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PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Significance denoted by: ns not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
A GIST-T1 and 882 cells were treated with different concentrations of IM for 24 h and the IC50 of IM was determined.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
B GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h in the absence or presence of Ferrostatin-1 (1 μM), and the cell viability was assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
C Cell morphology was observed via transmission electron microscopy after the cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h. The area of mitochondria are quantitatively analyzed by using the ImageJ software.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
D, E GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) in the absence or presence of Ferr-1 (1 μM) for 12 h, the relative lipid ROS levels were measured using a C11-BODIPY lipid peroxidation sensor via confocal microscope (D) and flow cytometry (E).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
In fluorescence images, the increase in lipid ROS levels resulted in the oxidation of the polyunsaturated butadienyl portion of C11-BODIPY, causing a shift in fluorescence from red to green.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Flow cytometry analysis utilized the FITC filter for oxidized C11-BODIPY (emission: 510 nm) and the PE-TexasRed filter for reduced C11-BODIPY (emission: 590 nm).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The results are presented as a ratio of oxidized to reduced C11-BODIPY.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
F GIST-T1 and 882 cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h, and the relative levels of Fe were assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Scale bar, 50 μm in D. Experiments were independently repeated three times.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Significance denoted by: ns not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The detection indicators of ferroptosis include changes in mitochondrial morphology, lipid ROS levels, Fe levels, and GSH levels.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
To clarify the relationship between IM and ferroptosis, we first investigated changes in the morphological characteristics of GIST cells after IM treatment by transmission electron microscopy.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The results showed that the mitochondrial volume of GIST cells was reduced after IM treatment (Fig. 1C).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Next, we measured the ROS and lipid ROS levels with the DCFH-DA or C11-BODIPY lipid peroxidation sensor.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
It was found that ROS and lipid-ROS levels in GIST-T1 and GIST-882 cells were increased after IM treatment and this effect could be reversed by Ferrostatin-1.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
However, Ferrostatin-1 itself did not affect ROS and lipid-ROS levels (Fig. S3 and 1D, E).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
In addition, The fluorescence intensity of FerroOrange was increased in GIST-T1 and GIST-882 cells treated with IM compared with that in untreated cells, illustrating that IM promoted the accumulation of Fe in GIST (Fig. 1F).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
In conclusion, IM could induce ferroptosis in GIST cells.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Knowing that ferroptosis caused by lipid peroxidation is regulated by the key components of the cysteine-glutamate antiporter known as systemxc- and the antioxidant enzyme glutathione peroxidase 4 (GPX4), we investigated how IM induced ferroptosis and the result showed that the GSH/GSSG ratio was decreased in GIST-T1 and GIST-882 cells treated with IM as compared with those in the untreated cells (Fig. 2A).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
To clarify the specific molecular mechanism of IM-induced ferroptosis, we examined the effects of different IM concentrations and treatment time on the expression of SLC7A11 and GPX4.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The results showed no significant effect on the expression of SLC7A11 at the transcriptional level and protein levels (Fig. 2B, D).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The expression of GPX4 increased in a dose- and time-dependent manner after IM treatment at the transcriptional level (Fig. 2C), whereas at the protein level, IM effectively induced the reduction of GPX4 protein level in GIST-T1 and GIST-882 cells (Fig. 2D).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Therefore, IM may regulate GPX4 expression in a post-transcriptional manner.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
To further clarify the role of GPX4 in the anti-tumor effect of IM, we overexpressed GPX4 in GIST-T1 and GIST-882 cells to detect the change of IM sensitivity and its effect on lipid ROS and GSH/GSSG ratio (Fig. 2E).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The results showed that GPX4 overexpression significantly reversed the anti-tumor effect of IM and the up-regulation of lipid ROS (Fig. 2F, G).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
In addition, GPX4 overexpression blocked the reduction of the GSH/GSSG ratio induced by IM (Fig. 2H).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
In conclusion, IM promotes ferroptosis in GIST by causing GPX4 protein degradation.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Fig. 2Imatinib (IM) induces ferroptosis by causing GPX4 degradation.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
A GIST-T1 and 882 cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h or treated with IM (50 nM for T1 and 200 nM for 882) for 0, 6, 12, or 24 h, and the relative GSH/GSSG ratios were assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
