Journal: OncoTargets and therapy

Article Title: Piwi-Interacting RNA1037 Enhances Chemoresistance and Motility in Human Oral Squamous Cell Carcinoma Cells

doi: 10.2147/OTT.S233322

Figure Lengend Snippet: Targeting the upregulated expression of piR-1037 induced by CDDP enhanced the sensitivity of OSCC cells to CDDP. ( A ) Confirmation of the suppressive effect of piRNA-1037 antisense oligonucleotides (piR-1037 complementary DNA oligonucleotides) (500 nM) on the expression of piRNA-1037 in SCC4 and SCC9 cells receiving the indicated treatments. ( B ) Western blot analysis and quantification of the effect of piR-1037 antisense oligonucleotides on the expression of the chemoresistance biomarkers MDR1 and α-enolase in SCC4 and SCC9 cells treated with CDDP (10 μM). ( C ) Decreased cell viability and increased apoptosis (TUNEL assay) ( D ) in OSCC cells transfected with piR-1037 antisense oligonucleotides and treated with CDDP compared with CDDP-treated cells transfected with a scrambled control. Cell apoptosis was detected using the TUNEL-based TiterTACS™ Colorimetric Apoptosis Detection Kit (Trevigen, USA). ( E ) Activity of caspase-3, caspase-8 and caspase-1 ( F ). The activity of caspases measured using Caspase-3 and Caspase-1 assay kits (Abcam, USA) and a Caspase-8 colorimetric kit (Sigma Aldrich, USA). The data represent the mean ±SD from at least three independent replicates. Statistical analysis was performed using one-way ANOVA analysis or unpaired student’s t -test: * p <0.05; ** p < 0.01.

Article Snippet: The membrane was then incubated with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 1 h. The membrane was washed once with TBST, followed by incubation with primary antibodies against MDR1 (Novus Biologicals, USA) (1:1000), α-enolase (Boster, China) (1:1000), XIAP (Abcam, USA) (1 μg/mL), cleaved caspase-3 (R&D systems, USA) (0,5 μg/mL), E-Cadherin (1:500), N-Cadherin (1:1000) (Abcam, USA), and β-actin (1:10,000) (Sigma Aldrich, USA).

Techniques: Expressing, Western Blot, TUNEL Assay, Transfection, Control, Activity Assay

Journal: Oncology Letters

Article Title: Phloretin induces apoptosis of human esophageal cancer via a mitochondria-dependent pathway

doi: 10.3892/ol.2017.7037

Figure Lengend Snippet: The relative expression level of cells treated with phloretin, as determined by western blot and relative quantification of (A) Smac/DIABLO, (B) APAF-1 and (C) XIAP. *P<0.05 between indicated groups, unpaired Student's t-test. Ct, control; DIABLO (Smac/DIABLO), direct binding protein with low PI; APAF-1, apoptotic protease activating factor 1; XIAP, X-linked inhibitor of apoptosis protein.

Article Snippet: Antibodies against BAX (#50599-2-Ig; dilution, 1:500), Bcl-2 (#12789-1-AP; dilution, 1:600), APAF-1 (#21710-1-AP; dilution, 1:200), DIABLO (#10434-1-AP; dilution, 1:1,000), XIAP (#10037-1-Ig; dilution, 1:400) and p53 (#10442-1-AP; dilution, 1:700) were purchased from ProteinTech Group, Inc. (Wuhan, China).

Techniques: Expressing, Western Blot, Quantitative Proteomics, Control, Binding Assay

Journal: Oncotarget

Article Title: Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

doi:

