Journal: Antioxidants

Article Title: Exploring the Leukemogenic Potential of GATA-1 S , the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency

doi: 10.3390/antiox10101603

Figure Lengend Snippet: Primer sequences used for quantitative Real-time PCR analysis.

Article Snippet: Primary antibodies were used at the following experimental conditions: FLAG (1:10,000 dilution; Sigma-Aldrich), GATA-1 (D24E4) (1:1000 dilution; Cell Signaling, Danvers, MA, USA #4589), SDHC (3E2) (1:500 dilution; Novus Biologicals Bio-Techne, Minneapolis, MN, USA #H00006391-M01), glutathione synthetase (1:20,000 dilution; Abcam, Cambridge, UK # ab124811), glutathione reductase (1:5000 dilution; Abcam, # ab124995).

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Amplification

Journal: Antioxidants

Article Title: Exploring the Leukemogenic Potential of GATA-1 S , the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency

doi: 10.3390/antiox10101603

Figure Lengend Snippet: Western blot analysis for SDHC expression levels in total protein extracts obtained from K562 cells over-expressing GATA-1 FL and GATA-1 S isoforms and from a mock control. ( a ) Representative image of three independent experiments showing the presence of two SDHC-positive protein bands on the membrane possibly representing different SDHC isoforms. ( b ) Densitometric analysis of Western blot results showing total SDHC levels markedly increased only in K562 cells over-expressing the GATA-1 S isoform. For each sample, band intensities of the two SDHC signals, taken as a whole, were quantified from three independent experiments and normalized to α-actin used as a loading control. ( c ) Schematic representation of the alternative splicing mechanism generating SDHC variants (ASVs). Solid boxes and bars indicate the deleted exons and the corresponding protein domains, respectively. ( d ) Quantitative real-time PCR analysis of SDHC mRNA variants in cells over-expressing GATA-1 isoforms and in a mock control. mRNA expression levels were normalized against GAPDH. Results showed increased total SDHC transcript levels in cells over-expressing GATA-1 S , thus confirming western blot analysis. Moreover, transcript-specific amplification revealed that SDHC abnormal expression in these cells was mostly due to the Δ5 ASV transcript. All data represent the mean ± SD of three independent experiments. Statistical analysis was performed by one-way ANOVA, followed by Dunnett’s multiple comparisons test, where appropriate. Differences were considered significant when p < 0.05 and highly significant when p < 0.0001. * p < 0.05, ** p < 0.0001 versus mock control.

Article Snippet: Primary antibodies were used at the following experimental conditions: FLAG (1:10,000 dilution; Sigma-Aldrich), GATA-1 (D24E4) (1:1000 dilution; Cell Signaling, Danvers, MA, USA #4589), SDHC (3E2) (1:500 dilution; Novus Biologicals Bio-Techne, Minneapolis, MN, USA #H00006391-M01), glutathione synthetase (1:20,000 dilution; Abcam, Cambridge, UK # ab124811), glutathione reductase (1:5000 dilution; Abcam, # ab124995).

Techniques: Western Blot, Expressing, Control, Membrane, Alternative Splicing, Real-time Polymerase Chain Reaction, Amplification

Journal: Antioxidants

Article Title: Exploring the Leukemogenic Potential of GATA-1 S , the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency

doi: 10.3390/antiox10101603

Figure Lengend Snippet: GATA-1 S knockdown experiments: ( a ) western blot analysis (10% SDS-page gel) of endogenous levels of GATA-1 isoforms and SDHC after K562 transfection with a custom GATA-1 S small interfering RNA (GATA-1 S siRNA) at final concentration of 50 and 100 nM. ( b ) Densitometric analysis of western blot results of GATA-1 S silenced protein. ( c ) Densitometric analysis of western blot results for SDHC after specific GATA-1 S siRNA transfection. ( d ) Quantitative real-time PCR analysis of SDHC mRNA variants in K562 cells previously transfected with two doses of specific GATA-1 S siRNA. mRNA expression levels were normalized against GAPDH and relative to negative control siRNA transfected cells. Results showed decreased total SDHC transcript levels in cells knocked down for GATA-1 S , thus confirming western blot analysis. In addition, transcript-specific amplification revealed a more significant dose-dependent reduction for the Δ5 ASV isoform of SDHC following GATA-1 S silencing. All data represent the mean ± SD of three independent experiments. Statistical analysis was performed by one-way ANOVA, followed by Dunnett’s multiple comparisons test, where appropriate. Differences were considered significant when p < 0.05 and highly significant when p < 0.0001. * p < 0.05, ** p < 0.0001 versus negative control; # p < 0.05 versus lower dose of siRNA transfection.

Article Snippet: Primary antibodies were used at the following experimental conditions: FLAG (1:10,000 dilution; Sigma-Aldrich), GATA-1 (D24E4) (1:1000 dilution; Cell Signaling, Danvers, MA, USA #4589), SDHC (3E2) (1:500 dilution; Novus Biologicals Bio-Techne, Minneapolis, MN, USA #H00006391-M01), glutathione synthetase (1:20,000 dilution; Abcam, Cambridge, UK # ab124811), glutathione reductase (1:5000 dilution; Abcam, # ab124995).

