Journal: Antioxidants

Article Title: Mitoferrin-1 Promotes Proliferation and Abrogates Protein Oxidation via the Glutathione Pathway in Glioblastoma

doi: 10.3390/antiox12020349

Figure Lengend Snippet: MFRN1 expression in stably transfected glioma cells. U251 cells were transfected with a vector expressing MFRN1 (pCMV6−CAT−-Myc−DDK) or the vector only (pCMV6−Myc−DDK) as a control and treated with G418 to produce stably transfected clones. ( A ), Quantitative analysis of MFRN1 mRNA by qPCR. Graphs represent the mean ± SEM of duplicate determinations from three independent experiments. **** p < 0.0001 calculated by Student t -test. ( B ), Representative Western blot depicting MFRN1 protein expression in mitochondrial extracts of select clones (9 and 10). Citrate synthase (CS) was used as a loading control. ( C ), Cell proliferation analysis in cell cultures of control and MFRN1-overexpressing cells. ( D , E ), Representative images ( D ) and quantitative analysis ( E ) of anchorage-independent colony formation in control and MFRN1-overexpressing cells. ( F ) OS in nude mice harboring orthotopic brain tumors generated by inoculation with vector control-transfected or MFRN1-overexpressing (clone 10) U251 cells (n = 6 per group). ( G ), Representative images of control and MFRN1 tumors stained for hematoxylin and eosin (H&E) and Ki-67. T, tumor; NT, Normal tissue. * p < 0.05, ** p < 0.01, and **** p < 0.0001 calculated using one-way ANOVA followed by Tukey’s multiple comparison test or unpaired, two-tailed Student t -test.

Article Snippet: U251 cells were electroporated with CMV6 plasmids containing Myc-DDK-epitope-tagged human MFRN1 or the pCMV6-Entry mammalian vector with a C-terminal Myc-DDK tag as the control (NM_016612.4, catalog # RC218413 and PS100001, respectively; OriGene Technologies, Rockville, MD, USA).

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Clone Assay, Western Blot, Generated, Staining, Two Tailed Test

Journal: Antioxidants

Article Title: Mitoferrin-1 Promotes Proliferation and Abrogates Protein Oxidation via the Glutathione Pathway in Glioblastoma

doi: 10.3390/antiox12020349

Figure Lengend Snippet: MFRN1 overexpression increases mitochondrial iron levels and the activity of ETC complexes. ( A ), MFRN1-overexpressing U251 cells were transfected with mCherry-TOMM20-N-10 (left panel) and stained with Mito-FerroGreen (middle panel) to confirm the mitochondrial localization of the detected iron (combined, right panel). ( B ), Quantification of the fluorescence intensity of the Mito-FerroGreen signal in MFRN1-overexpressing and vector-control expressing cells. ( C – G ), Activity of mitochondrial complex I ( C ), complexes II–III ( D ), CcO ( E ), citrate synthase ( F ), and complex V in MFRN1-overexpressing and vector control-expressing U251 cells. Graphs represent the mean ± SEM from duplicate determinations from at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 calculated using one-way ANOVA followed by Tukey’s multiple comparison test or unpaired, two-tailed Student t -test.

Article Snippet: U251 cells were electroporated with CMV6 plasmids containing Myc-DDK-epitope-tagged human MFRN1 or the pCMV6-Entry mammalian vector with a C-terminal Myc-DDK tag as the control (NM_016612.4, catalog # RC218413 and PS100001, respectively; OriGene Technologies, Rockville, MD, USA).

Techniques: Over Expression, Activity Assay, Transfection, Staining, Fluorescence, Plasmid Preparation, Expressing, Two Tailed Test

Journal: Antioxidants

Article Title: Mitoferrin-1 Promotes Proliferation and Abrogates Protein Oxidation via the Glutathione Pathway in Glioblastoma

doi: 10.3390/antiox12020349

Figure Lengend Snippet: MFRN1 overexpression protects glioma cells from 4-HNE-induced damage. ( A ), Level of labile iron pool in vector control-transfected and MFRN1-overexpressing U251 cells; ( B ), Relative levels of lipid peroxidation in vector control-transfected and MFRN1-overexpressing cells, assessed with BODIPY C11; ( C ), Representative images depicting Michael adducts on proteins (left immunoblot) and total protein detection (right gel) before and after 1 h of 4-HNE treatment at the indicated doses in vector control-transfected and MFRN1-overexpressing cells. ( D ), Bars depict the levels of key metabolites involved in the glutathione pathway in vector control-transfected and MFRN1-overexpressing cells. Results of six independent measurements. * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 calculated using one-way ANOVA followed by Tukey’s multiple comparison test or unpaired, two-tailed Student t -test. a.u., arbitrary units. GCS, glutamyl cysteine synthetase; GS, glutathione synthase; GSH, reduced glutathione.

