Journal: International Journal of Molecular Sciences

Article Title: Modulation of Cellular Senescence in HEK293 and HepG2 Cells by Ultrafiltrates UPla and ULu Is Partly Mediated by Modulation of Mitochondrial Homeostasis under Oxidative Stress

doi: 10.3390/ijms24076748

Figure Lengend Snippet: TaqMan gene assays.

Article Snippet: 25 , SDHC , Hs00818427_m1 , Mitochondrial protein.

Techniques: Marker

Journal: International Journal of Molecular Sciences

Article Title: Modulation of Cellular Senescence in HEK293 and HepG2 Cells by Ultrafiltrates UPla and ULu Is Partly Mediated by Modulation of Mitochondrial Homeostasis under Oxidative Stress

doi: 10.3390/ijms24076748

Figure Lengend Snippet: Genes significantly upregulated by H 2 O 2 in HepG2 senescence.

Article Snippet: 25 , SDHC , Hs00818427_m1 , Mitochondrial protein.

Techniques: Marker

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: bioRxiv

Article Title: Iron addicted colorectal cancers exploit Heme-Complex II axis to resist oxidative cell death

doi: 10.1101/2025.11.05.686813

Figure Lengend Snippet: A) Schematic showing the xenograft tumor model with subcutaneous implantation. B) Tumor weight from B) C) Lipid ROS are measured by C11-BODIPY581/591 in EPCAM positive cells from the tumors from B). D) Schematic showing breeding strategy to generate tamoxifen inducible intestine specific SDHC deficient mice. E) Western blot images showing cellular and mitochondrial protein expression of SDHC 5days post tamoxifen induction via 100mg/kg body weight I.P. injection in WT ( Sdhc +/+), Het ( Sdhc +/-) and KO ( Sdhc -/-) mice. F) Representative H&E images of Sdhc F/F and Sdhc ΔIE mice fed with 350 ppm or 1000ppm iron containing chow for 7 days. G) Inflammatory histology scored for small intestine and colon of Sdhc F/F and Sdhc ΔIE mice fed with 350 ppm or 1000ppm iron containing chow for 7 days. H) Representative immunohistochemistry images showing accumulation of 4 hydroxynonenal (4HNE) in small intestine and colon of Sdhc F/F and Sdhc ΔIE mice fed with 350 ppm or 1000ppm iron containing chow for 7 days. I) Schematic showing treatment protocol for colitis associated cancer model using AOM/DSS in Sdhc F/F (WT) and Sdhc Het IE (Het) mice. J) Tumor number measured in mice from I). K) Tumor burden measured in mice from I) L) Lipid ROS measured by C11-BODIPY581/591 in EPCAM positive epithelial cells derived from I). M) Schematic showing role of heme-SDHC-CoQ axis in mitigating iron induced cell death in CRC cells. All data are mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test for (B) and (G) and t test for (C), (J), (K) and (L). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Followed by blocking with 5% goat serum in PBS and sections were then probed with primary antibody against SDHC (Proteintech, 14575-AP) or 4HNE (R&D Systems, MAB3249-SP) diluted at 1:500 in the blocking solution.

Techniques: Western Blot, Expressing, Injection, Immunohistochemistry, Derivative Assay

Journal: Cell Research

Article Title: USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA

doi: 10.1038/cr.2016.125

Figure Lengend Snippet: USP18 recruits USP20 to deubiquitinate STING. (A) Immunoblot of HEK293 cells that were transfected to express FLAG-STING, HA-Ubiquitin, along with vector, USP18 or USP18 (C64S), lysed and immunoprecipitated with control IgG or anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-USP18. (B) Immunoblot of HEK293 cells that were transfected to express FLAG-STING, HA-Ubiquitin, along with vector, USP20 or USP20 (C560/563S), lysed and immunoprecipitated with control IgG or anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-USP20. (C) Immunoblot of HEK293 cells transfected with a control siRNA, siUSP18 (left panels) or siUSP20 (right panels) for 12 h followed by transfection of HA-STING and Myc-Ubiquitin, along with FLAG-USP20 (left panels) or pCMV-USP18 (right panels) for 24 h, lysed and immunoprecipitated with anti-HA (left) or anti-FLAG (right). Cell lysate was analyzed by immunoblot with anti-FLAG, anti-HA, anti-USP20, anti-USP18 or anti-β-Actin. The intensities of ubiquitin-modified STING were normalized to β-Actin (graphs below). (D) Immunoblot of Usp18+/+and Usp18−/− BMDCs infected with HSV-1 for 4-8 h, lysed and immunoprecipitated with anti-STING followed by immunoblot analysis with antibodies to the indicated proteins. (E) In vitro deubiquitination analysis of ubiquitin-modified STING eluted from the denature immunoprecipitates (anti-FLAG) from HEK293 cells transfected with FLAG-STING and HA-ubiquitin with FLAG peptide followed by incubation with in vitro generated USP18, USP18(C64S), USP20, USP20(C560/563S) by an in vitro transcription and translation kit. The mixtures were analyzed by immunoblot analysis with antibodies against HA, FLAG, USP18 or USP20. Data are three (A-C) or two (D-E) independent experiments.