Techniques: Knockdown, Western Blot, SDS Page, Transfection, Small Interfering RNA, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Negative Control, Amplification

Journal: Antioxidants

Article Title: Exploring the Leukemogenic Potential of GATA-1 S , the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency

doi: 10.3390/antiox10101603

Figure Lengend Snippet: Enzymatic activity of succinate CoQ oxidoreductase (SQR) in K562 cells over expressing GATA-1 isoforms. ( a ) SQR activity detected on total cell lysates is expressed as OD absorbance/min/mg total protein. Data represent mean ± SD from three independent experiments. Differences were considered significant when * p < 0.05 and highly significant when ** p < 0.0001 versus mock control; ( b , c ) Schematic representation of complex II disassembly induced by over-expression of the SDHC Δ5 variant lacking the heme binding site with impaired SQR activity and increased O 2 − production . (Created with BioRender.com, accessed on 6 August 2021).

Article Snippet: Primary antibodies were used at the following experimental conditions: FLAG (1:10,000 dilution; Sigma-Aldrich), GATA-1 (D24E4) (1:1000 dilution; Cell Signaling, Danvers, MA, USA #4589), SDHC (3E2) (1:500 dilution; Novus Biologicals Bio-Techne, Minneapolis, MN, USA #H00006391-M01), glutathione synthetase (1:20,000 dilution; Abcam, Cambridge, UK # ab124811), glutathione reductase (1:5000 dilution; Abcam, # ab124995).

Techniques: Activity Assay, Expressing, Control, Over Expression, Variant Assay, Binding Assay

Journal: Antioxidants

Article Title: Exploring the Leukemogenic Potential of GATA-1 S , the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency

doi: 10.3390/antiox10101603

Figure Lengend Snippet: ( a , b ) Quantitative analysis of SDHC mRNA variant transcripts and GATA-1 isoforms protein levels from bone marrow specimens of an AML patient at diagnosis and post-therapy stages relative to the remission values. mRNA expression levels were normalized against GAPDH; ( c ) Quantitative real-time PCR analysis of HIF-1α transcript levels, normalized against GAPDH, in bone marrow samples obtained from the AML patient at diagnosis, post-therapy and remission stages. Results showed significant increment of HIF-1α transcript levels at the diagnosis stage with respect to post-therapy and remission stages. All data represent the mean ± SD of three independent experiments. Statistical analysis was performed by one-way ANOVA, followed by Dunnett’s multiple comparisons test, where appropriate. Differences were considered significant when p < 0.05 and highly significant when p < 0.0001. * p < 0.05, ** p < 0.0001 versus control.

Article Snippet: Primary antibodies were used at the following experimental conditions: FLAG (1:10,000 dilution; Sigma-Aldrich), GATA-1 (D24E4) (1:1000 dilution; Cell Signaling, Danvers, MA, USA #4589), SDHC (3E2) (1:500 dilution; Novus Biologicals Bio-Techne, Minneapolis, MN, USA #H00006391-M01), glutathione synthetase (1:20,000 dilution; Abcam, Cambridge, UK # ab124811), glutathione reductase (1:5000 dilution; Abcam, # ab124995).

Techniques: Variant Assay, Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Cell Death and Differentiation

Article Title: Specific disintegration of complex II succinate:ubiquinone oxidoreductase links pH changes to oxidative stress for apoptosis induction

doi: 10.1038/cdd.2010.93

Figure Lengend Snippet: Effect of various anticancer drugs and cytokines and the shRNA-mediated downregulation on the enzymatic activities of complex II. (a) Effects of the various pro-apoptotic signals on the SQR and SDH activities of complex II. HeLa cells were treated with the indicated drugs (7 h) or cytokines (16 h). Mitochondria were isolated and SQR and SDH activities were assessed by the appropriate assay. *P<0.05 compared with the related control. (b, c) Downregulation of SDHA/B/C/D mRNA (b) and proteins (c) by shRNA. HeLa cells were transfected with an empty pSuper vector, a scrambled pSuper-shRNA or shRNA constructs targeting SDHA/B/C/D. After 72 h, mRNA levels (b) of the various complex II subunits were quantified by RT–PCR. Proteins levels (c) were analysed by western blot in mitochondrial fractions. Equal gel loading and transfer efficiency were checked with anti-Cox IV or Cyt C antibodies. (d) ShRNA-mediated downregulation of SDHA/B/C/D does not induce apoptosis. HeLa cells were transfected either with a luciferase vector, a scrambled shRNA or a specific SDHA/B/C/D shRNA. After 72 h, apoptosis was measured by flow cytometry using PI staining of the sub-G1 population. A wt SDHC expression vector was transfected as a positive control for apoptosis induction after 24 h. The same amount of a GFP plasmid was introduced in parallel to normalise the cell death induction to the transfection efficiency. Results are shown after subtraction of the luciferase-associated apoptosis background. #P<0.05. (e) Potent inhibition of SQR and SDH activity by shRNA-mediated downregulation of SDHA/B/C/D. SQR and SDH activities of complex II were measured in mitochondrial fractions isolated 72 h after transfection. Shown are the activities relative to the scrambled shRNA-transfected cells. *P<0.05 compared with the related scrambled shRNA-activity. (f) Reconstitution of SDHC expression in B9 cells sensitises cells for apoptosis. PcDNA3 clone 2 (control cells stably transfected with an empty pcDNA3 vector) and wt SDHC clone 4 (stably reconstituted cells) were treated with the indicated anticancer drugs for 48 h, at the concentrations shown in the Supplementary Table 1. Apoptosis was quantified as in (d). #P<0.05

