y 27632  (Santa Cruz Biotechnology)

Journal: International journal of molecular sciences

Article Title: Iron Accumulation and Lipid Peroxidation in Cellular Models of Nemaline Myopathies.

doi: 10.3390/ijms26041434

Figure Lengend Snippet: Figure 10. Actin staining by Rhodamine–Phalloidin and MitotrackerTM DeepRed of control fibroblasts treated with Y27632 or Cytochalasin D inhibitors. (A) Control fibroblasts (C1) were treated with 10 µM Y27632 for 3 h or with 20 µM Cytochalasin D for 24 h. Control fibroblasts were stained with Rhodamine–Phalloidin and MitotrackerTM DeepRed and visualized under a widefield fluorescence microscope. Nuclei were revealed by DAPI staining. Treated control fibroblasts presented smaller and unstructured actin filaments compared to untreated control fibroblasts. Images were taken using the 40× lens and processed by the ImageJ software (version 1.54f). (B) Measurement of the length of actin filaments (µm). The length of the actin filaments was measured in triplicate with the ImageJ software (version 1.54f) in 30 images. (C) Quantification of tubular and rounded percentage of mitochondria in control cells. Data represent the mean ± SD of three separate experiments (at least 100 cells for each condition and experiment were analyzed). *** p < 0.001 between treated and untreated control cells. Scale bar = 20 µm.

Article Snippet: Y27632 (sc-3536) and paraformaldehyde (PFA; sc-25326B) were purchased from Santa Cruz Biotechnology (California (CA), United States (USA)).

Techniques: Staining, Control, Fluorescence, Microscopy, Software

Journal: International journal of molecular sciences

Article Title: Iron Accumulation and Lipid Peroxidation in Cellular Models of Nemaline Myopathies.

doi: 10.3390/ijms26041434

Figure Lengend Snippet: Figure 11. Bioenergetic analysis of control cells untreated and treated with Y27632 inhibitor or Cytochalasin D. (A) Respiratory profile of untreated and treated control cells (C1). (B) Basal, maximal, and spare respiratory capacity and ATP production were determined in untreated and treated control fibroblast using the Seahorse analyzer. Control fibroblasts were treated with 10 µM of Y27632 for 24 h or with 20 µM of Cytochalasin D for 3 h. ** p < 0.01, *** p < 0.001 between untreated and treated control cells.

Article Snippet: Y27632 (sc-3536) and paraformaldehyde (PFA; sc-25326B) were purchased from Santa Cruz Biotechnology (California (CA), United States (USA)).

Techniques: Control

Journal: International journal of molecular sciences

Article Title: Iron Accumulation and Lipid Peroxidation in Cellular Models of Nemaline Myopathies.

doi: 10.3390/ijms26041434

Figure Lengend Snippet: Figure 12. Expression levels of iron metabolism-related proteins in control fibroblasts treated with acting depolymerizing agents. (A) Immunoblotting analysis of cellular extracts from control cells (C1) treated with 10 µM of Y27632 for 24 h or 20 µM Cytochalasin D for 3 h. Protein extracts (50 µg) were separated on a SDS polyacrylamide gel and immunostained with antibodies against TFR, DMT1, IRP1, Ferritin, Mitoferritin, Mitoferrin2, FXN, ISCU, Mt-ACP, and PANK2. Tubulin was used as a loading control. (B) Densitometry of Western blotting. Data represent the mean ± SD of three separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 between untreated and treated controls. A.U., arbitrary units.

Article Snippet: Y27632 (sc-3536) and paraformaldehyde (PFA; sc-25326B) were purchased from Santa Cruz Biotechnology (California (CA), United States (USA)).

Techniques: Expressing, Control, Western Blot

Journal: International journal of molecular sciences

Article Title: Iron Accumulation and Lipid Peroxidation in Cellular Models of Nemaline Myopathies.

doi: 10.3390/ijms26041434

Figure Lengend Snippet: Figure 13. Iron accumulation in control cells treated with Y27632 or Cytochalasin D inhibitors. (A) Prussian Blue staining of control (C1) (−) (−) and control cells treated with 10 µM Y27632 for 24 h, or with 20 µM Cytochalasin D for 3 h. Images were made in brightfield by an Axio Vert A1 inverted optical microscope (Zeiss, Oberkochen, Germany) with a 40× objective and were analyzed using Fiji-ImageJ software (version 2.9.0/1.53t) (National Institute of Health, Bethesda, MD, USA). Scale bar = 20 µm. (B) Quantification of Prussian Blue staining images was performed by the Image J software (version 1.54f). Data represent the mean ± SD of three separate experiments. ** p < 0.01, *** p < 0.001 between untreated and treated control cells. A.U., arbitrary units.

Article Snippet: Y27632 (sc-3536) and paraformaldehyde (PFA; sc-25326B) were purchased from Santa Cruz Biotechnology (California (CA), United States (USA)).

Techniques: Control, Staining, Microscopy, Software

Journal: International journal of molecular sciences

Article Title: Iron Accumulation and Lipid Peroxidation in Cellular Models of Nemaline Myopathies.

doi: 10.3390/ijms26041434

Figure Lengend Snippet: Figure 15. Cellular lipid peroxidation in control cells treated with Y27632 or Cytochalasin D inhibitors. (A) Control cells (C1) were treated with 10 µM Y27632 for 24 h, or with 20 µM Cytochalasin D for 3 h. The levels of cellular lipid peroxidation were measured using BODIPY® staining as detailed in the Material and Methods. (B) BODIPY® staining quantification was performed by using the Fiji software (version 2.9.0/1.53t). Control fibroblasts were treated with 500 µM Luperox (LUP, Tert-butyl hydroperoxide) for 15 min as positive control of lipid peroxidation. Scale bar = 20 µm. ** p < 0.01, *** p < 0.001 between treated and untreated control fibroblasts. Data represent the mean ± SD of four separate experiments. A.U., arbitrary units.

Article Snippet: Y27632 (sc-3536) and paraformaldehyde (PFA; sc-25326B) were purchased from Santa Cruz Biotechnology (California (CA), United States (USA)).

Techniques: Control, Staining, Software, Positive Control