magnetic separation kit  (Miltenyi Biotec)

Journal: The FASEB Journal

Article Title: Increased Expression of Desmin and Vimentin Impairs Mitochondrial Function and Reduces Contractility via G0S2 in Bladder Smooth Muscle

doi: 10.1096/fj.202502912R

Figure Lengend Snippet: Increased expression of desmin and vimentin does not cause mitochondrial structural abnormalities. (A) Human BSM cells were transduced with an adenovirus encoding desmin and vimentin, and GFP for 36 h. The expression levels of desmin and vimentin proteins were analyzed by immunoblotting following their overexpression. GAPDH served as a loading control. Representative immunoblots and corresponding quantifications are shown. (B–D) The expression levels of contractile proteins and desmin and vimentin proteins across passages (P3–P5) were analyzed by immunoblotting. GAPDH served as a loading control. Representative immunoblots and their corresponding quantifications are shown in panels B, C, and D. (E) TEM imaging : Human BSM cells overexpressing desmin and vimentin were fixed and processed for TEM. GFP‐expressing cells were used as a control group. The TEM images in panel E depict mitochondria from lower (upper panel) and higher (lower panel) magnifications. Scale bars = 500 nm. M denotes molecular weight marker. Data are presented as mean ± SD; n = 3–4 in A, n = 3 in B–D, and n = 2 in E. The ns indicates non‐significant. **** p < 0.0001.

Article Snippet: Mitochondria were isolated from human BSM cells and murine BSM tissue using the Mitochondria Isolation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions.

Techniques: Expressing, Transduction, Western Blot, Over Expression, Control, Imaging, Molecular Weight, Marker

Journal: The FASEB Journal

Article Title: Increased Expression of Desmin and Vimentin Impairs Mitochondrial Function and Reduces Contractility via G0S2 in Bladder Smooth Muscle

doi: 10.1096/fj.202502912R

Figure Lengend Snippet: Increased expression of desmin and vimentin reduces mitochondrial elongation and branching. (A) Human BSM cells expressing DsRed2‐Mito‐7 were transduced with adenovirus encoding GFP, desmin, and vimentin for 36 h, and the mitochondria in these cells were visualized via confocal microscopy. GFP‐expressing cells served as the control group. Confocal images of mitochondria were processed using Python‐based pipelines, as described in the Materials and Methods section. Representative confocal images are shown in panel A. The white color in panel A represents mitochondria. (B–F) Quantification of mitochondrial length and circularity, represented by aspect ratio and form factor, respectively. The quantification of the branching parameter is shown in panels D–F. Scale bars = 50 μm. Data are presented as mean ± SD; n > 75 cells per group. Ns indicates non‐significant. *** p < 0.001 and **** p < 0.0001.

Article Snippet: Mitochondria were isolated from human BSM cells and murine BSM tissue using the Mitochondria Isolation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions.

Techniques: Expressing, Transduction, Confocal, Microscopy, Control

Journal: The FASEB Journal

Article Title: Increased Expression of Desmin and Vimentin Impairs Mitochondrial Function and Reduces Contractility via G0S2 in Bladder Smooth Muscle

doi: 10.1096/fj.202502912R

Figure Lengend Snippet: Increased expression of desmin and vimentin enhances mitochondrial ROS and reduces oxygen consumption rate and ATP levels. Human BSM cells were transduced with an adenovirus encoding desmin and vimentin, and GFP for 36 h. Human BSM cells overexpressing desmin and vimentin were processed for ROS, OCR, and ATP measurements. GFP‐expressing cells served as a control group. (A) Mitochondrial ROS levels in live cells were assessed by MitoSOX fluorescence. Human BSM cells overexpressing desmin and vimentin were stained with MitoSOX (5 μM) and MitoTracker DeepRed (10 nM), then visualized by confocal microscopy. Images were captured using a 100× oil‐immersion objective on a Nikon AIR confocal microscope. Mitochondrial ROS levels were measured by fluorescence intensity of MitoSOX Red. GFP‐expressing human BSM cells were used as a control group. Scale bars = 50 μm. (B) Quantifications of MitoSOX fluorescent intensity. Live cell confocal images of mitochondria were processed using Python‐based pipelines, as described in the Materials and Methods section. (C) ROS levels in isolated mitochondria were assessed by measuring H 2 O 2 production using the Amplex Red Hydrogen Peroxide assay in the presence of superoxide dismutase. (D) OCR was measured using a Seahorse Bioscience extracellular flux analyzer. Basal and maximal respiration were determined. OCR values were normalized to the total number of cells. (E) Quantifications of total cellular ATP levels by a bioluminescence assay. ATP levels were measured based on firefly luciferin‐luciferase luminance intensity. Data are presented as mean ± SD, and n = 25 cells in each group in B, n = 3 in C and D, and n = 4 in E. ** p < 0.01 and *** p < 0.001.

Article Snippet: Mitochondria were isolated from human BSM cells and murine BSM tissue using the Mitochondria Isolation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions.