Article Snippet: USP18, USP18 (C64S), USP20 and USP20 (C560/563S) were obtained by a TNT in vitro transcription/translation kit (Promega).

Techniques: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Modification, Infection, In Vitro, Incubation, Generated

Journal: Cell Research

Article Title: USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA

doi: 10.1038/cr.2016.125

Figure Lengend Snippet: USP20 is involved in HSV-1-triggered signaling. (A) qRT-PCR analysis of IFNB, IL6, IP-10 and CCL5 mRNA in THP-1 cells (5 × 105) transfected with control siRNA (Control) or siRNA targeting USP20 (siUSP20) for 36 h left uninfected or untransfected, or infected with HSV-1, SeV or transfected with ISD45, HSV60, DNA90 and HSV120 (3 μg). (B) Immunoblot of total and phosphorylated (p-) IκBα and IRF3, total USP20 and β-Actin in ThP-1 cells (5 × 106) transfected with control siRNA (Con) or siUSP20 followed by HSV-1 infection for 0-8 h. (C) Flow cytometry analysis of GFP-tagged HSV-1 (MOI = 0.1) in THP-1 cells (5 × 105) transfected with control siRNA (Con) or siUSP20 followed by infection with HSV-1-GFP for 24 h. (D) qRT-PCR analysis of HSV-1 UL30 mRNA in THP-1 cells (5 × 105) transfected with control siRNA (Control) or siUSP20 infected with HSV-1 (MOI = 0.1) for 1 h followed by PBS wash twice and cultured with full medium for 24 h. *P < 0.05, **P < 0.01 and ***P < 0.001 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three (A, B) or two (C, D) independent experiments (mean ± SD in A and D).

Article Snippet: USP18, USP18 (C64S), USP20 and USP20 (C560/563S) were obtained by a TNT in vitro transcription/translation kit (Promega).

Techniques: Quantitative RT-PCR, Transfection, Infection, Western Blot, Flow Cytometry, Cell Culture

Journal: Cell Research

Article Title: USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA

doi: 10.1038/cr.2016.125

Figure Lengend Snippet: USP20 deconjuagates K33/48-linked ubiquitination of and stabilizes STING. (A) Immunoblot of HEK293 cells that were transfected to express FLAG-STING, pCMV-USP20, HA-tagged Ubi (WT) and various mutants, lysed and immunoprecipitated with anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-USP20. (B) Immunoblot of HEK293 cells that were transfected to express FLAG-STING, pCMV-USP20, HA-Ubi (WT), K33/48R mutant of HA-Ubi or AKR, lysed and immunoprecipitated with anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG anti-USP20. (C-D) Immunoblot of Usp18+/+and Usp18−/− BMDCs (5 × 106) (C) or THP-1 cells (5 × 106) transfected with control siRNA (siCon) or siUSP20 (D) infected with HSV-1 for 0-8 h in the presence of MG132 (10 μM), lysed and immunoprecipitated with anti-STING in the presence of NEM (20 mM) followed by immunoblot analysis with antibodies against the indicated proteins. (E, F) Immunoblot analysis of STING, Tubulin, USP18 or USP20 in Usp18+/+ and Usp18−/− BMDCs (2 × 106) (E) or in THP-1 cells (1 × 106) that were transfected with control siRNA (−) or siUSP20 (+) (F) followed by HSV-1 infection in the presence or absence of MG132 or 3MA for 6 h. (G, H) Immunoblot analysis of STING, Tubulin, USP18 or USP20 in Usp18+/+ and Usp18−/− BMDCs (2 × 106) (G) or in THP-1 cells (1 × 106) that were transfected with control siRNA (−) or siUSP20 (+) (H) followed by transfection with ISD45 in the presence or absence of MG132 or 3MA for 3 h. Data are representative of three independent experiments.

Article Snippet: USP18, USP18 (C64S), USP20 and USP20 (C560/563S) were obtained by a TNT in vitro transcription/translation kit (Promega).

Techniques: Western Blot, Transfection, Immunoprecipitation, Mutagenesis, Infection