Journal: Antioxidants

Article Title: ITPR1 Maintains Mitochondrial Redox Homeostasis to Drive Glioblastoma Progression Through Recruitment and Activation of DRP1

doi: 10.3390/antiox15050550

Figure Lengend Snippet: Knockdown suppresses GBM cell proliferation and induces apoptosis in vitro. ( A ) Western blot analysis of ITPR1 expression in LN229, U251, and U87 cells. ( B ) CCK8 assay showing a significant decrease in cell viability in LN229 and U251 cell lines after ITPR1 knockdown. ( C ) Representative images of EdU incorporation assays demonstrating reduced cell proliferation in LN229, U251, and U87 cells after ITPR1 knockdown (scale bar: 100 μm). ( D ) Quantification of the EdU incorporation assay showing a significant decrease in proliferating cells following ITPR1 knockdown in LN229, U251, and U87 cells. ( E ) Colony formation assay revealing reduced colony formation ability of LN229 and U251 cell lines upon ITPR1 knockdown (scale bar: 10 mm). Quantification of colony numbers is shown on the right. ( F ) Western blot analysis of apoptosis-related proteins in LN229 and U251 cell lines after ITPR1 knockdown. Increased cleaved caspase-3 and BAX expression and decreased BCL-2 expression was observed after ITPR1 knockdown. ( G ) Representative flow cytometry images showing increased apoptosis in LN229 and U251 cell lines upon ITPR1 knockdown. ( H ) Flow cytometry analysis of Annexin V and 7-AAD staining showing increased apoptosis in LN229 and U251 cell lines after ITPR1 knockdown. ( I ) Quantification of the percentage of apoptotic cells (Annexin V+/7-AAD+ cells) in LN229 and U251 cell lines following ITPR1 knockdown. Black represents the negative control (NC) group, red represents the sg-ITPR1#1 group, and pink represents the sg-ITPR1#2 group. (** p < 0.01, *** p < 0.001).

Article Snippet: The human cell lines LN229 and U251 correspond to ATCC ® CRL-2611TM and ATCC ® HTB-17TM, respectively.

Techniques: Knockdown, In Vitro, Western Blot, Expressing, CCK-8 Assay, Colony Assay, Flow Cytometry, Staining, Negative Control

Journal: Antioxidants

Article Title: ITPR1 Maintains Mitochondrial Redox Homeostasis to Drive Glioblastoma Progression Through Recruitment and Activation of DRP1

doi: 10.3390/antiox15050550

Figure Lengend Snippet: ITPR1 maintains ER–mitochondria Ca 2+ transfer and promotes DRP1-dependent mitochondrial fission and mitophagy. ( A ) Representative Fluo-4 AM fluorescence images (cellular calcium probe), including 3D surface plots and intensity analysis, demonstrating that ITPR1-knockdown decreases cytosolic Ca 2+ levels, including 3D surface plots and intensity analysis, demonstrating that ITPR1-knockdown decreases cytosolic Ca 2+ levels (scale bar: 20 μm). ( B ) Representative Mag-Fluo4 fluorescence images (ER calcium probe), with intensity analysis showing that ITPR1-knockdown slightly increases ER Ca 2+ storage (scale bar: 100 μm). ( C ) Representative Rhod2 fluorescence images (mitochondrial calcium probe), with intensity analysis showing that ITPR1 knockdown reduces mitochondrial Ca 2+ levels (scale bar: 100 μm). ( D ) Transmission electron microscopy (TEM) images illustrating the disruption of ER–mitochondrial contact sites and altered mitochondrial morphology upon ITPR1-knockdown (left scale bar: 1 μm; right scale bar: 0.5 μm). ( E ) Quantitative statistical analysis of MAM density in different groups of LN229 cells. ( F ) Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of ITPR1 immunoprecipitation combined with STRING protein-interaction network mapping identifies DNM1L/DRP1 as one of the high-confidence ITPR1-interacting proteins. ( G ) Molecular docking analysis using the murine ITPR1 structure predicts a stable interaction between ITPR1 and DRP1 in murine species (binding energy: −7.9 kcal/mol). In the docking illustration, purple represents the ITPR1 protein two-dimensional structure, and yellow represents the DRP1 protein two-dimensional structure. ( H ) Co-immunoprecipitation validation of endogenous ITPR1-DRP1 interaction in GBM cells. ( I ) Western blot showing decreased DRP1 phosphorylation at Ser616 upon ITPR1 knockdown. ( J ) Immunofluorescence analysis of DRP1 localization relative to mitochondria in LN229 cells under control (NC) and ITPR1 knockdown (sgITPR1#1 and sgITPR1#2) conditions. Scale bar: 10 μm. Line-scan analysis (right panels) shows the fluorescence intensity profiles of DRP1 and TOMM20 along the indicated regions. ( K ) Quantification of DRP1 and TOMM20 colocalization using Manders’ colocalization coefficient. ( L ) Western blot analysis of mitochondrial fractionation showing reduced DRP1 recruitment to mitochondria in LN229 and U251 cell lines after ITPR1 knockdown. ( M ) Western blot analysis showing decreased autophagy and mitophagy marker levels upon ITPR1 knockdown. (** p < 0.01, *** p < 0.001).

