PMCID string | Title string | Sentences string |
|---|---|---|
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | The KIT phosphorylation level (p-kit/KIT) was significantly lower in the imatinib-resistant group than in the normal group (**p<0.01). |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Compared with the imatinib-resistant group, p-kit/KIT in both the imatinib group and the CQ group increased significantly (#p<0.05), while the combination group showed an upward trend, but no significant change (p>0.05). |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Compared with the imatinib group, p-kit /KIT in the combination group was higher, but not significant (p>0.05). |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | ERK expression in the CQ group and the combination group increased significantly (p<0.05, p<0.01). |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | ERK expression was significantly higher in the combination group than in the imatinib group (p<0.05) (Figure 4B). |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Compared with the imatinib-resistant group, the ERK phosphorylation level (p-ERK/ERK) in the CQ group and combination group was lower than in the imatinib group, though not significant (p>0.05). |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | The p-ERK/ERK level in the combination group was significantly lower than in the imatinib group (p<0.05) (Figure 4B). |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Secondary resistance to imatinib is a well-described problem in GIST occurring with tumor progression after an initial period of stabilization or response to imatinib. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | The primary strategy for overcoming imatinib resistance is to switch to another tyrosine kinase inhibitor, such as sunitinib and regorafenib.6 It is hypothesized that an increased autophagic flux often favors tumor cell survival and growth. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Despite this potential for confusion, clinical interventions on autophagy regulation as a cancer therapy are already under way.19 Prior studies suggest that autophagy promotes the survival of quiet cancer cells and cancer recurrence.20 In 2018, a research showed that autophagy inhibitors were more useful as resistant cancer-eradicating drugs than the traditional chemotherapeutic agents.21 A combination of ERK and autophagy inhibition has been studied as a treatment approach for pancreatic cancer.22 Bryant et al found that the autophagy inhibitor, chloroquine and genetic or pharmacologic inhibition of specific autophagy regulators synergistically enhanced the ability of ERK inhibitors to mediate antitumor activity in KRAS-driven pancreatic ductal adenocarcinoma.22 In vitro and clinical models suggested that PI3K signaling, p53 status, RAS family status, and JAK–STAT activation may all play roles in the determination of autophagy dependence within pancreatic cancer cells.19,23 Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | RAS activates the MAPK cascade (RAF, MEK, and ERK), leading to changes in gene expression through MYC and ELK1.24 KIT signaling through the MAPK pathway maintains ETV1 activity. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Despite the finding of ubiquitous MAPK activation in primary GISTs and GIST cell lines, targeted inhibition of MAPK with a MEK1 and MEK2 inhibitor (U0126) had inconsistent effects on GIST cell line proliferation (5–40% inhibition) and did not induce apoptosis. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been provided. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | In addition, the RSK-independent effects of some RSK-inhibitors play a role in tumor suppression.25 The MAPK signaling pathway is shared by four distinct cascades including the extracellular signal-related kinases (ERK1/2), Jun amino-terminal kinases (JNK1/2/3), p38-MAPK, and ERK5. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | The MAPK/ERK pathway are reported to be associated with cell proliferation, differentiation, migration, senescence, and apoptosis.26 Targeting ERK is thought to be more valid than targeting BRAF or MEK in various types of acquired resistance, and may be more promising as cancer therapy.27 Resistance to the combined therapy in BRAF-mutant melanomas is mediated by mechanisms independent of ERK reactivation in many resistant cell lines and clinical samples.28 ERK1/2 mutation prompts a new question on how to deal with this resistance. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | In our study, we found that the phosphorylation level of ERK was related to autophagy, and the autophagy level of the combination group decreased significantly after adding CQ. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | In the future, large sample studies are needed to confirm. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | There are several limitations in our study. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Firstly, in this study, only one site of this pathway was detected. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Meanwhile, autophagy involves other complex pathways including PI3K/AKT/mTOR pathway. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Secondly, the small number of animal models may have affected our results. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Our data suggest that autophagy through the MAPK/ERK pathway may play a pivotal role in imatinib-resistant GIST proliferation. |
PMC7342409 | Chloroquine Combined with Imatinib Overcomes Imatinib Resistance in Gastrointestinal Stromal Tumors by Inhibiting Autophagy via the MAPK/ERK Pathway | Moreover, combining an autophagy inhibitor with imatinib may be a potential valuable strategy in overcoming acquired resistance in GIST patients. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the gastrointestinal tract and are characterized by activating mutations of c-KIT or PDGFRa receptor tyrosine kinases (RTKs). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Despite the clinical success of tyrosine kinase inhibitors (TKIs), more than half of GIST patients develop resistance due to a second mutation. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of CDK-activating kinase (CAK), and it plays an important role in the regulation of cell cycle transitions and gene transcription. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | THZ1, a CDK7 inhibitor, exhibits a dose-dependent inhibitory effect in various cancers. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Data from the public GEO database and tissue microarray were used to analyse the gene expression levels of CDKs in GISTs. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The impact of CDK7 knockdown and the CDK7 inhibitor THZ1 on GIST progression was investigated in vitro using CCK-8, colony formation, and flow cytometry assays and in vivo using a xenograft mouse model. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | RNA sequencing was performed to investigate the mechanism of GIST cell viability impairment mediated by THZ1 treatment. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Our study demonstrated that CDK7 is relatively overexpressed in high-risk GISTs and predicts a poor outcome. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | A low concentration of THZ1 exhibited a pronounced antineoplastic effect in GIST cells in vivo and in vitro. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Moreover, THZ1 exerted synergistic anticancer effects with imatinib. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | THZ1 treatment resulted in transcriptional modulation by inhibiting the phosphorylation of Ser2, Ser5, and Ser7 within RNA polymerase II (RNAPII). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | c-KIT, an oncogene driver of GIST, was transcriptionally repressed by THZ1 treatment or CDK7 knockdown. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Transcriptome sequencing analysis showed that OSR1 acts as a downstream target of CDK7 and regulates c-KIT expression. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Taken together, our results highlight elevated CDK7 expression as a predictor of poor outcome in GIST and present the combination of CDK7 and RTK inhibitors as a potent therapeutic strategy to improve the efficacy of GIST treatment. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Video abstract Video abstractGastrointestinal stromal tumours (GISTs) originate from interstitial cells of Cajal (ICCs) and are the most common malignant mesenchymal neoplasms of the gastrointestinal tract . |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The majority of GISTs harbour activating mutations in KIT (75–80%) or platelet-derived growth factor receptor alpha (PDGFRa) (10–15%) [3–5]. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The constitutive activation of these receptor tyrosine kinases (RTKs) in turn drives oncogenesis by activating downstream signalling pathways . |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | By inhibiting RTK signalling, tyrosine kinase inhibitors (TKIs) have been widely used as adjuvant therapy for GIST, and they significantly prolong survival [7–9]. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | However, despite initial responses, resistance to TKIs due to acquired secondary mutations limits their long-term benefit . |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Nearly half of patients with advanced GIST show tumour progression within the first two years of imatinib treatment, and the estimated 10-year progression-free survival and overall survival rates are 7–9% and 19.4–23%, respectively . |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Several core transcription factors have been revealed to play essential roles in driving GIST cell proliferation and metastases by binding to enhancers of GIST-associated genes and facilitating KIT gene expression [13–15]. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | FOXF1 is highly expressed in GISTs and colocalizes with ETV1 at enhancers to directly control the transcription of two major oncogenes, ETV1 and KIT . |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Therefore, characterization of transcription factor deregulation in GIST may provide innovative insights into its pathogenesis mechanisms and offer new therapeutic approaches. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Cyclin-dependent kinases (CDKs) catalyse the phosphorylation of cyclins and control cell cycle transitions. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | CDK7 is the catalytic subunit of CDK-activating kinase (CAK), which can stimulate cell cycle progression and activate multiple other CDKs, such as CDK1, CDK2, CDK4, and CDK6, through T-loop phosphorylation [16–18]. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | In addition, CDK7 is a component of the transcription factor TFIIH, which phosphorylates the C-terminal domain (CTD) of RNA polymerase II (RNAP II) and therefore activates transcription initiation and elongation . |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | CDK7 was found to be overexpressed and promote tumorigenesis in various cancers, such as breast cancer and osteosarcoma . |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | THZ1, a selective CDK7 inhibitor, covalently binds to CDK7 and suppresses its kinase activity based on modification of a unique cysteine residue . |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | THZ1 potently represses the transcription of several superenhancer-associated oncogenes and elicits a tumour inhibition effect in various cancers [21, 24–28]. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | However, the role of CDK7 in the progression of GIST has not yet been elucidated. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | In this study, we found that CDK7 expression was elevated in high-risk GISTs and related to a poor prognosis. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | CDK7 knockdown or THZ1 treatment inhibits GIST cell proliferation and leads to inhibition of transcriptional activity and protein expression of c-KIT. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Whole-transcriptome sequencing analysis was performed to decipher the mechanisms of CDK7 inhibition in GIST. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | THZ1 treatment led to inhibition of RNAPII phosphorylation, an indication of transcriptional inhibition, suggesting that targeting CDK7 may provide a mechanism to block transcriptional activation of c-KIT in GISTs. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Taken together, our findings revealed that targeting CDK7 may be a potent therapeutic strategy for GIST patients. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The GIST-T1 cell line was purchased from Cosmo Bio Co. Ltd. (Tokyo, Japan). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The GIST-882 cell line was kindly provided by Dr. Fletcher from Harvard Medical School. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | GIST-T1 and GIST-882 cells were cultured at 37 °C with 5% CO2 in RPMI-1640 medium (Gibco, USA) supplemented with 10% foetal bovine serum and 1% penicillin–streptomycin (Gibco, USA). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Antibodies against CDK7 (#2916), Rpb1 CTD (#2629), phospho-Rpb1 CTD (Ser2) (#13499), phospho-Rpb1 CTD (Ser5) (#13523), phospho-Rpb1 CTD (Ser7) (#13780), cyclin D1 (#55506), CDK4 (#12790), γ(p-)H2AX (#9718), cleaved PARP (#5625), c-KIT (#3074), phospho-c-KIT (#3073), ERK1/2 (#4695), phospho-ERK1/2 (#4370), AKT (#9272), phospho-AKT (#4060), FLAG (#14793) and Alexa Fluor conjugated anti-rabbit IgG (H + L) (#8889) were purchased from Cell Signaling Technology (CST, MA, USA). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | An antibody against Ki67 (ab16667) was purchased from Abcam (Cambridge, MA, USA). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | An antibody against OSR1 (sc-376545) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Antibodies against GAPDH (60004–1-Ig) and Caspase-3 (19677–1-AP) and HRP-conjugated secondary antibodies (SA00001-1 and SA00001-2) were purchased from Proteintech (Wuhan, China). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Imatinib was kindly provided by Novartis Pharmaceuticals (Basel, Switzerland). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | THZ1 cells (HY-80013A) were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Imatinib was dissolved in phosphate-buffered saline (PBS), and THZ1 was dissolved in dimethyl sulfoxide (DMSO) for the in vitro cell culture studies. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | For immunohistochemistry (IHC), we employed a constructed tissue microarray (TMA) containing 181 paraffin-embedded primary GIST surgical samples resected at Zhongshan Hospital between 2009 and 2012 with Institutional Review Board approval. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | IHC staining was performed with anti-CDK7 antibody (#2916, CST). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Two researchers separately evaluated the staining intensity and divided the samples into low expression and high expression groups. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Small interfering (si)RNAs targeting CDK7 and OSR1 and control siRNA were synthesized by Obio Technology (Shanghai, China). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Plasmids and vector plasmids as controls were synthesized by Shanghai GeneChem Co., Ltd. siRNA and plasmid transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | After transfection for 48 h, the knockdown efficiency was tested by western blotting. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The sequences of siRNAs were listed in Additional file 1. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Cell proliferation was examined using a Cell Counting Kit-8 (Yeasen, Shanghai, China) according to the instructions. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | GIST-T1 or GIST-882 cells were plated in 96-well plates at 1.5 × 10 cells in 100 μl of medium per well. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | After incubation overnight, the medium was replaced with 100 µl medium with 10 μl CCK-8 and incubated for 2 h. The absorbance value was detected with a microplate reader at 450 nm. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Similarly, the OD values were assessed at the indicated time points: 24, 48, and 72 h. For the drug inhibition assay, 5 × 10 GIST-T1 or GIST-882 cells/well were seeded and incubated at 37 °C overnight. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Cells were treated with THZ1 and imatinib at various doses, and the mean inhibitory concentration (IC50) was calculated using nonlinear regression analysis in GraphPad Prism 8.0. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The synergistic effect of the combination treatment was measured using CompuSyn software (ComboSyn, Inc. Paramus, NJ, USA) . |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The combination index (CI) was generated by CompuSyn software, and CI < 1, = 1, and > 1 indicate synergic, additive or antagonistic effects, respectively. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Approximately 1 × 10 cells were collected and fixed with 75% ethanol at 4 °C overnight. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | After centrifugation and resuspension in PBS, the cells were incubated with 500 µL propidium iodide (PI)/RNase staining solution (Absin, China) at 37 °C for 30 min. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Cell apoptosis was detected with an Annexin V-fluorescein isothiocyanate (FITC) apoptosis assay kit (Absin, China). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Briefly, cells in 6-well plates were harvested by EDTA-free trypsinization, washed twice with cold PBS buffer and then resuspended in binding buffer with 5 µl of Annexin V and 5 µl of PI for 15 min at RT in the dark. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The distribution of the cell cycle and apoptosis was determined in a BD Accuri C6 plus flow cytometer (BD Biosciences). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The results were analysed by ModFit 3.0 software (Verity software house, Topsham, ME, USA) and FlowJo software (FlowJo, LLC, Ashland, OR, USA). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The cells were seeded in 6-well plates at a density of 500 cells/well and cultured for 2 weeks. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The medium was replaced every five days. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | To examine colony formation, the cells were fixed with 4% paraformaldehyde for 20 min. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Next, crystal violet (0.5%) was used to stain the cells for 20 min. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The colonies were photographed and then counted using ImageJ software. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Total RNA was extracted using TRIzol Reagent (Yeasen Biotechnology Shanghai, China) and reverse transcribed into cDNA using cDNA Synthesis SuperMix (Yeasen) according to the manufacturer’s instructions. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Real-time PCR was performed using SYBR Green Master Mix (Yeasen) on a StepOne Real-Time PCR System (Applied Biosystems). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The qRT-PCRs were run in triplicate. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | GAPDH was used as an endogenous control. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The sequences of primers were listed in Additional file 1. |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | Total protein was extracted from the cells with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology Shanghai, China) supplemented with protease inhibitors (Beyotime) and phosphatase inhibitors (Beyotime). |
PMC9454178 | THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours | The membranes were then blocked with 5% skim milk for 90 min at room temperature and incubated with the primary antibodies overnight at 4 °C. |
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