Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Sensory Denervation Causes Epithelial Ferroptosis and Delayed Healing in Mice

doi: 10.1167/iovs.66.6.28

Figure Lengend Snippet: Nerve degeneration induces the ferroptosis of corneal epithelial cells. ( A ) The corneal epithelial defect of surgical denervated mice was observed with slit-lamp and fluorescein sodium staining. ( B ) Corneal nerve degeneration was detected by whole-mount βIII-tubulin staining, and the percentage of nerve-covered area was analyzed by ImageJ. ( C ) Corneal sensation was measured using a Cochet–Bonnet esthesiometer. ( D ) Intracellular GSH was detected by monochlorobimane staining and analyzed by ImageJ. ( E ) The levels of Gpx4 , Gstm1 , Gsta2 , Gsta3 , and Slc7a11 were analyzed by quantitative PCR. ( F – H ) The results of immunofluorescence in the expression of GPX4 and GSTM1 proteins in the corneal epithelium of the denervated mice. The expression of GPX4 and GSTM1 in the corneal epithelium of surgical corneal denervated mice (F) and patients with neurotrophic keratitis ( G ) was detected by immunofluorescence staining, and the fluorescence intensity was quantified by ImageJ ( H ). ( I ) The mitochondrial morphology of corneal epithelium was observed by transmission electron microscopy after surgical corneal denervation. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Then, the sections were incubated with GPX4 antibodies (DF6701; Affinity Biosciences, Changzhou, China), GSTM1 antibodies (12412-1-AP; Proteintech, Rosemont, IL, USA), p53 antibodies (10442-1-AP; Proteintech), p-AKT antibody (4060; Cell Signaling Technology, Danvers, MA, USA), or p-mTOR antibodies (ab109268; Abcam, Cambridge, UK) overnight at 4°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Immunofluorescence, Expressing, Fluorescence, Transmission Assay, Electron Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Sensory Denervation Causes Epithelial Ferroptosis and Delayed Healing in Mice

doi: 10.1167/iovs.66.6.28

Figure Lengend Snippet: TRPV1 + sensory degeneration triggers the ferroptosis of corneal epithelial cells. ( A ) The ocular surface of capsaicin (Caps)-treated mice was observed with slit-lamp and fluorescein sodium staining. ( B ) Corneal nerve degeneration was detected by βIII-tubulin staining and the percentage of nerve-covered area was analyzed by ImageJ. ( C ) Corneal sensation was measured using a Cochet–Bonnet esthesiometer. ( D ) Intracellular GSH was detected by monochlorobimane staining and analyzed by ImageJ. ( E ) The levels of Gpx4 , Gstm1 , Gsta2 , Gsta3 , and Slc7a11 were analyzed by quantitative PCR. ( F ) The expression of GPX4 and GSTM1 in the corneal epithelium of capsaicin-treated mice was detected by immunofluorescence staining and quantified by ImageJ. ( G ) The mitochondrial morphology of corneal epithelium was observed by transmission electron microscopy after capsaicin treatment. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Then, the sections were incubated with GPX4 antibodies (DF6701; Affinity Biosciences, Changzhou, China), GSTM1 antibodies (12412-1-AP; Proteintech, Rosemont, IL, USA), p53 antibodies (10442-1-AP; Proteintech), p-AKT antibody (4060; Cell Signaling Technology, Danvers, MA, USA), or p-mTOR antibodies (ab109268; Abcam, Cambridge, UK) overnight at 4°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Transmission Assay, Electron Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells

doi: 10.3390/ijms22137080

Figure Lengend Snippet: Glutathione S-transferase (GST) activity and GST subfamily member protein expression in glioblastoma cell lines. ( A ) The reaction mixture contained 100 µg of cytosolic protein of each glioblastoma multiforme (GBM) cell line. Values are expressed as mean ± SD ( n = 3). * p < 0.05 relative to T98G cells. ( B ) Western blot analysis of GSTM1, GSTM2, GSTM3, GSTM4, GSTM5, GSTP1, and GSTK1 of human fetal glial cell line SVGp12 and human glioblastoma cell lines U87MG, T98G, U138MG, A172, and GBM8401. β-Actin was used as the loading control.

