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