Techniques: Expressing, Transduction, Control, Fluorescence, Staining, Confocal, Microscopy, Isolation, Amplex Red Hydrogen Peroxide Assay, ATP Bioluminescent Assay, Luciferase

Journal: The FASEB Journal

Article Title: Increased Expression of Desmin and Vimentin Impairs Mitochondrial Function and Reduces Contractility via G0S2 in Bladder Smooth Muscle

doi: 10.1096/fj.202502912R

Figure Lengend Snippet: Overexpression of MnSOD did not rescue the decreased contractile response and reduced ATP levels in human BSM cells with elevated levels of desmin and vimentin. Human BSM cells were transfected with MnSOD cDNA and transduced with adenoviruses encoding desmin and vimentin. (A) Gel contraction : For gel contraction studies, cells were seeded in a collagen gel as described in the Materials and Methods section, and gel contraction was measured in response to 120 mM KCl stimulation. The gels were subsequently imaged. The size of collagen gel in each image was quantified using Fiji (ImageJ/NIH) software, and the percentage change was calculated. (B) Measurement of mitochondrial ROS levels : ROS levels in cells were measured using isolated mitochondria with the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit. (C) Quantifications of total cellular ATP levels : ATP levels were measured based on firefly luciferin‐luciferase luminescence intensity. (D, E) Effect of antimycin on desmin and vimentin expression . Human BSM cells were treated with the indicated concentrations of antimycin A for 36 h. The expression levels of desmin and vimentin proteins were analyzed by immunoblot. GAPDH served as a loading control. Representative immunoblots and their corresponding quantification are shown in D, E panels. Control cells received an equivalent volume of DMSO to vehicle treatment, and the final DMSO concentration was maintained at ≤ 0.1%. (F) Measurement of mitochondrial ROS : ROS levels in control and antimycin A‐treated human BSM cells were measured using isolated mitochondria with the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit. Data are presented as mean ± SD, and n = 3. The ns indicates non‐significant. * p < 0.05 and ** p < 0.01.

Article Snippet: Mitochondria were isolated from human BSM cells and murine BSM tissue using the Mitochondria Isolation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions.

Techniques: Over Expression, Transfection, Transduction, Software, Isolation, Luciferase, Expressing, Western Blot, Control, Concentration Assay

Journal: The FASEB Journal

Article Title: Increased Expression of Desmin and Vimentin Impairs Mitochondrial Function and Reduces Contractility via G0S2 in Bladder Smooth Muscle

doi: 10.1096/fj.202502912R

Figure Lengend Snippet: Increased expression of desmin and vimentin induces mitochondrial and contractile dysfunction via G0S2 in human BSM cells. (A) Microarray analysis : Gene expression was analyzed using microarray‐based transcriptomics. A heatmap generated by hierarchical clustering illustrates trends in gene expression in human BSM cells overexpressing desmin or vimentin proteins compared with GFP‐expressing control cells. Each row represents a single mRNA probe, and the accompanying dendrogram shows the hierarchical clustering tree of the gene expression data. The colored bar at the bottom represents changes in gene expression on a linear scale, with blue indicating down‐regulation and red indicating up‐regulation. (B) G0S2 interaction with VDAC : Mitochondrial proteins from GFP‐, desmin‐, and vimentin‐overexpressing human BSM cells were subjected to co‐immunoprecipitation. Mitochondrial proteins were immunoprecipitated using an anti‐G0S2 antibody. The resulting immunoprecipitate was separated by SDS‐PAGE and probed with anti‐G0S2 and anti‐VDAC antibodies. (C) The protein expression levels of CAV1 were analyzed by immunoblotting. Representative immunoblot and its corresponding quantification are shown in panel C. (D) The levels of IL‐6 and IL‐1β were analyzed by ELISA. (E) The expression levels of VDAC protein across passages (P3–P5) were analyzed by immunoblotting. Cytochrome c oxidase IV (COXIV) served as a loading control. (F–I) Human BSM cells were transduced with adenovirus encoding GFP, desmin (Des), and vimentin (Vim) in the presence of scrambled (Scr) shRNA or G0S2 shRNA. (F, G) ROS levels in cells were measured using isolated mitochondria with the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit. (H) Quantification of total cellular ATP levels . ATP levels were measured based on firefly luciferin‐luciferase luminescence intensity. (I) BSM gel contraction : For gel contraction studies, cells were seeded in a collagen gel as described in Materials and Methods, and gel contraction was measured in response to 120 mM KCl stimulation. The gels were subsequently imaged. The size of the collagen gel in each image was quantified using Fiji (ImageJ/NIH) software, and the percentage change was calculated. Data are presented as mean ± SD, with n = 3 in A, n = 3 in C, F, G, and I, and n = 2 in D, n = 4 in H. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Mitochondria were isolated from human BSM cells and murine BSM tissue using the Mitochondria Isolation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions.

Techniques: Expressing, Microarray, Analysis, Gene Expression, Transcriptomics, Generated, Control, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Transduction, shRNA, Isolation, Luciferase, Software

Journal: The FASEB Journal

Article Title: Increased Expression of Desmin and Vimentin Impairs Mitochondrial Function and Reduces Contractility via G0S2 in Bladder Smooth Muscle

doi: 10.1096/fj.202502912R

Figure Lengend Snippet: Increased expression of desmin and vimentin induces contractile and mitochondrial dysfunction via G0S2 in murine BSM tissue. BSM strips from WT and G0S2 KO mice were transduced with an adenovirus encoding GFP, desmin, and vimentin for 36 h. BSM contraction : (A–D) The contractile responses of murine BSM strips overexpressing desmin and vimentin in response to KCl (120 mM) and carbachol (10 −9 to 10 −4 M) stimulation were determined. (E) Quantifications of ROS levels : ROS levels in murine BSM strips were measured using isolated mitochondria with the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit. (F) Quantifications of total cellular ATP levels . ATP levels were measured based on firefly luciferin‐luciferase luminance intensity. Data are presented as mean ± SD and n = 5 in A–F.

Article Snippet: Mitochondria were isolated from human BSM cells and murine BSM tissue using the Mitochondria Isolation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions.

Techniques: Expressing, Tissue, Transduction, Isolation, Luciferase