Article Snippet: GSTM3 Ab was procured from ProteinTech Group (Rosemont, IL, USA).

Techniques: Activity Assay, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells

doi: 10.3390/ijms22137080

Figure Lengend Snippet: Knockdown of GSTM3 T98G cells. ( a ) Protein expression of GSTM3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in siNC and siGSTM3 T98G cells. β-Actin was used as the loading control. ( b ) Morphology of small interfering RNA (siRNA) negative control (siNC) and GSTM3 (siGSTM3) T98G glioblastoma. Cells were subjected to microscopy at 40× magnification. ( c ) GST activity of siNC and GSTM3 T98G cells. The reaction mixture contained 100 µg of cytosolic protein in 0.1 M KPO4 buffer, pH 7.5, containing 1 mM GSH and 1 mM CDNB at 25 °C. Values are expressed as mean ± SD ( n = 3). ** p < 0.001 relative to siNC. ( d ) MTT assays were performed on Mock, siNC, and GSTM3 T98G cells. The MTT assay of T98G cells after 72 h of TMZ treatment. Values are expressed as mean ± SD ( n = 6). * p < 0.05 relative to siNC. # p < 0.05 relative to mock cells.

Article Snippet: GSTM3 Ab was procured from ProteinTech Group (Rosemont, IL, USA).

Techniques: Knockdown, Expressing, Control, Small Interfering RNA, Negative Control, Microscopy, Activity Assay, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells

doi: 10.3390/ijms22137080

Figure Lengend Snippet: OCR and ECAR values from a glycolysis stress test in GSTM3-knockdown T98G cells. T98G cells treated with siRNA knockdown were depleted 72 h after transfection. The ECAR was measured and normalized to cell number by using the Seahorse XF Analyzer in cells. ( a ) Real-time measurement of the OCR as mitochondrial respiration. Oligomycin (1 µM), FCCP (0.5 µM), and rotenone/antimycin A (0.5 µM) were injected at the indicated times. Values are expressed as mean ± SD ( n = 3). ( b ) Real-time measurement of the ECAR as the glycolysis rate. Glucose (10 mM), oligomycin (1 µM), and 2-DG (50 mM) were injected at the indicated times. Values are expressed as mean ± SD ( n = 3). ( c ) The glycolytic rate, glycolytic capacity, and glycolytic reserve were analyzed using standard methods. Values are expressed as the mean ± SD ( n = 3). *** p < 0.001 relative to siNC.

Article Snippet: GSTM3 Ab was procured from ProteinTech Group (Rosemont, IL, USA).

Techniques: Knockdown, Transfection, Injection

Journal: International Journal of Molecular Sciences

Article Title: Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells

doi: 10.3390/ijms22137080

Figure Lengend Snippet: GSTM3 downregulation decreases the invasion ability of T98G glioblastoma cells. ( a ) Cell migration was measured using a collagen-I-coated Transwell chamber (8-µm pore) for evaluating the migration of siNC and GSTM3 siRNA-treated cells. Migrated cells were stained with Giemsa solution (magnification 200×). ( b ) Cells on the underside of the Transwell insert were counted per file. Data are presented as mean ± SD ( n = 3). *** p < 0.001 relative to siNC.

Article Snippet: GSTM3 Ab was procured from ProteinTech Group (Rosemont, IL, USA).

Techniques: Migration, Staining

Journal: International Journal of Molecular Sciences

Article Title: Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells

doi: 10.3390/ijms22137080

Figure Lengend Snippet: Differential gene expression patterns between GSTM3 inhibited or negative control of T98G glioblastoma cells. The volcano plot of log2 (fold change) versus −log10 ( q -value) reveals differentially upregulated (right upper quadrant) and downregulated (left upper quadrant) genes expressed in GSTM3-inhibited cells versus negative control cells. Genes with q-values >0.05 and >2-fold changes are plotted in blue (downregulated) and red (upregulated), respectively.

Article Snippet: GSTM3 Ab was procured from ProteinTech Group (Rosemont, IL, USA).