Article Snippet: U251 cells were electroporated with CMV6 plasmids containing Myc-DDK-epitope-tagged human MFRN1 or the pCMV6-Entry mammalian vector with a C-terminal Myc-DDK tag as the control (NM_016612.4, catalog # RC218413 and PS100001, respectively; OriGene Technologies, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Transfection, Western Blot, Two Tailed Test

Journal: Antioxidants

Article Title: Mitoferrin-1 Promotes Proliferation and Abrogates Protein Oxidation via the Glutathione Pathway in Glioblastoma

doi: 10.3390/antiox12020349

Figure Lengend Snippet: Inhibition of glutathione production facilitates 4-HNE-mediated protein damage in MFRN1-overexpressing glioma cells. ( A ), Quantification of intracellular GSH content in MFRN1-overexpressing U251 cells treated with 5 mM BSO for 24 h. ( B ), Representative images depicting the effects of BSO pretreatment (5 mM, 24 h) on the formation of 4-HNE-induced Michael adducts on proteins (left immunoblot) and total protein detection (right gel) in MFRN1-overexpressing cells. ( C ), Quantification of intracellular GSH content in MFRN1-overexpressing cells treated with 20 µM erastin for 24 h. (D) Representative images depicting the effects of erastin pretreatment (20 µM, 24 h) on the formation of 4-HNE-induced Michael adducts after 1 in MFRN1-overexpressing cells. **** p < 0.0001 calculated using one-way ANOVA followed by Tukey’s multiple comparison test or unpaired, two-tailed Student t -test.

Article Snippet: U251 cells were electroporated with CMV6 plasmids containing Myc-DDK-epitope-tagged human MFRN1 or the pCMV6-Entry mammalian vector with a C-terminal Myc-DDK tag as the control (NM_016612.4, catalog # RC218413 and PS100001, respectively; OriGene Technologies, Rockville, MD, USA).

Techniques: Inhibition, Western Blot, Two Tailed Test

Journal: Acta Neuropathologica

Article Title: Expression of the progenitor marker NG2/ CSPG4 predicts poor survival and resistance to ionising radiation in glioblastoma

doi: 10.1007/s00401-011-0867-2

Figure Lengend Snippet: PRDX-1 knockdown sensitises IR resistant NG2 positive cells in vivo. a Schematic of an intracerebral tumour-bearing mouse implanted with 125 I seeds. A gradient IR dose ( fading line ) was delivered to the tumour bed. B brain, S guide-screw, T tumour, n number of animals (adapted from ). b Tumour sizes in vivo before IR treatment ( top panel ) and after treatment with 125 I ( bottom panel ). c Histological staining at 19 weeks with H&E and anti-NG2 antibodies. Note the vascular lakes in the U251-NG2 control shRNA and numerous mitotic figures ( arrowheads ) in U251-NG2/PRDX-1 shRNA tumours, giant multinucleated cells ( arrowheads ). Scale bars in all (100 μm; magnification 400×), except rows stained for NG2 (200 μm; magnification 200×). d Densitometric quantified, Fold PRDX-1 change in tumour compared to normal brain tissue of mice receiving no treatment or ionizing radiation

Article Snippet: Retroviral PRDX-1 and irrelevant GFP control shRNA constructs were purchased from Origene (Rockville, MD), and used to generate the stable U251-NG2 shPRDX-1 and U251-NG2-shCTRL cell lines as well control U251 shPRDX-1 and U251 shCTRL cell lines.

Techniques: In Vivo, Staining, shRNA

Journal: Acta Neuropathologica

Article Title: Expression of the progenitor marker NG2/ CSPG4 predicts poor survival and resistance to ionising radiation in glioblastoma

doi: 10.1007/s00401-011-0867-2

Figure Lengend Snippet: NG2 positive GBM cells activate G2 checkpoint post irradiation, and this effect is partly abrogated by PRDX-1 knockdown. Activation of G2 checkpoint post 5 Gy irradiation was assessed by flow cytometry. Plots display phospho-Histone H3 staining intensity versus DNA Content/cell from one representative of three independent experiments. The percentages of mitotic cells indicate an efficient G2 arrest in the U251-NG2 positive cells and the U251-NG2control shRNA cells ( middle panels ), as fewer cells enter mitosis post irradiation compared to the U251 ( left panels ). Knockdown of PRDX-1 partially abrogates the effect of NG2 on G2 cell cycle checkpoint activity by allowing transition to M-phase post-irradiation ( right panels )

Article Snippet: Retroviral PRDX-1 and irrelevant GFP control shRNA constructs were purchased from Origene (Rockville, MD), and used to generate the stable U251-NG2 shPRDX-1 and U251-NG2-shCTRL cell lines as well control U251 shPRDX-1 and U251 shCTRL cell lines.

Techniques: Irradiation, Activation Assay, Flow Cytometry, Staining, shRNA, Activity Assay