Figure Lengend Snippet: (A) In PC3 (1 × 10 5 ) cells, uPA activity is reduced following transfection with a PN1 expression vector (2 μg) ( N = 4, t -test, * P < 0.01). (B) Knock-down of PN1 via (10nM) siRNA increases uPA activity ( N = 4, one-way ANOVA, * P < 0.01). Mock treatment and scrambled siRNA (NEG) used as controls. (C) 1U or 5U of recombinant uPA proteins were added to the medium of PC3 cells (1 × 10 5 ) for 24 h and xiap mRNA transcripts were measured ( N = 4, one-way ANOVA, * P < 0.05). (D) Immunoblotting of proteins from PC3 conditioned media treated with 10nM control siRNA (NEG) or siRNA uPA (siuPA) and with or without transfection of PN1 (i) and quantitation of xiap transcripts (ii). (Two-way ANOVA; N = 3, * P < 0.01). (E) PC3 cells transfected with control or expression vectors (2 μg) for WT-PN1 or PN1-LRP binding mutant (mLRP) or RCL binding mutant (mRCL), and measurement of xiap expression by ELISA ( N = 4, one-way ANOVA, * P < 0.01). (F) PC3 cells transfected with control or expression vectors (2 μg) for WT-PN1 or treated with anti-LRP (50 μg/ml), anti-uPAR (50 μg/ml) blocking antibody for 24 h, and measurement of xiap expression by ELISA ( N = 4, one-way ANOVA with Tukey Test, * P < 0.01; ** refer to similarly significant comparisons between specific groups as denoted by the horizontal lines over the bar graph). (G) Immunoblotting of PC3 lysates transfected with PN1 vector or treated with anti-LRP or anti-uPAR blocking antibody for 24 h.

Article Snippet: All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC (R&D Systems, DYC822) according to factory instructions.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Knockdown, Recombinant, Western Blot, Control, Quantitation Assay, Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal:

Article Title: Chemically synthesized human survivin does not inhibit caspase-3

doi: 10.1110/ps.036145.108

Figure Lengend Snippet: (A) Ligation of (1–45)αCOSR and (46–142) at 1.5 h. The reaction was monitored by analytical HPLC on a Waters XBridge C18 column (4.6 × 150 mm, 3.5 μM) running a 30-min gradient of 25%–45% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. (Insets) Mass spectra of (1–45)αCOSR and (46–142) determined by ESI-MS. (B) Ligated full-length survivin characterized by C18 RP-HPLC and ESI-MS. HPLC conditions: Waters symmetry 300 C18 column (4.6 × 150 mm, 5 μM) running a 30-min gradient of 5%–65% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. (C) CD spectra of synthetic survivin at 5 μM in 5 mM phosphate buffer containing 0.1 mM TCEP, pH 7.5 (thin line), and at 25 μM in 5 mM phosphate buffer containing 0.1 mM TCEP and 50 μM Zn2+, pH 7.5 (thick line). (D) Representative size-exclusion chromatograms of synthetic survivin (3) and molecular mass standards (1, 2, 4, 5). Linear regression analysis of the correlation between logarithmic M r and retention time is illustrated in the inset. (E) Representative raw data from the hydrolysis of Ac-DEVD-AMC by caspase-3 in the absence and presence of different concentrations of XIAP and synthetic survivin. (F) Dose-dependent percent inhibition of caspase-3 by XIAP (filled circles), synthetic survivin without Zn2+ (empty squares), and synthetic survivin in the presence of Zn2+ (filled squares). Each curve is the mean of three independent experiments.

Article Snippet: EnzChek Caspase-3 assay kit #1 was purchased from Invitrogen; recombinant caspase-3 was obtained from Calbiochem and recombinant human XIAP, from R&D Systems.

Techniques: Ligation, Inhibition

Journal: The FEBS journal

Article Title: Cell-penetrating peptide-conjugated XIAP-inhibitory cyclic hexapeptides enter into Jurkat cells and inhibit cell proliferation.

doi: 10.1111/j.1742-4658.2008.06730.x

Figure Lengend Snippet: Fig. 8. Binding and competition assay during the interaction of CHCPP 1 with the BIR2 domain. (A) The diffusion time in the BIR2 binding and competition assay was measured by the FCS system. The BIR2 domain of recombinant human XIAP (1.29 lg) was incu- bated with 0.2 lM of unlabelled CHCPP 1 or Npys-free CH 1 for 30 min prior to the addition of FITC-labelled CHCPP 1 (0.2 lM). The grey column indicates the absence of BIR2 and a competitor. (B) The percentage of complex formation was determined by the fluo- rescence fluctuation analysis, as described in Experimental proce- dures. Each value represents the mean ± SE of at least three experiments. *P < 0.05 versus the absence of a competitor.

Article Snippet: For binding assays, FITC-labelled CHCPP 1 (0.2 lm) was mixed with recombinant human XIAP (BIR2 domain) (R & D Systems, Minneapolis, MN, USA), and the mixture was incubated for 30 min at room temperature.

Techniques: Binding Assay, Competitive Binding Assay, Diffusion-based Assay, Recombinant