Journal: Pharmaceuticals

Article Title: Antitumor Mechanism of the Essential Oils from Two Succulent Plants in Multidrug Resistance Leukemia Cell

doi: 10.3390/ph12030124

Figure Lengend Snippet: Western blotting analysis of the levels of Bcl-2, XIAP and Survivin, in HL-60 R cells treated for 24 h with C. juttae or K. beharensis essential oils (40 μg/mL). On the left the results of a representative experiment; on the right, the results expressed as mean ± standard error of two different experiments. Differences when treatments are compared to the control: * p < 0.05; ** p < 0.01.

Article Snippet: The specific primers used were: Survivin Hs00153353, XIAP Hs00236913, Bcl-2 Hs00236329, ABCB1 Hs00184005 (Applied Biosystems Life Technologies Inc., Foster City, CA, USA).

Techniques: Western Blot, Control

Journal: Oncology reports

Article Title: Tumor necrosis factor‑related apoptosis‑inducing ligand as a therapeutic option in urothelial cancer cells with acquired resistance against first‑line chemotherapy.

doi: 10.3892/or.2020.7487

Figure Lengend Snippet: Figure 4. Effects of selective depletion of IAPs and Mcl‑1 on cancer cell viability. Effects of siRNA‑mediated depletion of cIAP1, cIAP2, XIAP and Mcl‑1 on TRAIL sensitivity in (A) RT112, (B) RT112rGEMCI20 and (C) RT112rCDDP1000 cells following incubation for 48 h. Western blots (left) showing depletion of the knockdown protein. Cell viability was measured via the MTT assay (right), demonstrating the effect of siRNA depletion of the proteins cIAP1, cIAP2, XIAP and Mcl‑1 on sensitivity to TRAIL treatment. The results shown are representative of three independent experiments. *P<0.05 relative to control. TRAIL, tumor necrosis factor‑related apoptosis‑inducing ligand; IAPs, inhibitors of apoptosis; cIAP, cellular inhibitor of apoptosis protein; XIAP, x‑linked inhibitor of apoptosis protein; Mcl‑1, myeloid leukemia cell differentiation protein 1; RT112rGEMCI20, gemcitabine‑resistant RT112 subline; RT112rCDDP1000, cisplatin‑resistant RT112 subline.

Article Snippet: Proteins were detected using specific antibodies against β-actin (Sigma-Aldrich, Merck KGaA, cat. no. A2228, 1:5,000), TRAILR1 (DR4, EnzoLifeSciences, cat. no. ALX‐804‐912‐ 0100, 1:5,000), TRAILR2 (DR5, EnzoLifeSciences, cat. no. ALX-210-743-C200, 1:1, 0 0 0), T R A I LR3 ( DCR1, EnzoLi feSciences, cat. no. ALX-804-667-C100, 1:500), TRAILR4 (DCR2, EnzoLifeSciences, cat. no. ADI-AAP-371-E, 1:1,000), XIAP (Cell Signaling via New England Biolabs, cat. no. 14334, 1:1,000), cIAP1 (Cell Signaling via New England Biolabs, cat. no. 7065, 1:1,000), cIAP2 (Cell Signaling via New England Biolabs, cat. no. 3130, 1:1,000), and Mcl-1 (Cell Signaling via New England Biolabs, cat. no. 94296, 1:1,000) and were visualized by enhanced chemiluminescence using a commercially available kit (GE Healthcare).

Techniques: Incubation, Western Blot, Knockdown, MTT Assay, Control, Cell Differentiation

Journal: IUBMB life

Article Title: Activating transcription factor 3 regulates survivability and migration of vascular smooth muscle cells.

doi: 10.1002/iub.416

Figure Lengend Snippet: Figure 2. Knockdown of ATF3 induces apoptosis of VSMCs. A: VSMCs were transfected with ATF3 plasmid or pcDNA3.1 vector as a negative control. Proliferation of the cells was determined by counting cells. The error bars indicated standard deviations from three independent experiments. Whole cell extracts were prepared and used for Western blot with anti-ATF3 antibody. b-actin was used as loading control. B: VSMCs were transducted with shATF3 adenovirus (shATF3-1 and 2) or shlacZ adenovirus (Con) as described under Materials and Methods Section. The cells were harvested at indicated time (48, 72, and 96 h), and total cell number was determined by counting cells. *, P \ 0.05 versus control. RT-PCR was performed to indicate the efficacy of ATF3 knockdown. Glyceraldehyde-3-phosphate dehydrogenase was used as a control. C: VSMCs were transducted with shATF3-1 adenovirus or shlacZ adenovirus (Con). At 72 h, the cells were harvested for apoptosis assay. Data are mean 6 SD from three independent experiments. *, P \ 0.05 versus control. D: VSMCs were transducted with shATF3-1 adenovirus or shlacZ adenovirus (Con). The transducted cells were harvested at 72 h for immunoblotting with anti-ATF3, anti-caspase-3, and anti-XIAP antibodies. b-actin was used as loading control. The figure is a representative of three independent experiments. E: VSMCs were transducted with shATF3-1 virus and the cells were incubated with or without Z-VAD-FMK (20 lM). At 72 h, the cells were harvested and subjected to apoptosis assay. F: VSMCs were transducted with shATF3-1 adenovirus or shlacZ adenovirus (Con) as described above. At 72 h, the mitochondrial and cytosolic proteins were isolated for immunoblotting with anti-cytochrome c antibody. b-actin was used as loading control. The figure is a representative of three independent experiments.