Article Snippet: 18 Goat polyclonal Abs against SDHB and SDHD were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), mouse mAbs against SDHA and COX IV from Abcam (Cambridge, UK), and mouse mAbs against Cyt c and SDHC from BD Pharmingen (Oxford, UK) and Abnova (Stratech Scientific, Newmarket, UK), respectively.

Techniques: shRNA, Isolation, Control, Transfection, Plasmid Preparation, Construct, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Flow Cytometry, Staining, Expressing, Positive Control, Inhibition, Activity Assay, Stable Transfection

Journal: Cell Death and Differentiation

Article Title: Specific disintegration of complex II succinate:ubiquinone oxidoreductase links pH changes to oxidative stress for apoptosis induction

doi: 10.1038/cdd.2010.93

Figure Lengend Snippet: Specific complex II disintegration by pH change. (a) Effects of pHi acidification and NHE1 inhibition on complex II enzymatic activities. (Left panel) Isolated mitochondria were incubated in a phosphate buffer at normal pH 7.3 or acidic pH 6.7 and the appropriate assay was performed. (Right panel) HeLa cells were treated for 7 h with EIPA (20 μM), mitochondria were isolated, and SQR and SDH activities were assessed by specific enzymatic assays. #P<0.05. (b) Integrity of complex II as revealed by immunoblots of blue native PAGE. (Left panel) Isolated mitochondria from HeLa cells were incubated in phosphate buffers with the indicated pH for 30 min and complex II solubilisation was performed as described in Materials and Methods. (Right panel) HeLa cells were incubated for 7 h with 10 μM As2O3 or 1.4 μM doxorubicin. Whole-cell lysates were prepared and their proteins solubilised as described in Materials and Methods. Equal amounts of protein were loaded onto a native gel, blotted onto a membrane, which was probed for SDHA. (c) Overexpression of NHE1 reduces drug-induced complex II dissociation. HeLa cells were transfected either with pcDNA3 or with a NHE1 expression plasmid. After 24 h, the cells were treated with 1.4 μM doxorubicin (left panel) or 10 μM As2O3 (right panel) for 7 h. Proteins from whole-cell lysates were processed as in (b) and loaded onto a native gel. For (b) and (c), the percentage of SDHA/B subcomplex dissociation was determined using densitometric analyses of at least four independent experiments and shown as graphs under the related blots. *P<0.01 compared with the related control; #P<0.01

Article Snippet: 18 Goat polyclonal Abs against SDHB and SDHD were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), mouse mAbs against SDHA and COX IV from Abcam (Cambridge, UK), and mouse mAbs against Cyt c and SDHC from BD Pharmingen (Oxford, UK) and Abnova (Stratech Scientific, Newmarket, UK), respectively.

Techniques: Inhibition, Isolation, Incubation, Western Blot, Blue Native PAGE, Membrane, Over Expression, Transfection, Expressing, Plasmid Preparation, Control

Journal: Cell Death and Differentiation

Article Title: Specific disintegration of complex II succinate:ubiquinone oxidoreductase links pH changes to oxidative stress for apoptosis induction

doi: 10.1038/cdd.2010.93

Figure Lengend Snippet: Model for the role of specific complex II inhibition for apoptosis induction by various pro-apoptotic compounds. (a) In healthy cells, complex II serves to funnel electrons derived from the Krebs cycle to the respiratory chain. SDHA-mediated oxidation of succinate to fumarate by the succinate dehydrogenase activity (SDH), as part of the Krebs cycle, provides electrons to complex II. They are transferred to the iron-sulfur centres of the SDHB subunit and finally to the CoQ reduction site at the interface between SDHC and SDHD, the two transmembrane subunits of complex II. The succinate CoQ oxidoreductase (SQR) reaction comprises, in addition, the transfer of the electrons to CoQ. (b) Pro-apoptotic compounds, such as various anticancer drugs, FasL, or TNF-α, induce intracellular (pHi) and mitochondrial (pHM) acidification. These pH changes lead to the dissociation of the SDHA/B subunits from complex II and finally to the partial inhibition of the SQR activity without any impairment of the SDH reaction. This specific inhibition leads to complex II uncoupling, superoxide production, and apoptosis

Article Snippet: 18 Goat polyclonal Abs against SDHB and SDHD were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), mouse mAbs against SDHA and COX IV from Abcam (Cambridge, UK), and mouse mAbs against Cyt c and SDHC from BD Pharmingen (Oxford, UK) and Abnova (Stratech Scientific, Newmarket, UK), respectively.

Techniques: Inhibition, Derivative Assay, Activity Assay