Journal:

Article Title: Protein Kinase C- α and ERK1/2 Mediate Mitochondrial Dysfunction, Decreases in Active Na + Transport, and Cisplatin-induced Apoptosis in Renal Cells

doi: 10.1074/jbc.M206373200

Figure Lengend Snippet: The monolayers were treated and RPTC functions analyzed as described under “Experimental Procedures.” White columns, controls; black columns, 50 μm cisplatin; light gray columns, 50 μm PD98059 + 50 μm cisplatin; dark gray columns, 10 nm Go6976 + 50 μm cisplatin; hatched columns, 3 μm UO126 + 50 μm cisplatin; striped columns, 50 μm zVAD-fmk + 50 μm cisplatin. Results are the average ± S.E. of three to five independent experiments (RPTC isolations).

Article Snippet: PKC- α inhibitor (Go6976) and MEK inhibitors (PD98059 and UO126) were supplied by Calbiochem (La Jolla, CA). zVAD-fmk was purchased from Biomol (Plymouth Meeting, PA).

Techniques:

Journal: Nature Communications

Article Title: Cancer cells dying from ferroptosis impede dendritic cell-mediated anti-tumor immunity

doi: 10.1038/s41467-022-31218-2

Figure Lengend Snippet: a Prophylactic vaccination model assessing the immunogenicity of ferroptosis. ML162-induced ferroptosis (5 μM, 14 h), in comparison with apoptosis (1000 IU/ml TNF + 10 μM TAK1i, 14 h) and necroptosis (1000 IU/ml TNF + 10 μM TAK1i + 10 μM zVAD.fmk, 24 h). OVA-expressing non-tumorigenic MEF cells (BM1-OVA) were used as a vaccine and live ovalbumin expressing melanoma cells (B16-OVA) were used as challenge. Kaplan–Meier curves represent the effectiveness of ferroptotic cells in preventing the tumor growth at the challenge site. Data were analyzed by Kaplan–Meier simple survival analysis. b Tumor size of B16-OVA derived melanoma after vaccination with BM1-OVA cells. Data presented as mean ± SEM. c Scheme of the therapeutic vaccination experiment. Conventional dendritic cells type 1 (cDC1) carrying ferroptotic or necroptotic cargo were intradermally injected in melanoma tumor-bearing mice. d B16-OVA tumor size progression of animals receiving either cDC1 with ferroptotic (5 μM, 14 h) or necroptotic (1000 IU/ml TNF + 10 μM TAK1i + 10 μM zVAD.fmk, 24 h) cargo. Data presented as mean ± SEM. Statistical significance was determined by two-sided t-test on each day of measurement. e Comparison of euthanasia time determined by the size of the tumor. Kaplan–Meier curves show the time of euthanasia, Data analyzed by Kaplan–Meier simple survival analysis. f Prophylactic vaccination model using either Mitoxantrone-treated MCA205 cells (1 μM, 24 h) or Mitoxantrone-treated MCA205 mixed with ML162-killed cells (0.5 µM, 14 h). Kaplan–Meier curves show the percentage of tumor-free mice after the challenge with live cancer cells. Data were analyzed by Kaplan–Meier simple survival analysis.

Article Snippet: Cell death inhibitors zVAD.fmk (#BACE4026865.0005, VWR International, Belgium) and Nec-1s (synthesized by the Laboratory of Medicinal Chemistry; University of Antwerp, Belgium) were used at 10 μM, Fer1 (#M60042-2s, Xcess Biosciences, USA) was used at 0,5 μM and DFO (#D-9533, Sigma-Aldrich, USA) at 50 μM.

Techniques: Expressing, Derivative Assay, Injection

Journal: Redox Biology

Article Title: Superoxide induced inhibition of death receptor signaling is mediated via induced expression of apoptosis inhibitory protein cFLIP

doi: 10.1016/j.redox.2019.101403

Figure Lengend Snippet: Death receptor sensitization upon GW is associated with decrease in intracellular O 2 •- . (A) CEM/Neo and CEM/Bcl-2 cells were pretreated for 1hr with 50 μM ZVAD-fmk before triggering apoptosis with anti-Fas (0.25 μg/ml for 18 hrs) in culture medium or in GW conditions and PI staining was used to assess DNA fragmentation (sub-G1 fraction) as described in Materials and Methods. (B) Cells were incubated for 4 hrs with 0.25 μg/ml of anti-Fas in normal medium or GW conditions and caspase 8 and (C) caspase 9 and 3 activities were determined in whole cell lysates using fluorometric assays that detect cleavage of specific substrates, as described in Materials and Methods. Enzyme activity are shown as fold increase over the untreated cells. (D) Cells were pretreated with PMA (62.5 ng/ml) for 1hr before treatment for 4 hrs with anti-Fas (0.25 μg/ml) in normal cell culture medium or GW medium and apoptosis was assessed by PI staining (sub-G1 fraction). (E) Caspase 8 activity was determined in the lysates from cells treated with anti-Fas in the presence and absence of PMA using a fluorometric assay and presented as fold increase over untreated cells. (F) Processing of capase-3 and 8 was determined by Western blot analysis using specific antibodies as described in Materials and Methods. Where applicable, data are Mean ± S.D. of three independent experiments and P values (* <0.05; ** <0.01; *** <0.005) were calculated by Ordinary one-way ANOVA using GraphPad Prism.

Article Snippet: Death receptor sensitization upon GW is associated with decrease in intracellular O 2 •- . (A) CEM/Neo and CEM/Bcl-2 cells were pretreated for 1hr with 50 μM ZVAD-fmk before triggering apoptosis with anti-Fas (0.25 μg/ml for 18 hrs) in culture medium or in GW conditions and PI staining was used to assess DNA fragmentation (sub-G1 fraction) as described in Materials and Methods. (B) Cells were incubated for 4 hrs with 0.25 μg/ml of anti-Fas in normal medium or GW conditions and caspase 8 and (C) caspase 9 and 3 activities were determined in whole cell lysates using fluorometric assays that detect cleavage of specific substrates, as described in Materials and Methods.

Techniques: Staining, Incubation, Activity Assay, Cell Culture, Western Blot