Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hyperglycemia Induces Endoplasmic Reticulum Stress in Atrial Cardiomyocytes, and Mitofusin-2 Downregulation Prevents Mitochondrial Dysfunction and Subsequent Cell Death

doi: 10.1155/2020/6569728

Figure Lengend Snippet: Mfn2 siRNA inhibited endoplasmic reticulum- (ER-) mitochondria-dependent apoptosis. (a) Cell apoptosis was evaluated by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. (b) HL-1 cells under TM stimulated for 24 hours. Mfn siRNA inhibited the Bax/Bcl-2 ratio and inhibited caspase3 and caspase9 activation (c). Data are mean ± SEM, n = 4 independent experiments, ∗ p < 0.05, # p < 0.01, ANOVA with Bonferroni posttest.

Article Snippet: Proteins (60 μ g) were separated by SDS-PAGE and transferred to PVDF membranes, blocked with TBST containing 5% nonfat milk and incubated overnight at 4°C with the following antibodies: anti-CHOP (#2895), anti-caspase9 (#9504S), anti-caspase3 (#9662S), anti-cleaved-caspase9 (#9509S), anti-cleaved-caspase3 (Asp175) (#9661S), and anti-MCU (D2Z3B) (#14997S) from Cell Signaling Technology; anti-GRP78 (ab21685), anti-Mfn2 (ab56889), anti-VDAC1 (ab15895), anti-Bax (ab32503), anti-Bcl-2 (ab59348), and anti-Mn-SOD (ab13533) from Abcam; and anti- β -actin antibody as a loading control from Proteintech.

Techniques: Flow Cytometry, Staining, Activation Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Maresin-1 Inhibits Oxidative Stress and Inflammation and Promotes Apoptosis in a Mouse Model of Caerulein-Induced Acute Pancreatitis

doi: 10.12659/MSM.917380

Figure Lengend Snippet: Mice with caerulein-induced acute pancreatitis (AP) treated with maresin-1 (MaR1) showed increased expression of apoptosis-associated proteins. Western blot shows the expression levels of Bcl-2, Bax, cleaved caspase-3 and cleaved caspase-9. *** P<0.001 vs. control; ## P<0.01, ### P<0.001 vs. AP. MaR1 – maresin-1; AP – acute pancreatitis

Article Snippet: The primary antibodies were obtained from Cell Signaling Technology (Boston, MA, USA) and included anti-Bcl-2 (3498S), anti-Bax (14796S), anti-cleaved caspase-3 (9664S), anti-cleaved caspase-9 (9509T), anti-TLR4 (14358S), anti-MyD88 (4283S), anti-phospho-NF-κB p65 (3033S), anti-NF-κB p65 (8242S), and anti-GAPDH (5174S).

Techniques: Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Specific Inhibition of SOD1 Selectively Promotes Apoptosis of Cancer Cells via Regulation of the ROS Signaling Network

doi: 10.1155/2019/9706792

Figure Lengend Snippet: The specific SOD1 inhibition activates mitochondria-dependent apoptosis of cancer cells. (a, b) RT-qPCR (a) and western blotting (b) examination of p53 mRNA levels and protein content in 50 (RT-qPCR) or 0-100 μ M (western blotting) LD100-treated HeLa, DU145, and RWPE-1 cells. (c) The protein levels of p21 and p19 in HeLa and DU145 cells are evaluated by western blotting analysis, after the cells are incubated with varied concentrations of LD100 for 24 h. β -Actin is used as an internal control (b, c). (d) The protein levels of proto- and cleaved caspase-9 and -3 in HeLa cells are evaluated by western blotting analysis, after HeLa cells are incubated with varied concentrations of LD100 for 24 h. (e) The time dependence of the protein levels of proto- and cleaved caspase-9 is evaluated by western blotting analysis in 50 μ M LD100-treated HeLa cells. (f, h) HeLa cells are incubated with varied concentrations of LD100 for 24 h prior to western blotting evaluation of Bcl-2 (f) and cleaved PARP1 (h) protein levels. (g) Rh123 fluorescence that is positively correlated with mitochondrial membrane potential is measured by flow cytometry in HeLa, DU145, and RWPE-1 cells after incubated with varied concentrations of LD100 for 24 h. (i) DNA fragmentation in HeLa cells is assayed using agarose gel electrophoresis after incubated with varied concentrations of LD100 for 24 h. (j) The peroxynitrite content in HeLa cells is presented by DHR123 fluorescence measured with flow cytometry after incubation for 24 h, respectively, with LD100 (0, 50, and 100 μ M) and L-NMMA (0, 1 mM), a cell membrane-permeable competitive NOS inhibitor. (k) After incubation for 24 h, respectively, with LD100 (0, 100 μ M) and L-NMMA (1 mM L-NMMA), flow cytometric analysis of HeLa cell apoptosis is carried out with Annexin V and propidium iodide staining. To obtain more significant results, 100 μ M LD100 was used here. The relative intensity of protein bands (means ± SD) in the western blotting is quantified by using the ImageJ software and normalized through the negative control, respectively (b–g). Representative results from three independent experiments are shown (b–g and k). Data are mean of triplicate samples ± SD (ns: not statistically significant; ∗ P < 0.05; unpaired Student's t -test), and all error bars are SD.

