Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: Cardiac-specific Dsg2 knockout induced cardiac fibrosis. ( A ) Masson staining of heart sections in WT and CS-Dsg2 −/− (−/−) mice. Arrow shows cardiac fibrosis. Collagen volume fraction in the hearts of WT and CS-Dsg2 −/− mice was assessed. ( B ) Representative Western blots from mouse left ventricular (LV), interventricular septum (IVS), and right ventricle (RV). DSG2, PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. ( C ) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse LV and RV are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT.

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Knock-Out, Staining, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: Effects of Dsg2 siRNA and Stat3 siRNA on the expression levels of fibrotic markers in HL-1 cells. ( A , B ) HL-1 cells were transfected with control siRNA or Dsg2 siRNA. ( A ) Representative Western blots for DSG2, pSTAT3, α-SMA, and Collagen I were detected using specific antibodies. STAT3 and β-actin were used as loading controls. ( B ) Results of quantitative PCR analysis of Dsg2, α-SMA, and Collagen I mRNA levels in HL-1 cells treated with control or Dsg2 siRNA are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 3. * p < 0.05, ** p < 0.01 vs. control. ( C , D ) HL-1 cells were transfected with control siRNA or Stat3 siRNA. ( C ) Representative Western blots for STAT3 and α-SMA were detected using specific antibodies. β-actin were used as loading controls. ( D ) Results of quantitative PCR analysis of α-SMA and Collagen I mRNA levels in HL-1 cells treated with control or Stat3 siRNA are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 3. * p < 0.05, ** p < 0.01, vs. control.

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: Fenofibrate alleviated cardiac fibrosis in CS-Dsg2 −/− mice. ( A ) Masson staining of heart sections in WT, CS-Dsg2 −/− mice, and CS-Dsg2 −/− mice treated with fenofibrate (Dsg2 −/− F). Collagen volume fraction in the hearts of WT, CS-Dsg2 −/− , and Dsg2 −/− F mice were assessed. ( B ) Representative Western blots from ventricles of WT, CS-Dsg2 −/− , and Dsg2 −/− F mice. DSG2, PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. ( C ) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse ventricles are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Staining, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: AAV9-Pparα alleviated cardiac fibrosis in CS-Dsg2 −/− mice. ( A ) Masson staining of heart sections in CS-Dsg2 −/− mice and CS-Dsg2 −/− mice received AAV9-Pparα (Dsg2 −/− P). Collagen volume fraction in the hearts of CS-Dsg2 −/− and Dsg2 −/− P mice were assessed. ( B ) Representative Western blots from ventricles of CS-Dsg2 −/− and Dsg2 −/− P mice. PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. ( C ) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse ventricles are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CS-Dsg2 −/− .

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Staining, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: BMC Cancer

Article Title: Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B Leukemic Cells

doi: 10.1186/1471-2407-11-268

Figure Lengend Snippet: JSI-124-induced apoptosis was not mediated by c-Jun activation in B-cell malignancies . A . BJAB, I-83, and NALM-6 cells were pre-treated with 20 μM SP600125 followed by 1 μM JSI-124. Decreased levels of phospho-and total c-Jun protein levels were detected by western blotting in the cells treated with the combination of SP600125 and JSI-124 compared to SP600125 alone. Representative original data from three independent experiments are shown. The XIAP and STAT3proteins level, as determined by immunoblotting with antibodies against XIAP and serine phosphor-STAT3/STAT3, did not change with this treatment B . Apoptotic cell population was measured using flow cytometric analysis for AnnexinV/PI staining for apoptosis after 24 hour treatment with JSI-124, either alone or in combination with SP600125. DMSO-treated cells were taken as the control. Cells that were 7-AAD-negative and Annexin V-positive were undergoing apoptosis. The percentage of cells in each quadrant is indicated in the quadrant. Representative original data are shown. C . Cells were treated with same as above and were analyzed for cell cycle by FACS as described in Materials and Methods. Experiments were done at least three times and representative original data are shown (* represents significant difference of p value of < 0.05 between JSI-124 treated and untreated cells and ** represents a p value of < 0.01).

Article Snippet: Rabbit polyclonal antibodies against phosphorylated or total c-Jun, JNK, p38, phosphor and total Erk1/2, XIAP and STAT3 antibodies, as well as siRNA for c-Jun (#6203) and Stat3 siRNA I (#6580) and control siRNA (#6201 fluorescein conjugate) were purchased from Cell Signaling Technology, VEGF antibody was purchased from ThermoScientific and Santa Cruz.

