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: Experimental and Therapeutic Medicine

Article Title: Preserving hepatic artery flow during portal triad blood occlusion improves regeneration of the remnant liver in rats with obstructive jaundice following partial hepatectomy

doi: 10.3892/etm.2018.6402

Figure Lengend Snippet: Protein expression in the remnant liver of rats with obstructive jaundice that underwent different intraoperative hepatic blood inflow occlusions and hepatectomy. Western blot analysis of PCNA, caspase-3 and 9 expressions at (A) 6 h, (C) 24 h and (E) 72 h following hepatectomy. The quantified results of (B) PCNA, (D) caspase-3 and (F) caspase-9 are presented. # P<0.05 vs. the C group; *P<0.05 vs. the OPT group. n=3 rats per group per time point. C, control group without portal blood occlusion; OPT, occlusion of the portal triad; OPV, occlusion of the portal vein and preservation of the hepatic artery; PCNA, proliferating cell nuclear antigen.

Article Snippet: Membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies: A mouse monoclonal antibody of anti-proliferating cell nuclear antigen (PCNA; 1:200; cat. no. sc-56; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit anti-caspase-3 (Asp175) (5A1E) monoclonal antibody (cat. no. 9664) and rabbit anti-caspase-9 (Asp353) polyclonal antibody (cat. no. 9507) (both 1:1,000; Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot, Preserving

Journal: FEBS letters

Article Title: Uncoupling phototoxicity-elicited neural dysmorphology and death by insidious function and selective impairment of Ran-binding protein 2 (Ranbp2)

doi: 10.1016/j.febslet.2015.11.037

Figure Lengend Snippet: Apoptotic cell bodies of photoreceptors of 24-week-old Ranbp2−/−::Tg-Ranbp2CLDm-HA and wild type mice lack differences in caspases’ activations (cleaved caspases) induced by light-stress. There are no changes in TUNEL+Casp3+, TUNEL+Casp7+, TUNEL+Casp8+, TUNEL+Casp9+ in cell bodies of photoreceptors between age-matched genotypes by light stress (A, B). Arrows show TUNEL+ cell bodies without caspases’ activations, whereas arrowheads show TUNEL+Casp+ cell bodies. (B) are quantitative analyses of representative images in (A). Retinal sections were counterstained with DAPI. Data shown represent the mean ± S.D., n=3. Legend: +/+, wild type; −/−::Tg-Ranbp2CLDm-HA, Ranbp2−/−::Tg-Ranbp2CLDm-HA; n.s., non-significant; Scale bar: 20 μm.

Article Snippet: The following primary antibodies were used for immunohistochemistry: rabbit monoclonal anti-cleaved caspase-3 Asp175 (Cell Signaling), rabbit polyclonal anti-cleaved-caspase-7 Asp353 (Cell Signaling), rabbit anti-cleaved caspase-8 Asp391 (Cell Signaling), rabbit anti-cleaved caspase-9 Asp 330 (Cell Signaling), mouse anti-ubiquitin (Santa Cruz Biotechnology), rabbit anti-hsc70 (ENZO Life Science), rabbit anti-S1 subunit of the 19 S proteasome (Thermo Scientific); rabbit anti-HDAC4 (Santa Cruz Biotechnology); mouse anti-Ubc9 (BD Biosciences), Alexa-conjugated secondary antibodies (408, 488, 568 and Cy5) were from Invitrogen (Carlsbad, CA).

Techniques: TUNEL Assay

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan Synthase 2 Protects Skin Fibroblasts against Apoptosis Induced by Environmental Stress

doi: 10.1074/jbc.M114.578377

Figure Lengend Snippet: Attenuated apoptotic responses in Has1/3 null fibroblasts involve reduced caspase activation. A, representative Western blots of cleaved caspase-9, cleaved caspase-3, and full-length and cleaved PARP in skin fibroblasts at different times after UVB exposure (32 mJ/cm2). GAPDH was used as a loading control. B, Western blots as in A, from fibroblasts harvested at different times after serum starvation. Data in the table below these blots are the mean from densitometric scans in the corresponding lane, averaged from two independent experiments; the variance was less than 20% of the mean in all cases.

Article Snippet: Western Blot Analysis The primary antibodies for Western analyses were polyclonal rabbit anti-cleaved caspase-9 antibody (Cell Signaling Technology, catalogue no. 9509), polyclonal rabbit anti-active caspase-3 antibody (BioVision, catalog no. 3015-100), polyclonal rabbit anti-PARP antibody (Cell Signaling Technology, catalog no. 9542), and polyclonal rabbit anti-GAPDH antibody (Santa Cruz Biotechnology, Inc., catalog no. sc-25778).