Article Snippet: Antibodies against X-linked inhibitor of apoptosis protein (XIAP) and Caspase-3 were obtained from Cell Signaling (Beverly, MA).

Techniques: Knockdown, Transfection, Plasmid Preparation, Negative Control, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Apoptosis Assay, Virus, Incubation, Isolation

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: Chronic ethanol intake leads to structural and molecular alterations in the rat endometrium.

doi: 10.1016/j.alcohol.2016.02.002

Figure Lengend Snippet: Fig. 2. Immunohistochemical images of the uterine horn of Control (A, D, G, J, M), UChA (B, E, H, K, N), and UChB (C, F, I, L, O) groups. Caspase-3 (A, B, C); Bcl2 (D, E, F); Xiap (G, H, I); Ki-67 (J, K, L); and IGFR-1 (M, N, O). UChA rats showed an intense positive reaction to caspase-3 and a moderate reaction to Bcl2 and Xiap. A strong reaction to Ki-67 and IGFR-1 was observed in the UCh rats. Bar ¼ 20 mm.

Article Snippet: Rabbit primary antibodies (1:100 Santa Cruz Biotechnology, CA, USA) were used for IGFR-1, Ki-67, Bcl2, and Xiap analysis, and rabbit primary antibodies (1:100 Biocare, USA) were used for caspace-3 analysis, according to the manufacturers’ procedures (n 1⁄4 5/ group).

Techniques: Immunohistochemical staining, Control

Journal: British Journal of Cancer

Article Title: X-chromosome-linked inhibitor of apoptosis as a key factor for chemoresistance in clear cell carcinoma of the ovary

doi: 10.1038/bjc.2014.255

Figure Lengend Snippet: Downregulation of XIAP by transfection with XIAP siRNA and sensitivity to cisplatin in vitro . ( A ) Clear cell carcinoma cell line, KK, was transfected with nonspecific siRNA (KK-C), XIAP-specific siRNA I (KK-I) and XIAP-specific siRNA II (KK-II). Expression ratio of XIAP protein compared with KK cells without transfection was as follows: 73.14±12.7% in KK-C, 20.6±3.94% in KK-I and 19.54±6.67% in KK-II, respectively. Phospho-Akt expression was decreased in KK-I and KK-II cells compared with KK-C: 62.9±11.0% in KK-I, 64.5±4.6% in KK-II. ( B ) After transfection, these cells were treated with for 24 h at a dose of 10 μ M . Expression levels of cleaved caspase-3 and cleaved PARP increased in both KK-I and KK-II cells compared with KK or KK-C. ( C ) Apoptosis fractions induced by 24-h treatment of cisplatin in KK-C, KK-I and KK-II were shown. Ratio of apoptotic fraction was higher in KK-I and KK-II compared with that of KK-C at each concentration. The data represented the mean ± s.d. of at least four times. * P <0.01.

Article Snippet: Nonspecific control siRNA, XIAP-specific siRNA (Signal Science XIAP siRNA, #6446) and XIAP-specific siRNA II (Signal Science XIAP siRNA, #6550) were purchased from Cell Signaling Technology.

Techniques: Transfection, In Vitro, Expressing, Concentration Assay

Journal: Journal of Clinical Investigation

Article Title: Bacterial CagA protein compromises tumor suppressor mechanisms in gastric epithelial cells

doi: 10.1172/jci130015

Figure Lengend Snippet: Figure 6. Activation of the PI3K/Akt pathway by H. pylori augments phosphorylation of XIAP leading to degradation of Siva1. (A) Western blot analysis of Siva1 protein in AGS cells treated with chemical inhibitors (final concentration 10 μM) for the indicated enzymes and cocultured with H. pylori strain 7.13. The graph panel shows quantification of Siva1 protein by densitometry, normalized to actin (n = 3). Expression of Siva1 protein in control cells treated with vehicle (DMSO) was arbitrarily set at 1. (B) Western blot analysis of Siva1 and XIAP proteins in AGS cells transfected with PI3K siRNA or scrambled siRNA and then either left uninfected or cocultured with H. pylori strain 7.13 for 4 hours (n = 3). (C) The same as B, but siRNA against Akt protein was used (n = 3). Statistical significance was calculated using unpaired 2-tailed t tests and P value corrected by Bonferroni’s multiple comparison adjustment. Data are displayed as mean ± SE and are representative of 3 independent experiments. **P < 0.01; ***P < 0.001.

Article Snippet: PI3K (catalog sc-37269), XIAP (catalog sc-37508), Cbl-b (catalog sc-29950), and Akt (catalog sc-43609) siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Concentration Assay, Expressing, Control, Transfection, Comparison