Techniques: Activation Assay, Western Blot, Staining, Control

Journal: BMC Cancer

Article Title: Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B Leukemic Cells

doi: 10.1186/1471-2407-11-268

Figure Lengend Snippet: JSI-124 mediated c-Jun activation was not dependent on STAT3 expression in B-cell leukemia . A and B , Cells were transfected with siRNA-STAT or siRNA-c-Jun or siRNA non-target control. Then cells were treated with JSI-124 for additional 6 h. Cell extract was assayed for Western blotting for STAT3 and c-Jun. Knock-down of STAT3 or c-Jun was confirmed by western blotting with antibody to STAT3 or c-Jun. C , The same transfected cells were treated with JSI-124 for an additional 24 hours and then cells were analyzed for cell cycle analysis for G1 and G2 phase fractions in the cells transfected with siRNA-c-Jun or non-targeting control siRNA. Standard error was determined on the basis of three independent experiments and an asterisk represents significant difference (a p value of < 0.05) between JSI-124 treated and untreated controls. D , Cells were stimulated with JSI-124 and nuclear extracts incubated with the AP1 probe. Pulled down with streptavidin beads, the bound proteins were detected by SDS/polyacrylamide gel electrophoresis and western blotting. To control for specificity, a biotin-labeled scrambled probe was used along with no probe beads control. Blots were probed with antibody to c-Jun.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated or total c-Jun, JNK, p38, phosphor and total Erk1/2, XIAP and STAT3 antibodies, as well as siRNA for c-Jun (#6203) and Stat3 siRNA I (#6580) and control siRNA (#6201 fluorescein conjugate) were purchased from Cell Signaling Technology, VEGF antibody was purchased from ThermoScientific and Santa Cruz.

Techniques: Activation Assay, Expressing, Transfection, Control, Western Blot, Knockdown, Cell Cycle Assay, Incubation, Polyacrylamide Gel Electrophoresis, Labeling

Journal: International journal of oncology

Article Title: B-cell CLL/lymphoma 3 promotes glioma cell proliferation and inhibits apoptosis through the oncogenic STAT3 pathway.

doi: 10.3892/ijo.2016.3729

Figure Lengend Snippet: Figure 8. STAT3 knockdown reverses the oncogenic role of BCL3 in glioma cells. (A) Western blot analyses confirmed that STAT3 expression was knocked down by siRNA in BCL3 overexpressing U87 cells. n=3. (B) STAT3 knockdown evidently reduced proliferation in BCL3 overexpressing U87 cells. n=3, *P<0.05. (C) The proliferative ability of BCL3 overexpressing U87 cells were prominently reduced after STAT3 knockdown. n=3, *P<0.05. (D) STAT3 knockdown led to G1-phase arrest in BCL3 overexpressing U87 cells. n=3, *P<0.05. (E) STAT3 knockdown resulted in an obviously increased percentage of apoptotic cells in BCL3 overexpressing U87 cells. n=3, *P<0.05.

Article Snippet: A STAT3 specific siRNA and scrambled siRNA was obtained from OriGene Technologies, Inc. (Shanghai, China).

Techniques: Knockdown, Western Blot, Expressing

Journal: Frontiers in Oncology

Article Title: RelA Is an Essential Target for Enhancing Cellular Responses to the DNA Repair/Ref-1 Redox Signaling Protein and Restoring Perturbated Cellular Redox Homeostasis in Mouse PDAC Cells

doi: 10.3389/fonc.2022.826617

Figure Lengend Snippet: STAT3 is not a primary target altering cellular sensitivity to Ref-1 inhibitors in murine PDAC Kras G12D cells. (A) STAT3 was knocked down in KC3590 cells that are RelA deficient (Vector) and clone with functional RelA add back (C13) and knockdown efficiency was assessed by Western bot. Vinculin was used as loading control. The cells were challenged for 48 hours with (B) APX3330, (C) APX2009, (D) APX2014, and (E) RN7-58. Cytotoxicity was measured by alamarBlue. At least three independent experiments were performed (N = 3).

Article Snippet: Cells were transfected by lipofectamine RNAiMax (Invitrogen, CA, USA) with PRDX1 (SR405074, OriGene Technologies, MD, USA), RelA (SR417160, OriGene Technologies, MD, USA), STAT3 (SR427487, OriGene Technologies, MD, USA), and universal scrambled control (SCR) (SR30004, OriGene Technologies, MD, USA) siRNAs.

Techniques: Plasmid Preparation, Functional Assay, Knockdown, Western Blot, Control