B and C The relative RNA level of SLC7A11 and GPX4 was measured via quantitative real-time polymerase chain reaction after GIST-T1 and 882 cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h or treated with IM (50 nM for T1 and 200 nM for 882) for 0, 6, 12, or 24 h. D Representative Western blot analysis of SLC7A11 and GPX4 protein levels in GIST-T1 and 882 cells.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h or treated with IM (50 nM for T1 and 200 nM for 882) for 0, 12, 24, or 48 h. The histograms indicate the levels of the protein determined from 3 independent experiments expressed as the mean ratio relative to that in the untreated control after normalization to GAPDH.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
E Western blot analysis of GPX4 in GIST-T1 and 882 cells with GPX4 overexpression.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
F GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h with or without the overexpression of GPX4, and cell viability was assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
G GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 12 h with or without the overexpression of GPX4, and the relative lipid ROS levels were assayed via flow cytometry.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Flow cytometry analysis was performed using the FITC filter for oxidized C11-BODIPY (emission: 510 nm) and the PE-TexasRed filter for reduced C11-BODIPY (emission: 590 nm).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The results are displayed as a ratio of oxidized/reduced C11-BODIPY.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
H GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h with or without the overexpression of GPX4.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The relative GSH/GSSG ratios were assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Experiments were independently repeated three times.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Significance denoted by: ns not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
A GIST-T1 and 882 cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h or treated with IM (50 nM for T1 and 200 nM for 882) for 0, 6, 12, or 24 h, and the relative GSH/GSSG ratios were assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
B and C The relative RNA level of SLC7A11 and GPX4 was measured via quantitative real-time polymerase chain reaction after GIST-T1 and 882 cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h or treated with IM (50 nM for T1 and 200 nM for 882) for 0, 6, 12, or 24 h. D Representative Western blot analysis of SLC7A11 and GPX4 protein levels in GIST-T1 and 882 cells.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h or treated with IM (50 nM for T1 and 200 nM for 882) for 0, 12, 24, or 48 h. The histograms indicate the levels of the protein determined from 3 independent experiments expressed as the mean ratio relative to that in the untreated control after normalization to GAPDH.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
E Western blot analysis of GPX4 in GIST-T1 and 882 cells with GPX4 overexpression.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
F GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h with or without the overexpression of GPX4, and cell viability was assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
G GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 12 h with or without the overexpression of GPX4, and the relative lipid ROS levels were assayed via flow cytometry.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Flow cytometry analysis was performed using the FITC filter for oxidized C11-BODIPY (emission: 510 nm) and the PE-TexasRed filter for reduced C11-BODIPY (emission: 590 nm).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The results are displayed as a ratio of oxidized/reduced C11-BODIPY.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
H GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h with or without the overexpression of GPX4.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The relative GSH/GSSG ratios were assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Experiments were independently repeated three times.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Significance denoted by: ns not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
To clarify the specific molecular mechanism of IM in promoting GPX4 protein degradation, we conducted CO-IP experiments to detect the effect of IM on the ubiquitination of GPX4 and the result showed that IM effectively induced the ubiquitination of GPX4 (Fig. 3A).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Next, we used the ubiquitin ligase prediction website (http://ubibrowser.ncpsb.org.cn) to predict the ubiquitin ligase E3 of GPX4 (Fig. 3B and Table S3).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Based on DRUGSURV database analysis (the first computational tool to estimate the potential effects of a drug) , we found that IM could indirectly target STUB1 (Fig. 3C, D).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
To detect the regulatory effect of IM on STUB1, we tested the effect of IM on STUB1 expression.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The results showed that IM promoted STUB1 at both transcriptional and protein levels (Fig. 3E, F).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Therefore, we speculated that STUB1 may play a role in IM-induced ferroptosis.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
To further clarify the effect of STUB1 on the anti-tumor activity of IM, STUB1 was knocked down in GIST-T1 and GIST-882 cells (Fig. 3G).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The results showed that STUB1 knockdown effectively blocked the anti-tumor effect of IM and reduced the promotion of lipid-ROS mediated by IM (Fig. 3I, H).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
In addition, STUB1 knockdown blocked the reduction of the GSH/GSSG ratio induced by IM (Fig. 3J).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Our results suggest that IM may promote ferroptosis by regulating STUB1 expression in GIST.Fig.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
