PMCID string | Title string | Sentences string |
|---|---|---|
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The Golgi complex is a specialized organelle for protein processing and trafficking . |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Currently, little is known about the function of the Golgi complex, specifically as a platform for initiating receptor tyrosine kinase (RTK) signaling. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | It was demonstrated that MT-KIT directly recruits P85 and GRB2 to the Golgi complex, and its loss in the Golgi complex drastically perturbs downstream signaling. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Herein, we propose a Golgi-based signaling model for MT-KIT. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Since the Golgi complex is not central to protein quality control, this unique form of signaling that hides RTK in the Golgi complex would be advantageous for cancer cells, as there is always a risk that mutant RTKs in the ER or PM are degraded through ERQC and PMQC. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Mutations in KIT are most frequently detected in exon 11 (juxtamembrane domain, 70%), followed by exon 9 (extracellular domain, 10%) and less frequently in exon 13 (adenosine triphosphate (ATP)-binding pocket, 1%), or in exon 17 (activation loop, 1%) . |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Since GIST430, GIST882, GIST48, and GIST430-V654A cells commonly displayed Golgi-localization of MT-KIT regardless of mutation type, we suspected that GIST might have acquired a complementary mechanism for Golgi-retention of MT-KIT. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | BLZF1, a Golgi resident protein , was found to be important in tethering MT-KIT in the Golgi complex and thereby for GIST survival. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Considering its selective and high expression in GIST, BLZF1 should be further characterized as a druggable target for GIST treatment. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | To rationalize the localization of MT-KIT in the Golgi complex, ERQC bypass of MT-KIT should be preceded. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | As a large solid tumor, GIST undergoes chronic ER stress conditions, such as insufficient nutrients and hypoxia, that eventually induce ERQC and cell death [30–32]. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Cancer cells alleviate ER stress by initiating the UPR system that usually leads to mutant protein degradation by ERQC. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | However, this mechanism might be disadvantageous for cancer cells because of the loss of driver oncoproteins, such as MT-KIT . |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Therefore, promoting folding rather than degrading mutant proteins may be favorable for long-term cancer progression. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Results of this study show evidence that GIST has developed an ATF6-dependent UPR activation mechanism to ameliorate ER stress. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Sustained activation of ATF6 was detected in all GIST cell lines and frequently in the patients’ tissues. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | This unique ER stress-protective environment allows GISTs to resist ER stress to some extent. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Pharmacological inhibition of ATF6 significantly decreased chaperone expression, including HSP90, and resensitized GIST cells, even under mild ER stress. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The ATF6-HSP90 axis is especially beneficial for GIST tumorigenesis in a manner of MT-KIT folding and ERQC bypass of MT-KIT. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | This finding is consistent with previous research showing that 17AAG and TAS-116, HSP90 inhibitors, effectively suppress GIST growth . |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | HSP90 inhibitors have a different mechanism of action from kinase activity inhibition, such as that seen with imatinib. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Instead, they fundamentally downregulate mutant KIT expression. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | This mechanism sets them apart from imatinib, as it makes these drugs irrelevant to the generation of secondary mutations that can weaken the antitumor effect. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | By targeting mutant KIT expression itself, HSP90 inhibitors offer a promising approach for GIST treatment that may overcome imatinib resistance mechanisms associated with secondary mutations. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Moreover, previous studies have suggested that sustained activation of ATF6 is a prosurvival mechanism in various cancer types such as melanoma, glioblastoma, and hepatocellular carcinoma [35–37]. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Based on these findings we propose that HSP90 inhibitors, either alone or in combination with ATF6 inhibitors, hold great promise as alternative treatments for GIST. