Journal: Antioxidants

Article Title: Actin Polymerization Defects Induce Mitochondrial Dysfunction in Cellular Models of Nemaline Myopathies

doi: 10.3390/antiox12122023

Figure Lengend Snippet: Mitochondrial polarization and network in control and NM cells. ( A ) Representative images of control (C1) and NM fibroblasts (P1, P2, P3, and P4) stained with MitoTracker TM Red CMXRos and visualized under a widefield fluorescence microscope. Nuclei were revealed by DAPI staining; 100 µM CCCP was used for 4 h as a positive control of mitochondrial depolarization in control cells. Rounded small mitochondria are marked with white arrows and tubular mitochondria are marked with green arrows in P3 and P4. Images were taken using a 100× lens and processed by ImageJ software. Scale bar = 20 µm. ( B ) Fluorescence quantification of MitoTracker signal. Data represent the mean ± SD of three separate experiments (at least 100 cells for each condition and experiment were analyzed). ( C ) Quantification of tubular and rounded percentages of mitochondria in control and NM fibroblasts. Data represent the mean ± SD of three separate experiments (at least 100 cells for each condition and experiment were analyzed). ( D ) Cellular extracts from controls (C1 and C2) and NM patient cell lines P1, P2, P3, and P4 were subjected to immunoblotting analysis. An SDS polyacrylamide gel was used to separate protein extracts (50 μg), then the samples were immunostained using antibodies against DRP1, OPA1, and α-tubulin, which was used as a loading control. ( E ) Densitometry of the Western blotting. For controls cells (C1 and C2), data are the mean ± SD of the two control cell lines. Data represent the mean ± SD of three separate experiments. ** p < 0.01, *** p < 0.001 between NM cells and controls; a p < 0.05, aaa p < 0.001 between the presence and the absence of CCCP. a.u.: arbitrary units.

Article Snippet: Optic atrophy type 1 (OPA1) (HPA036926) was acquired from Sigma-Aldrich (San Luis, MO, USA).

Techniques: Staining, Fluorescence, Microscopy, Positive Control, Software, Western Blot

Journal: Antioxidants

Article Title: Actin Polymerization Defects Induce Mitochondrial Dysfunction in Cellular Models of Nemaline Myopathies

doi: 10.3390/antiox12122023

Figure Lengend Snippet: Effect of LA and LCAR on mitochondrial protein expression levels in control and NM cells. Control (C1) and NM fibroblasts (P1, P2, P3, and P4) were treated with 5 µM LA and 10 µM LCAR individually or in combination (Cocktail, CK) for 7 days. ( A ) Cellular extracts from control and NM patient cell lines P1 and P2 were subjected to immunoblotting analysis. ( B ) Cellular extracts from control and NM patient cell lines P3 and P4 were subjected to immunoblotting analysis. An SDS polyacrylamide gel was used to separate protein extracts (50 μg), then the samples were immunostained using antibodies against NDUFA9, SDHB, UQCR2, Mt-CO2, ATP5F1A, VDAC, OPA1, and DRP1. α-Tubulin was used as a loading control. (Densitometry of the Western blotting ).

Article Snippet: Optic atrophy type 1 (OPA1) (HPA036926) was acquired from Sigma-Aldrich (San Luis, MO, USA).

Techniques: Expressing, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: Optineurin E50K triggers BDNF deficiency-mediated mitochondrial dysfunction in retinal photoreceptor cell line

doi: 10.1016/j.bbrc.2018.08.025

Figure Lengend Snippet: BDNF deficiency results in bioenergetic dysfunction but did not alter mitochondrial dynamics-related protein expression in RGC-5 cells. Protein expression of DRP1, p-DRP1 S616, OPA1, and Mfn1 and 2 in RGC-5 cells transfected with BNDF siRNA. For each determination, the actin level was normalized to a value of 1.0. Data are shown as the mean ± S.D.

Article Snippet: Primary antibodies included Bax (6A7; 1:1,000; Santa Cruz Biotechnology, US), BDNF (1:1,000; Santa Cruz Biotechnology), phosphocyclic adenosine monophosphate response element-binding protein (CREB, Serine 133) (1:1,000; Life Technologies, Grand Island, US), cyclophilin D (CypD) antibody (1:1,000; Life Technologies), dynamin-related protein 1 (DRP1) (1:5,000, BD Transduction Laboratories, US), phospho-DRP1 (Serine 616) (1:1,000, Cell Signaling, US), microtubule-associated protein 1A/1B-Iight chain 3 (LC3) (1:3,000; MBL International, US), OPTN (1:1,000; Santa Cruz Biotechnology), total oxidative phosphorylation (OXPHOS) complex (Cx) (containing a mixture of antibodies to CxI-IV and ATP synthase, 1:4000; Life Technologies), optic atrophy type 1 (OPA1) (1:5,000; BD Transduction Laboratories), mitofusin (Mfn)1 and 2 (1:3,000; Abcam), huntingtin (HTT) (1:2,000, Millipore) and actin (1:10,000; Millipore, US).

Techniques: Expressing, Transfection