Journal: The FASEB Journal

Article Title: Cystathionine β-synthase regulates mitochondrial morphogenesis in ovarian cancer

doi: 10.1096/fj.201701095R

Figure Lengend Snippet: CBS regulates MFN2 expression. A) Expression of mitochondrial fusion and fission markers MFN2, MFN1, Opa1, Drp1, and Fis1 were determined by immunoblotting in CP20 and OV90 cells, either transfected with scrambled siRNA (siCTL) or siRNA against the CBS gene for 72 h. Efficient gene silencing was determined by immunoblotting with the CBS antibody, and GAPDH served as a loading control. B) Immunoblotting of CBS, MFN2, and MFN1 from tumor tissue of orthotopically implanted control (shCTL) and CBS knockdown (shCBS) A2780 cells. GAPDH is used as a loading control. T1–T3, tumors 1-3. C) Mitochondrial morphology was depicted by immunofluorescence of siCTL and siCBS OV90 cells, stained with the Mitotracker Red CMXRos. Original scale bars, 10 µm. D) Representative images from each group stained with the mitochondrial marker COXIV were selected to illustrate the analysis process. Color channels were separated from each image so that only mitochondria (green) were present and then skeletonized for network analysis. Mitochondrial networks are defined as any segment with at least 1 additional branch (blue arrows), whereas individual mitochondria are defined as unbranched (yellow arrows). E) Images were analyzed using a publicly available ImageJ macro designed by Valente et al. (32). A Student’s t test (α = 0.05) was used to compare CBS knockdown siCBS (n = 16) with control siCTL (n = 20) CP20 cells. All experiments were performed 3 times in triplicates, and values are represented as mean fold change ± sd. P ≤ 0.05 is considered statistically significant.

Article Snippet: Reagents, cell lines, and culture The following antibodies were used for immunoblotting: rabbit polyclonal CBS antibody (H-300, sc-67154; Santa Cruz Biotechnology, Dallas, TX, USA), mouse β-actin mAb (A-2228; Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal to MFN2 (D2D10 rabbit mAb; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal MFN1 antibody (ab126575; Abcam, Cambridge, United Kingdom), rabbit polyclonal OPA1 antibody (ab42364; Abcam), rabbit polyclonal to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; G9545; Sigma-Aldrich), and anti-DRP1 (ab56788; Abcam).

Techniques: Expressing, Western Blot, Transfection, Control, Knockdown, Immunofluorescence, Staining, Marker

Journal: The FASEB Journal

Article Title: Cystathionine β-synthase regulates mitochondrial morphogenesis in ovarian cancer

doi: 10.1096/fj.201701095R

Figure Lengend Snippet: Clinicopathological significance of CBS and MFN2 in cancer. A) Expression of CBS, MFN1/2, HUWE1, and Parkin in normal (OSE, HOSE, FTE) vs. OvCa cells, as determined by immunoblotting. B, C) Kaplan-Meier overall survival curves in CBS (B) and MFN2 (C) low- and high-expression (mRNA data) OvCa cases from The Cancer Genome Atlas database. The percent probability of survival is plotted vs. time since diagnosis in months. OS, overall survival. D) Panel represents scatterplot of CBS vs. MFN2 with overlaid linear regression line among tumor tissue samples. Spearman correlation coefficient is 0.155 (P = 0.003).

Article Snippet: Reagents, cell lines, and culture The following antibodies were used for immunoblotting: rabbit polyclonal CBS antibody (H-300, sc-67154; Santa Cruz Biotechnology, Dallas, TX, USA), mouse β-actin mAb (A-2228; Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal to MFN2 (D2D10 rabbit mAb; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal MFN1 antibody (ab126575; Abcam, Cambridge, United Kingdom), rabbit polyclonal OPA1 antibody (ab42364; Abcam), rabbit polyclonal to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; G9545; Sigma-Aldrich), and anti-DRP1 (ab56788; Abcam).