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | In vitro and in vivo experiments clearly demonstrated that ATF6 inhibition is a promising strategy for GIST treatment, and combined treatment with ER stress-inducing drugs further enhances the antitumor efficacy. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | This treatment strategy showed invariable effects even on imatinib-resistant GIST, which is a major challenge in clinical practice. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Although there are no FDA-approved regimens for targeting ATF6 in cancers, three reported ATF6 inhibitors with different modes of action have shown great efficacy . |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Notably, melatonin, a relatively safe dietary supplement, effectively suppressed GIST growth at a generally safe concentration (1 mM) and showed synergistic effects with ER stress-inducing drugs. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Although melatonin use for GIST treatment requires intensive in vivo and clinical validation, this strategy could be practical since no severe side effects were reported for melatonin. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Considering that sustained activation of ATF6 is exclusively observed in GIST and that the ATF6-HSP90 axis plays a crucial role in MT-KIT stabilization, we propose that ATF6 is a promising multi-effect target for GIST treatment, at least for imatinib-resistant patients with no other therapeutic options. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | In conclusion, this study provides evidence that GIST has developed highly coordinated survival strategies of hiding MT-KIT in the quality control-free Golgi complex, and developing an intrinsic ER stress resistance mechanism (summarized in Fig. 6). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Our findings provide insights into future GIST treatment strategies. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Therapeutic antibodies targeting membranous MT-KIT might be challenging. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | BLZF1 and ATF6 pathway could be promising drug targets for GIST treatment. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Fig. 6Schematic representation of novel GIST tumorigenesis mechanisms via the Golgi-localized MT-KIT-mediated oncogenic signaling and constant activation of pro-survival ATF6 dependent UPR signaling. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Schematic representation of novel GIST tumorigenesis mechanisms via the Golgi-localized MT-KIT-mediated oncogenic signaling and constant activation of pro-survival ATF6 dependent UPR signaling. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | In-depth mechanistic and clinical studies are crucial to enhance our understanding of whether targeting BLZF1 and ATF6 could overcome the limitations of conventional therapy in actual patients with GISTs. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Additionally, investigating the relationship between BLZF1 and ATF6 would provide valuable insights into their cooperative roles in GIST pathogenesis. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | It would be beneficial to utilize genomic and transcriptomic analysis data from patients with GIST to further advance our understanding. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Furthermore, leveraging the differential protein trafficking regulation between murine and human cells can be a valuable strategy for future studies in this field . |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | By incorporating these approaches, we can gain valuable insights that may lead to the development of improved therapeutic strategies for GIST patients. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | GIST430 (exon 11 in-frame deletion) and GIST430-V654A (exon 11 in-frame deletion and V654A) cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 15% fetal calf serum (Life Technologies, Carlsbad, CA, USA). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | GIST48 (V560D) cells were cultured in IMDM supplemented with 15% fetal bovine serum (FBS; Life Technologies). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | GIST882 (homozygous exon 13 K642E point mutation) cells were cultured in RPMI1640 with 15% FBS. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | GIST cell lines were established by Prof. Jonathan Fletcher (Boston, MA, USA). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Colon cancer cell lines (DLD-1, LS174T, Colo320DM) and small-cell lung cancer cell lines (H69, H128, H209) were cultured in RPMI1640 medium supplemented with 10% FBS. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The leukemia cell lines K562 and Kasumi-1 were cultured in RPMI1640 medium supplemented with 10% FBS. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Another leukemia cell line, HMC-1, was cultured in IMDM medium supplemented with 15% FBS. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | A cervical cancer cell line (HeLa), pancreatic cancer cell line (Capan-1), and breast cancer cell line (MDA-MB-231) were used as KIT-negative cell lines. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | HeLa cells were cultured in Minimum Essential Medium supplemented with 10% FBS. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Capan-1 and MDA-MB-231 cells were cultured in RPMI1640 medium supplemented with 10% FBS. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Cell lines were authenticated by KIT sequencing and microscopic examinations. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | RT-qPCR and western blot was performed to monitor morphology, growth patterns, mutation status, and KIT expression. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Cell lines were screened bi-monthly to monitor for mycoplasma infections. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | For treatment reagents, GIST cells were treated with thapsigargin (TG; Sigma, St. Louis, MO, USA), bortezomib (Sigma), or 17AAG (Sigma) to mimic endoplasmic reticulum (ER) stress; Brefeldin A (BFA; Sigma) to inhibit ER-to-Golgi trafficking; Bafilomycin A1 (BAF; Sigma) to inhibit lysosomal degradation; cycloheximide CHX) to block translation; 30N12 (Chembridge, San Diego, CA, USA) to inhibit trans-Golgi-to-PM trafficking. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | PF429242 (Tocris, Ellisville, MO, USA), Ceapin-A7 (Sigma), and melatonin (Sigma) were used to inhibit ATF6 activation. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Imatinib mesylate (Sigma) was used as the control drug for GIST treatment. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | To construct GRB2 and p85 expression vectors with a hemagglutinin (HA) tag, GRB2 and p85 coding regions were amplified by PCR using cDNA from GIST430 cells, then cloned into a pCMV vector with an HA tag. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The primers used to construct GRB2 and P85 expression vectors are listed in Supplementary Table S3. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Whole cell lysates were prepared using passive lysis buffer (Promega, Madison, WI, USA) with a protease inhibitor cocktail (Roche, Basel, Switzerland). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The proteins were electrophoresed and transferred onto a membrane. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The membranes were incubated overnight at 4 °C with primary antibodies against GAPDH (Trevigen, Gaithersburg, MD, USA), KIT (Dako or Cell Signaling Technology, Beverly, MA, USA), HA (Cell Signaling Technology), phospho-KIT (Cell Signaling Technology), AKT (Cell Signaling Technology), phospho-AKT (Cell Signaling Technology), ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-ERK (Santa Cruz Biotechnology), P85 (Cell Signaling Technology), GRB2 (Cell Signaling Technology), GBF1 (BD Biosciences; NJ, USA), GM130 (BD Biosciences), Golgin97 (MyBioSource, San Diego, CA, USA), TGN38 (Bio-Rad Laboratories, Hercules, CA, USA), GRASP55 (Abcam), GRASP65 (Invitrogen, Carlsbad, CA, USA), BLZF1 (Novus Biologicals, Littleton, CO, USA), STX3 (Abcam), STX6 (Cell Signaling Technology), GOLPH3 (LifeSpan Biosciences, Seattle, WA, USA), ATF6 (Abcam), IRE1α (Cell Signaling Technology), phospho-IRE1α (Novus Biologicals), PERK (Cell Signaling Technology), phospho-PERK (Cell Signaling Technology), CHOP (Cell Signaling Technology), BIP (Cell Signaling Technology), GRP94 (Cell Signaling Technology), HSP70 (Abcam) and HSP90 (Cell Signaling Technology). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The membranes were washed and incubated with the appropriate secondary antibodies for 1 h at room temperature. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Western blot images were analyzed using an LAS 4000 mini camera (Fujifilm, Tokyo, Japan). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | For immunoprecipitation assays, immune complexes of MT-KIT were collected by mixing 500 µg of GIST cell lysate with a KIT antibody (Cell Signaling Technology) and protein A/G PLUS-Agarose (Santa Cruz Biotechnology), then gently rocking on an orbital shaker at 4 °C overnight. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Immune complexes bound to agarose beads were washed and boiled with 3× NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) to elute the complexes. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The relative density of each band was quantified using ImageJ software (Ver 1.53 K; National Institutes of Health, MD, USA). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Formalin-fixed paraffin-embedded (FFPE) GIST tissue samples from 42 patients were used. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | All specimens were obtained via surgical resection, and some of the clinicopathological findings of the 42 GIST samples have been previously reported . |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The specimens were obtained from the archives of the Department of Pathology, Yonsei University, Seoul, Korea and the Liver Cancer Specimen Bank of the National Research Resource Bank Program of the Korea Science and Engineering Foundation of the Ministry of Science and Technology. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Authorization for the use of these tissues for research purposes was obtained from the Institutional Review Board of Yonsei University, College of Medicine (IRB 4-2015-0227). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | For IHC analysis, 4 µm-thick sections were obtained from FFPE GIST tissue specimens obtained from 42 patients. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | IHC was performed using the Ventana Discovery XT autoimmunostainer (Ventana, Tucson, AZ, USA) with antibodies against KIT (1:200, Dako) and ATF6 (1:200, Abcam) according to the manufacturer’s standard protocol. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | IHC results were evaluated using an H-score method. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The H-score method, which assigns an IHC H-score to each case on a continuous scale of 0–300 based on the percentage of cells at different staining intensities, was used for the interpretation of KIT and ATF6 results. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The staining intensity was scored as follows: 0 = no staining; 1+ = weak staining visible at high magnification; 2+ = intermediate staining; 3+ = strong staining. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The percentage of cells at different staining intensities was determined by visual assessment with scores calculated using the following formula: 1(% of 1+ cells) +2 (% of 2+ cells) +3 (% of 3+ cells). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Results were then classified as low (H-score ≤10) or high (H-score >10) protein level. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | DLD-1, Colo320DM, GIST430, and GIST882 cells were biotin-labeled for 30 min, washed extensively, and harvested for further analysis. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | To analyze membranous MT-KIT stability, GIST cells were harvested at predefined incubation times. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Subsequently, the labeled proteins were isolated and eluted using the Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Because of the low expression of membranous MT-KIT in GIST cells, biotinylation assays for GIST cells were performed with twice the amount of lysate compared to those used for DLD-1 and Colo320DM cells. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Cells grown on slides were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde or 100% methanol for 15 min, and permeabilized in 0.2% Triton X-100 in PBS. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The slides were incubated overnight at 4 °C with primary antibodies against KIT (Cell Signaling Technology), calnexin (Cell Signaling Technology), GM130 (Cell Signaling Technology), mannosidase II (Abcam), Golgin-97 (Cell Signaling Technology), HA (Cell Signaling Technology), BLZF1 (Novus Biologicals), LAMP1 (Cell Signaling Technology), and ATF6 (Abcam). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The slides were then incubated for 1 h with the appropriate fluorescence-labeled secondary antibody (Life Technologies). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | All images were captured using an LSM700 confocal microscope (Carl Zeiss, Oberkochen, Germany). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Immunofluorescence intensity and colocalization among proteins were quantified using ImageJ software (NIH). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | GIST cells grown on slides were washed three times with ice-cold serum-free medium and incubated with KIT antibody conjugated with a fluorescent dye (Alexa Fluor 488, Cell Signaling Technology) in ice-cold serum-free medium for 30 min. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | The cells were washed with cold serum-free medium to remove excess antibodies and incubated at 37 °C for various timepoints. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Cells were fixed in 4% paraformaldehyde in PBS and processed for immunofluorescence analysis. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Colocalization of MT-KIT with EEA1 (Abcam), LAMP1 (Cell Signaling Technology), and GM130 (Cell Signaling Technology) was examined. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | GIST cells were seeded in 96-well plates at a density of 3 × 10 cells/well and treated with PF429242, Ceapin-A7, or melatonin with or without thapsigargin, bortezomib, 17AAG, or imatinib for 72 h. MTT (5 mg ml) solution was then added to the plates and incubated at 37 °C for 4 h. After the formazan crystals were dissolved in DMSO, the OD was measured at 570 nm using a microplate reader. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | This experiment was performed with or without TG. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Highest single agent (HSA) synergy scores were calculated using SynergyFinder 2.0 (10.1093/nar/gkaa216). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | Short hairpin RNA (shRNA) against BLZF1 (gene ID: 8548) was purchased from the shRNA library of the RNAi Consortium (TRC) provided by the Yonsei Genome Center (Seoul, Korea). |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | GIST cells were transfected with the TRC2-pLKO-puro vector containing shRNA for BLZF1, and the MTT assay was performed 72 h after transfection. |
PMC10589262 | Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT | All animal modeling procedures were approved by the Institutional Animal Care and Use Committee of the Catholic University of Korea (No. 2022-0037-05). |
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