Article Snippet: Rabbit polyclonal anti-caspase-9 (Cat# 9502; RRID:AB_2068621) and rabbit polyclonal anti-cleaved caspase-9 (Asp353) (Cat# 9509; RRID:AB_2073476) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Quantitative RT-PCR, Western Blot, Incubation, Fluorescence, Flow Cytometry, Agarose Gel Electrophoresis, Staining, Software, Negative Control

Journal: PLoS ONE

Article Title: Differential Regulation of Cutaneous Oncoprotein HPVE6 by wtp53, Mutant p53R248W and ΔNp63α is HPV Type Dependent

doi: 10.1371/journal.pone.0035540

Figure Lengend Snippet: Western blot analysis of cell lysates from pLXSN-flagHPV20E6- and pLXSN-NIKS cells after transfection with siRNAp53 (10 nM) or siRNA-NK (negative control siRNA, 10 nM) and incubation for 16 h with or without MG132 (10 µm). MG132 treatment induced activation of apaf-1, caspase-8, caspase-9 and caspase-3, as well as the autophagy marker LC3BII. Histograms represent HPV20E6 and wtp53 protein levels normalized against β-actin.

Article Snippet: Primary antibodies against Apaf-1 (cat#4452), caspase-3 (cat#9662), caspase-9 (cat#9509), caspase-8 (cat#9746), LC3B (cat#4108), Bak (cat#3792), Bax (cat#2774), phospho-c-Jun (ser63) (cat#9261) and c-Jun (cat#9165) were purchased from Cell Signaling (Frankfurt am Main, Germany).

Techniques: Western Blot, Transfection, Negative Control, Incubation, Activation Assay, Marker

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Short Isoform of DNAJB6 Protects against 1-Methyl-4-phenylpridinium Ion-Induced Apoptosis in LN18 Cells via Inhibiting Both ROS Formation and Mitochondrial Membrane Potential Loss

doi: 10.1155/2017/7982389

Figure Lengend Snippet: DNAJB6(S) inhibits p18 Bax cleavage and caspase-9 activation in MPP + -treated cells. LN18 cells stably transfected by pMOCK or pDNAJB6(S) were treated with or without 500 µ M MPP + for 48 h. The cells were harvested, and mitochondrial and cytosolic fractions (F) were prepared. The expression levels of p18 Bax and caspase-9 cleavage were analyzed by western blotting. An anti-Bax antibody detected both full-length Bax (20 kDa) and p18 Bax. β -Actin was used as a loading control and COX-II was used for mitochondrial marker.

Article Snippet: FBS and Mitochondrial Isolation kit are from Thermo Scientific (Waltham, MA), glutamine and penicillin/streptomycin are from Welgene (Kyungsan, Korea), MPP + and β -actin antibody are bought from Sigma-Aldrich (St. Louis, MO, USA), restriction enzymes Bam HI and Hin dIII (NEB) and RNeasy Mini Spin Columns are from Qiagen (Hiden, Germany), Transcriptor First-Strand cDNA Synthesis kit is from Roche Diagnostics (Basel, Switzerland), anti-DNAJB6 antibody (1 : 1000) is from Abnova (Taipei, Taiwan, H00010049-M01), anti-c-myc antibody (1 : 3000) is from Invitrogen (R950-25), anti-Bax antibody (1 : 1,000) is from Cell Signaling (2772), anti-COX-2 antibody (1 : 2000) is from Cell Signaling (4842), and anticleaved caspase-9 antibody (1 : 1,000) is from Cell Signaling (9509).

Techniques: Activation Assay, Stable Transfection, Transfection, Expressing, Western Blot, Marker