Techniques: Activation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan Synthase 2 Protects Skin Fibroblasts against Apoptosis Induced by Environmental Stress

doi: 10.1074/jbc.M114.578377

Figure Lengend Snippet: Apoptosis resistance of skin fibroblasts is not mediated by HA in the extracellular matrix. A, extracellular HA in skin fibroblast cultures treated with S. dysgalactiae hyaluronidase (0.3 unit/ml, 10 min at 37 °C) (+Hyal) or in untreated control cultures (−Hyal) and then immunostained with bHABP/streptavidin-Alexa Fluor 488. Scale bar, 100 μm. B, representative Western blots of cleaved caspase-9 and -3 and cleaved PARP in skin fibroblasts treated with either the hyaluronidase (Hyal) or UVB irradiation (UVB) or both (UVB + Hyal), versus untreated normal controls (NC). C, Western blots of cleaved caspase-9 and -3 and cleaved PARP in skin fibroblasts treated with either Hyal or serum starvation (SS) or both (Hyal + SS), versus untreated normal controls (NC). GAPDH was examined as a loading control.

Article Snippet: Western Blot Analysis The primary antibodies for Western analyses were polyclonal rabbit anti-cleaved caspase-9 antibody (Cell Signaling Technology, catalogue no. 9509), polyclonal rabbit anti-active caspase-3 antibody (BioVision, catalog no. 3015-100), polyclonal rabbit anti-PARP antibody (Cell Signaling Technology, catalog no. 9542), and polyclonal rabbit anti-GAPDH antibody (Santa Cruz Biotechnology, Inc., catalog no. sc-25778).

Techniques: Western Blot, Irradiation

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan Synthase 2 Protects Skin Fibroblasts against Apoptosis Induced by Environmental Stress

doi: 10.1074/jbc.M114.578377

Figure Lengend Snippet: 4-MU treatment sensitizes skin fibroblasts to apoptosis. A, HA levels in the cell layer (A) or culture media (B) of fibroblasts treated with 4-MU (1 mm) for either 6 or 12 h were quantified by FACE analysis. C, quantitation of fluorescence intensity of extracellular HA (left) and intracellular HA (right) staining with bHABP, in skin fibroblasts treated with 4-MU for 6 h and analyzed by image processing. Gray bars, no 4-MU; black bars, +4-MU. *, p < 0.05 by Student's t test (significant difference from vehicle-treated cells). Changes in Has2 mRNA levels in both types of fibroblasts (D) or in Has1 and Has3 mRNA levels in WT fibroblasts (E), treated with 4-MU (1 mm) or vehicle alone for 6 h, were analyzed by qPCR. Each data point is the mean ± S.D. of two independent experiments. F, representative Western blots of cleaved caspase-9 and -3 and cleaved PARP in skin fibroblasts treated with 4-MU or vehicle control for 6 h, followed by 32 mJ/cm2 UVB (4-MU + UVB and Veh + UVB). G, Western blots of cleaved caspase-9 and -3 and cleaved PARP in skin fibroblasts treated with either 4-MU or vehicle control for 6 h followed by 4 h of serum starvation (4-MU + SS and Veh + SS). GAPDH, loading control.

Article Snippet: Western Blot Analysis The primary antibodies for Western analyses were polyclonal rabbit anti-cleaved caspase-9 antibody (Cell Signaling Technology, catalogue no. 9509), polyclonal rabbit anti-active caspase-3 antibody (BioVision, catalog no. 3015-100), polyclonal rabbit anti-PARP antibody (Cell Signaling Technology, catalog no. 9542), and polyclonal rabbit anti-GAPDH antibody (Santa Cruz Biotechnology, Inc., catalog no. sc-25778).

Techniques: Quantitation Assay, Fluorescence, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan Synthase 2 Protects Skin Fibroblasts against Apoptosis Induced by Environmental Stress

doi: 10.1074/jbc.M114.578377

Figure Lengend Snippet: Knockdown of Has2 gene expression by Has2 siRNA transfection sensitizes skin fibroblasts to apoptosis. mRNA expression levels of Has2 (A), Has1 (B), and Has3 (C) in either WT or Has1/3 null fibroblasts, analyzed by qPCR. No Rx control, untreated normal control; Scrambled siRNA, transfected with non-targeted scrambled siRNA; Has2 siRNA, transfected with Has2 siRNA. Each bar represents the mean ± S.D. (error bars) of two independent experiments. D, HA levels in the cell layer (left) or media (right) from fibroblast cultures, treated as described for A–C and analyzed by FACE. Each bar is the mean ± S.D. of two independent experiments. E and F, Western blots of skin fibroblasts after UVB (32 mJ/cm2) or serum starvation (4 h), respectively, showing effects upon the expression of cleaved caspase-9 and -3, relative to GAPDH loading controls, in cells transfected with siRNA against Has2 (Has2 siRNA), non-targeted scrambled siRNA (Scrambled), or untreated normal control (NC).

Article Snippet: Western Blot Analysis The primary antibodies for Western analyses were polyclonal rabbit anti-cleaved caspase-9 antibody (Cell Signaling Technology, catalogue no. 9509), polyclonal rabbit anti-active caspase-3 antibody (BioVision, catalog no. 3015-100), polyclonal rabbit anti-PARP antibody (Cell Signaling Technology, catalog no. 9542), and polyclonal rabbit anti-GAPDH antibody (Santa Cruz Biotechnology, Inc., catalog no. sc-25778).

Techniques: Expressing, Transfection, Western Blot