Article Snippet: The human cell lines LN229 and U251 correspond to ATCC ® CRL-2611TM and ATCC ® HTB-17TM, respectively.

Techniques: Fluorescence, Knockdown, Transmission Assay, Electron Microscopy, Disruption, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Binding Assay, Biomarker Discovery, Western Blot, Phospho-proteomics, Immunofluorescence, Control, Fractionation, Marker

Journal: Antioxidants

Article Title: ITPR1 Maintains Mitochondrial Redox Homeostasis to Drive Glioblastoma Progression Through Recruitment and Activation of DRP1

doi: 10.3390/antiox15050550

Figure Lengend Snippet: ITPR1 knockdown induces mitochondrial depolarization, reactive oxygen species (ROS) accumulation, and intrinsic apoptosis in GBM cells. ( A ) Representative images of mitochondrial ROS measurement by MitoTracker and MitoSOX staining (scale bar: 50 μm). ( B ) Quantification of mitochondrial ROS in different groups of GBM cells. ( C ) Quantification of mitochondrial membrane potential in LN229 and U251 cells under control (NC) and ITPR1 knockdown (sgITPR1#1 and sgITPR1#2) conditions using tetramethylrhodamine methyl ester (TMRM) staining. ( D ) Representative images of mitochondrial membrane potential (ΔΨm) analysis by flow cytometry using JC-1 staining in NC and sgITPR1 cells. ( E ) Quantification of mitochondrial membrane potential (ΔΨm) using JC-1 staining in NC and sgITPR1 cells. ( F ) Intracellular ROS levels measured by DCFH-DA demonstrating elevated ROS accumulation after ITPR1 knockdown. (*** p < 0.001).

Article Snippet: The human cell lines LN229 and U251 correspond to ATCC ® CRL-2611TM and ATCC ® HTB-17TM, respectively.

Techniques: Knockdown, Staining, Membrane, Control, Flow Cytometry

Journal: Antioxidants

Article Title: ITPR1 Maintains Mitochondrial Redox Homeostasis to Drive Glioblastoma Progression Through Recruitment and Activation of DRP1

doi: 10.3390/antiox15050550

Figure Lengend Snippet: DRP1 is required for ITPR1-mediated regulation of mitochondrial function and autophagy in GBM cells. ( A ) Western blot analysis showing autophagy markers in control, ITPR1-overexpressing, DRP1-knockdown, and DRP1-knockdown ITPR1-overexpressing cells. ( B ) Representative images of mitochondrial reactive oxygen species (ROS) measurement by MitoTracker and MitoSOX staining in the indicated cell groups (scale bar: 50 μm). ( C ) Quantification of mitochondrial ROS measurement by MitoTracker and MitoSOX staining in the indicated cell groups. ( D ) Representative confocal images of LN229 and U251 cells stained with TMRM under the indicated conditions (Laz+ctrl, OE-ITPR1+ctrl, Laz+siDRP1, OE-ITPR1+siDRP1) (Scale bar: 50 μm). ( E ) Quantification of TMRM fluorescence intensity as an indicator of mitochondrial membrane potential. ( F ) Quantification of mitochondrial membrane potential (ΔΨm) levels using JC-1 staining in control, ITPR1-overexpressing, DRP1-knockdown and DRP1-knockdown ITPR1-overexpressing cells. ( G ) Representative histograms of cellular ROS levels detected by DCFH-DA in different cell groups. ( H ) Representative confocal images of mitochondrial morphology in LN229 and U251 cells stained with MitoTracker Green under the indicated conditions (Laz+ctrl, OE-ITPR1+ctrl, Laz+siDRP1, OE-ITPR1+siDRP1). Insets show enlarged views of mitochondrial structures. Scale bars: 50 μm (upper panels) and 5 μm (lower panels). ( I ) Quantification of mitochondrial mean branch length. Data are presented as relative mean branch length normalized to the control. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The human cell lines LN229 and U251 correspond to ATCC ® CRL-2611TM and ATCC ® HTB-17TM, respectively.

Techniques: Western Blot, Control, Knockdown, Staining, Fluorescence, Membrane