Techniques: Gene Expression, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells

doi: 10.3390/ijms22137080

Figure Lengend Snippet: The upregulated mRNAs revealed in GSTM3 knockdown T98G.

Article Snippet: GSTM3 Ab was procured from ProteinTech Group (Rosemont, IL, USA).

Techniques: Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells

doi: 10.3390/ijms22137080

Figure Lengend Snippet: The downregulated mRNAs revealed in GSTM3 knockdown T98G.

Article Snippet: GSTM3 Ab was procured from ProteinTech Group (Rosemont, IL, USA).

Techniques: Knockdown

Journal: Cancers

Article Title: ZEB2/TWIST1/PRMT5/NuRD Multicomplex Contributes to the Epigenetic Regulation of EMT and Metastasis in Colorectal Carcinoma

doi: 10.3390/cancers14143426

Figure Lengend Snippet: ZEB2 interacts with TWIST1, PRMT5, and NuRD complex to form a functional multicomplex. Purification of ZEB2-containing protein complexes in SW480 cells. Cellular extracts from SW480 cells expressing HA (control) or HA-ZEB2 were immunopurified with anti-HA affinity beads. Eluates were analyzed by SDS-PAGE and mass spectrometry. The detailed data are provided in . ( A ) Association of ZEB2 with TWIST1, PRMT5, and NuRD. Whole-cell lysates from SW480 cells (TWIST1, ZEB2, and TWIST1+ZEB2) were immunoprecipitated with antibodies and the precipitated proteins were blotted with antibodies against the indicated proteins. IgG served as a negative control. ( B ) Co-immunoprecipitation of FLAG-tagged TWIST1 and HA-tagged ZEB2 transiently transfected in SW480 cells. Extracts were immunoprecipitated with FLAG antibody, and bound ZEB2 was detected by Western blotting. ( C – E ) GST pull-down assays were performed by mixing GST-fused proteins and 6*His-fused proteins.

Article Snippet: The following commercial antibodies and reagents were used: ZEB2 (ab138222, 1:500; Abcam, Cambridge, UK); TWIST1 (46702S, 1:500; Cell Signaling Technology, Danvers, MA, USA); E-cadherin (610181, 1:1000; BD Biosciences, San Diego, CA, USA); HDAC2 (5113T, 1:1000; Cell Signaling Technology); GAPDH (60004-1-Ig, 1:2000; Proteintech, Wuhan, China); ACTIN (66009-1-Ig, 1:2000; Proteintech); Tublin (11224-1-AP, 1:2000; Proteintech); GST (10000-0-Ig, 1:1000; Proteintech); HIS (66005-1-Ig, 1:1000; Proteintech); Flag (20543-1-AP, 1:1000; Proteintech); PRMT5 (18436-1-AP, 1:1000; Proteintech); RbAp48 (20364-1-AP, 1:1000; Proteintech); MTA2 (66195-1-Ig, 1:1000; Proteintech); Vimentin (10366-1-AP, 1:1000; Proteintech); Fibronectin (15613-1-AP, 1:1000; Proteintech); H3K56ac (39281, 5 μg/ChIP; Active Motif, Carlsbad, CA, USA); H4R3me2S (61187, 5 μg/ChIP; Active Motif). siRNAs and shRNAs were synthesized by GenePharma Co., Ltd. (Shanghai, China).

Techniques: Functional Assay, Purification, Expressing, Control, SDS Page, Mass Spectrometry, Immunoprecipitation, Negative Control, Transfection, Western Blot

Journal: iScience

Article Title: Inositol polyphosphate multikinase modulates redox signaling through nuclear factor erythroid 2-related factor 2 and glutathione metabolism

doi: 10.1016/j.isci.2023.107199

Figure Lengend Snippet:

Article Snippet: Anti-GSTA2 (1:1000) , Proteintech , Cat# 10505-1-AP, RRID:AB_2248094.

Techniques: Recombinant, Modification, Transfection, Protease Inhibitor, Membrane, Fractionation, Glutathione Assay, Gene Expression, Concentration Assay, Plasmid Preparation, Construct, Software