Techniques: Expressing, Western Blot, Biomarker Discovery

Journal: The FASEB Journal

Article Title: Cystathionine β-synthase regulates mitochondrial morphogenesis in ovarian cancer

doi: 10.1096/fj.201701095R

Figure Lengend Snippet: CBS silencing increases oxidative stress and leads to MFN2 degradation by the ubiquitin-proteasome system. A) qRT-PCR was performed to determine relative mRNA expression of CBS, MFN1, and MFN2 in CP20 and OV90 cells transfected for 48 h with scrambled siRNA (siCTL) or siRNA against the CBS gene (siCBS). 36β4 was used as the internal control. B) CP20 cells were transfected as in A, and an equal amount of extracted protein was subjected to immunoprecipitation (IP) assay with MFN2 antibodies crosslinked with the agarose resin and further immunoprobed (IB) with ubiquitin and MFN2 antibodies. C) CP20 and OV90 cells were transfected with siCTL or siCBS and then treated with MG132 (5 µM/12 h) or left untreated. Expression of MFN2 and CBS was determined by immunoblot, and GAPDH was used to indicate equal protein loading. D) Total cellular ROS was determined by 2′,7′-dichlorofluorescein assay and quantified by fluorescence measurement in CP20 or OV90 cells transfected with scrambled siRNA (siCTL) or CBS siRNA (siCBS) and represented as relative fold change over control. E) CP20 and OV90 cells were transfected with siCTL or siCBS for 48 h and then treated with the antioxidant (Antx.; mitoTEMPO; 10 µM/12 h), and the expression of MFN2 and CBS was determined by immunoblotting. F) Expression of JNK, phosphorylated (p)p38, pERK1/2, and CBS was determined by immunoblotting of OV90 and CP20 cells, transfected for 72 h with siCTL or siCBS. G) CP20 and OV90 cells were transfected with siCTL or siCBS and then treated with SP600125 (SP; 20 µM/12 h) or left untreated. Expression of MFN2 and CBS was determined by immunoblot, and GAPDH was used to indicate equal protein loading. H) Equal amount of extracted protein from siCTL or siCBS CP20 cells were subjected to immunoprecipitation assay with MFN2 antibodies crosslinked with the agarose resin and further immunoprobed with HUWE1 (HUWE), CBS, p-Ser, and MFN2 antibodies. I) Equal amount of extracted protein from siCBS CP20 cells treated with vehicle or SP600125 (20 µM/12 h) were subjected to immunoprecipitation assay with MFN2 antibodies crosslinked with the agarose resin and further immunoprobed with p-Ser antibody. All experiments were performed 3 times in triplicates, and values are represented as mean fold change ± sd. *P ≤ 0.05 is considered statistically significant. J) Illustration depicts proposed mechanism of the CBS modulation of MFN2 stability. CBS regulates redox homeostasis, which if disrupted, activates the oxidative stress-sensing MAPK, JNK. Activated JNK phosphorylates MFN2, which recruits the E3 ligase, HUWE1, which in turn, ubiquitinates MFN2, thereby leading to its proteasomal degradation and mitochondrial fragmentation.

Article Snippet: Reagents, cell lines, and culture The following antibodies were used for immunoblotting: rabbit polyclonal CBS antibody (H-300, sc-67154; Santa Cruz Biotechnology, Dallas, TX, USA), mouse β-actin mAb (A-2228; Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal to MFN2 (D2D10 rabbit mAb; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal MFN1 antibody (ab126575; Abcam, Cambridge, United Kingdom), rabbit polyclonal OPA1 antibody (ab42364; Abcam), rabbit polyclonal to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; G9545; Sigma-Aldrich), and anti-DRP1 (ab56788; Abcam).

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Expressing, Transfection, Control, Immunoprecipitation, Western Blot, Dichlorofluorescein Assay, Fluorescence

Journal: Mediators of Inflammation

Article Title: Markers of Inflammation and Fibrosis in the Orbital Fat/Connective Tissue of Patients with Graves' Orbitopathy: Clinical Implications

doi: 10.1155/2014/412158

Figure Lengend Snippet: Antibodies used in the study for immunohistochemical staining.

Article Snippet: FGF β , Rabbit polyclonal FGF-1 antibody (H-125): sc-7910 Santa Cruz Biotechnology , 1 : 100.

Techniques: Immunohistochemical staining, Staining