Journal: American Journal of Cancer Research

Article Title: PDE3A inhibitor anagrelide activates death signaling pathway genes and synergizes with cell death-inducing cytokines to selectively inhibit cancer cell growth

doi:

Figure Lengend Snippet: Effects of ANA on the expressions of cell cycle and cell death regulating genes. (A) Immunoblotting analyses of effects of ANA on cyclinD1 and P21 protein expression in the three types of cells. The cells were treated with ANA for the indicated time periods and the cell lysates were processed for immunoblotting with indicated antibodies. α-tubulin antibody was used as a control for equal protein loading. (B) Effects of ANA on the expressions of Bcl-XL and Bcl-2 proteins (left) and the effects of SLFN12 siRNA on the ANA-induced protein level changes of Bcl-XL and Bcl-2 (right). H4 cells were treated with or without ANA (100 nM), or with or without SLFN12 siRNA as indicate for 12 or 24 hours, and the cell lysates were processed for immunoblotting analyses using antibodies as indicated. (C and D) Effects of ANA on the expressions of TNF-α, DR4, and DR5 mRNAs and the effects of SLFN12 siRNA on the ANA-induced mRNA level changes of TNF-α, DR4, and DR5 in H4 cells. The H4 cells were treated with or without ANA (100nM), or with or without the SLFN12 siRNA as indicated for 18 h, and the cell lysates were processed and the mRNA levels of TNF-α (C), DR4, DR5 (D) were measured by quantitative RT-PCR analyses. (E) Effects of ANA on DR4 and DR5 protein expressions in the three types of cells. The cells were treated with ANA (100 nM) for indicated time periods and the cell lysates were processed for immunoblotting analyses using the indicated antibodies. Anti-tubulin antibody was used as a control for equal protein loading. (F) Effects of ANA or ANA plus SLFN12 siRNA on the activation of caspase 8 and caspase 9, protein expressions of DR4 and DR5, and cleavage of PARP. The H4 cells were treated with ANA, a combination of ANA and SLFN12 siRNA, for the indicated time periods, and the cells were processed for immunoblotting analyses using antibodies as indicated. Anti-tubulin antibody was used as a control for equal protein loading.

Article Snippet: Anti-PDE3A (#99236), anti-DR5 (#ab199357), anti-GAPDH (#8245) antibodies were purchased from Abcam; anti-Bcl-2 (#2876), anti-Bcl-XL (#2764), anti-Caspase-8 (#4790), anti-Caspase-9 (#9508), anti-Cleaved-PARP (#9541), anti-Cyclin D1 (#2978) antibodies were purchased from CST; anti-DR4 (#24063-1-AP) antibody was purchased from Proteintech; anti-α-tubulin (#5286) antibody was purchased from Santa Cruz; anti-PDE3B (#138892) antibody was purchased from Absin; anti-PARP (#556494) antibody was purchased from BD.

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Impact of Extracellular pH on Apoptotic and Non-Apoptotic TRAIL-Induced Signaling in Pancreatic Ductal Adenocarcinoma Cells

doi: 10.3389/fcell.2022.768579

Figure Lengend Snippet: pH e influences the expression of TRAIL death receptors. (A,B) PDAC cells were grown for 24 h, then stained with APC-conjugated anti-TRAIL-Rs antibodies to measure their cell surface expression by flow cytometry. (C,D) PDAC cells were grown for 24 h, then either not treated (−) or treated (+) with 200 ng/ml TRAIL for 24 h, lysed, and subjected to western blot analyses for TRAIL-R1 and TRAIL-R2. Blots are representative and (E,F) show densitometric quantification normalized to loading control and the respective level of untreated cells cultured under pH e 7.4 conditions. Data are shown as mean with S.E.M error bars, of at least three independent experiments per cell line. **<0.01, ***<0.001, and ****<0.0001: significant difference between untreated and treated cells using two-way ANOVA with Sidak’s multiple-comparison test or between pH e conditions using two-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: The supernatant was discarded, and cells were incubated for 30 min with the following APC-conjugated antibodies with the concentration 1:3 in 0.6% BSA/PBS: TRAIL-R1 (FAB347A), TRAIL-R2 (FAB6311A), TRAIL-R3 (FAB6302A), and TRAIL-R4 (FAB633A) all purchased from R&D Systems GmbH, Wiesbaden, Germany.

Techniques: Expressing, Staining, Flow Cytometry, Western Blot, Control, Cell Culture, Comparison

Journal: Clinical Cancer Research

Article Title: Drozitumab, a Human Antibody to Death Receptor 5, Has Potent Antitumor Activity against Rhabdomyosarcoma with the Expression of Caspase-8 Predictive of Response

doi: 10.1158/1078-0432.ccr-10-2874

Figure Lengend Snippet: Figure 1. TRAIL reduces RMS cell viability mediated via the DR5 receptor. A, cell viability analysis of RMS cell lines treated with TRAIL for 72 hours, followed by ATPLite assay. A highly sensitive breast cancer cell line, MDA-231, was used as a reference. B, sensitivity of RMS cell lines to mouse monoclonal antibody against DR4 (top) or DR5 (bottom). The cells were treated with the indicated concentrations of these antibodies for 72 hours prior to the viability assay. C, the expression of DR4 and DR5 in the panel of RMS cell lines was determined by immunoblotting. D, the cell surface expression of DR4 and DR5 was analyzed by FACS in selected TRAIL and DR5 resistant (R) or sensitive (S) cells. The purple signal was from the control nonspecific antibody whereas the green signal was from either the DR4 or DR5 specific antibody. Increased intensity of the green signal over the purple reflects a specific expression of the analyte.

Article Snippet: Recombinant human TRAIL, mouse antibodies against human DR4 (MAB347), and DR5 (MAB631) for cell-based assays were purchased from R&D Systems.

Techniques: Viability Assay, Expressing, Western Blot, Control