secondary antibody staining at room temperature for 1 h. Then, 40 g/ml RNase A and 200 g/ml PI were added for 30 min, followed by flow cytometry analysis.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The Buffy coats from healthy voluteers were used to isolate human monocytes.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Ficoll-Hypaque (Pharmacia Corporation) was utilized to isolate peripheral blood monocytes followed by density gradient centrifugation for 50 min at 400 g. Cells were seeded into 24-well plates at a density of 2 × 10 cells/ml in RPMI 1640 medium plus 10% heat-inactivated human AB serum, 50 U penicillin and streptomycin/ml, 2 mM L-glutamine, and 100 ng/ml human macrophage colony-stimulating factor, which was aimed at stimulating the differentiation of macrophages.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Warm medium was used to wash away the cells that did not adhere gently and repeatedly after 6 days of culture.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
CD14 macrophages constituted over 95% of the adherent cells using this approach.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
For the activation of M1 macrophages in vitro, 2 × 10 cells/l of the isolated cells (as described above) were treated for 1 day with 25 μg/ml LPS (lipopolysaccharide; Sigma) to the above-isolated cells, while for the activation of M2-polarized macrophages, 45 ng/ml recombinant human interleukin-4 (IL-4, R&D) was used.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Flow cytometry was conducted to check the differentiated macrophages from monocytes.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Conditioned media for the next round of in vitro experiments was obtained from PBS washing of cells followed by incubation in medium minus supplements for another 24 h. Endothelial cell tube formation was performed as previously described.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Incubation of the GIST cell lines in DMEM minus serum was done as indicated overnight.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Ninety-six-well plates with Matrigel were seeded with 20,000 HUVECs per well along with the above-described conditioned medium.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
After incubation, imaging of tube formation by microscopy and quantification the branch number in the formed HUVEC tubes were performed.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
After treatment in DMEM minus serum overnight as indicated, conditioned medium was collected from the cells for VEGFA-enzyme-linked immunosorbent assay (ELISA) analysis in accordance with the prescribed protocols of the manufacturer.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The absorbance at 470 nm was recorded for the OD values.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The assay for wound healing involved six-well plates used for seeding GIST cells.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
A sterile 200-μm plastic pipette tip was employed to softly scratch the monolayer once cells were 95% confluent, followed by documentation of the wound with photographs.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The Transwell invasion assay involved seeding of 4 × 10 cells suspended in medium lacking serum in the membranes of the upper chamber without/with Matrigel (BD Biosciences) coating.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The lower chamber received 600 μl medium plus 10% FBS.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
After 24 h, the membrane bottom was subjected to 30-min fixation and 0.1% crystal violet staining.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Following the use of a cotton swab to wipe the interior of the membrane, microscopy was utilized to quantify the cells.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Six-well plates were used to culture 500–1000 cells/well that has been exposed to the aforementioned chemicals and combinations.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Renewal of the medium was performed every third day untill the 14th day, after which cells were fixed and stained with crystal violet.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Cells that had been exposed to drugs were compared to control cells exposed to DMSO, and assays were completed in triplicate.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The Animal Care and Use Committee of the Second Hospital of Jilin University issued approval for the following experiments.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Balb/c nude mice (famale, 4 weeks in age) were randomly divided into two groups (n = 6).
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The animals received subcutaneous administration of GIST-882 cells that expressed the vector or BRD4 resuspended in PBS with Matrigel (1:1).
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The tumor volumes were computed with the following formula expression once a visible tumor mass had developed in the animals: (length × width)/2.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Animals were sacrificed 19 days after injection.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
Prism 5.0 (GraphPad software) was employed for analyses.
PMC6901583
RETRACTED ARTICLE: BRD4 promotes tumor progression and NF-κB/CCL2-dependent tumor-associated macrophage recruitment in GIST
The mean±s.d.