3Imatinib (IM) induces ferroptosis by regulating the E3 ubiquitin ligase STUB1.A GIST-T1 cells were treated with IM (50 nM) for 24 h with or without MG132 (5 μM), and GPX4 ubiquitination was measured via CO-IP.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
B Bioinformatics prediction of the interaction between the GPX4 and multiple E3 ubiquitin ligases using the UbiBrowser website (http://ubibrowser.ncpsb.org.cn).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
C Venn diagram depicting that the predicted E3 ubiquitin ligase STUB1, CBL, and HSPA8 of GPX4 protein may be the targets of IM.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
D DRUGSURV database (http://www.bioprofiling.de/GEO/DRUGSURV/) analysis revealed that STUB1 is an indirect target of IM.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
E GIST-T1 and 882 cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h, and RNA expression of STUB1 were measured.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
F Representative Western blot analysis of STUB1 protein levels in GIST-T1 and 882 cells.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h, and subjected to Western blot analysis.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The histograms indicate the levels of the protein determined from three independent experiments expressed as the mean ratio relative to that in the untreated control after normalization to GAPDH.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
G Western blot analysis of STUB1 in GIST-T1 and 882 cells transfected with siRNA-control, siRNA-STUB1–1 and siRNA-STUB1–2.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
H GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 12 h with or without STUB1 knockdown, and the relative lipid ROS levels were assayed via flow cytometry.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Flow cytometry analysis was performed using the FITC filter for oxidized C11-BODIPY (emission: 510 nm) and the PE-TexasRed filter for reduced C11-BODIPY (emission: 590 nm).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The results are displayed as a ratio of oxidized/reduced C11-BODIPY.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
I GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h with or without STUB1 knockdown.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Cell viability was assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
J GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h with or without STUB1 knockdown.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The relative GSH/GSSG ratios were assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Experiments were independently repeated three times.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Significance denoted by: ns not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
A GIST-T1 cells were treated with IM (50 nM) for 24 h with or without MG132 (5 μM), and GPX4 ubiquitination was measured via CO-IP.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
B Bioinformatics prediction of the interaction between the GPX4 and multiple E3 ubiquitin ligases using the UbiBrowser website (http://ubibrowser.ncpsb.org.cn).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
C Venn diagram depicting that the predicted E3 ubiquitin ligase STUB1, CBL, and HSPA8 of GPX4 protein may be the targets of IM.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
D DRUGSURV database (http://www.bioprofiling.de/GEO/DRUGSURV/) analysis revealed that STUB1 is an indirect target of IM.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
E GIST-T1 and 882 cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h, and RNA expression of STUB1 were measured.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
F Representative Western blot analysis of STUB1 protein levels in GIST-T1 and 882 cells.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The cells were treated with IM at 0, 25, 50, and 100 nM and 0, 100, 200, and 400 nM for 24 h, and subjected to Western blot analysis.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The histograms indicate the levels of the protein determined from three independent experiments expressed as the mean ratio relative to that in the untreated control after normalization to GAPDH.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
G Western blot analysis of STUB1 in GIST-T1 and 882 cells transfected with siRNA-control, siRNA-STUB1–1 and siRNA-STUB1–2.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
H GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 12 h with or without STUB1 knockdown, and the relative lipid ROS levels were assayed via flow cytometry.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Flow cytometry analysis was performed using the FITC filter for oxidized C11-BODIPY (emission: 510 nm) and the PE-TexasRed filter for reduced C11-BODIPY (emission: 590 nm).
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The results are displayed as a ratio of oxidized/reduced C11-BODIPY.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
I GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h with or without STUB1 knockdown.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Cell viability was assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
J GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) for 24 h with or without STUB1 knockdown.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
The relative GSH/GSSG ratios were assayed.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Experiments were independently repeated three times.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Significance denoted by: ns not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.
PMC10728200
Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination
Knowing that STUB1 and GPX4 play important roles in IM-induced ferroptosis, we first examined the localization of STUB1 and GPX4 in GIST cells to determine whether STUB1 was the E3 ubiquitin ligase of GPX4.