Journal: Science signaling

Article Title: The injury response to DNA damage in live tumor cells promotes anti-tumor immunity

doi: 10.1126/scisignal.abc4764

Figure Lengend Snippet: (A) Left, percentage of B16-Ova tumor cells displaying surface calreticulin 24 hours after the indicated treatment from a representative of two experiments. Results from an additional representative experiment using a second antibody is shown in fig. S2A . Middle and right, levels of HMGB1 and ATP in the culture media measured 24 to 48 hours after the indicated treatment. Results are from 4 independent experiments, with error bars indicating SEM. * P <0.05, ** P <0.01, *** P <0.005, and **** P <0.0001 compared to DMSO-treated control by ANOVA followed by Dunnett’s multiple comparisons test. “ns” indicates P >0.05 by one-way ANOVA. (B) Schematic of the siRNA experiment testing the role of B16-Ova cell calreticulin in BMDC-mediated IFN-γ induction in T cells. (C) Left, quantification of the fold change in %IFN-γ + CD8 + T-cells from the experiment in (B). Data for each calreticulin knockdown condition is normalized to the respective drug-treated control knockdown condition. Results represent 3 independent experiments with error bars indicating SEM. Data were analyzed by ANOVA followed by Sidak’s multiple comparisons test comparing drug-treated knockdown to drug-treated controls. * P <0.05 and **** P <0.0001. Right, quantification of calreticulin knockdown efficiency by Western blotting. First lane, “-”, defined as in Fig. 1C . (D to G) Quantification of the fold change in %IFN-γ + CD8 + T cells induced by BMDC after incubation with etoposide- or mitoxantrone-treated B16-Ova (D and E) or MC-38-Ova (F and G) cells that were co-treated with the indicated DNA damaging agent plus either Z-VAD, necrostatin-1 (Nec-1), Bay 11-7085 (inhibitor of NF-κB signaling) or SB202190 (p38 MAPK inhibitor). First lane (−) defined as in Fig. 1C . Data are normalized to the condition in which B16-Ova cells are treated with etoposide alone or mitoxantrone alone, respectively. Results from 3 independent experiments with error bars indicating SEM. * P <0.05 and **** P <0.0001 wherein each co-treatment condition (etoposide or mitoxantrone + either Z-VAD, Nec-1, NF-κBi or p38i) is compared with the etoposide or mitoxantrone alone condition using ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: Necrostatin-1 and Z-VAD were purchased from Invivogen.

Techniques: Control, Knockdown, Western Blot, Incubation

Journal: Cell Death Discovery

Article Title: Smac-mimetic enhances antitumor effect of standard chemotherapy in ovarian cancer models via Caspase 8-independent mechanism

doi: 10.1038/s41420-021-00511-2

Figure Lengend Snippet: A Overall survival in patients with high (above the median) or low (below the median) Caspase 8 in two datasets shows that low Caspase 8 consistently relates to shorter overall survival. B Ovcar3 cells transduced with either control shRNA (Caspase 8 WT) or Caspase 8 shRNA (Caspase 8 KD) were treated with increasing concentrations of combined cisplatin and paclitaxel in the presence or absence of birinapant (12.5 μM) for 72 h. Cell viability was measured by XTT assay. Representative results are expressed as average ± S.E.M., n = 4 and shown as percentage of change from vehicle control. C Ovcar3 cells expressing either control (Caspase 8 WT) or Caspase 8 shRNA (Caspase 8 KD) were exposed to combined cisplatin (2.5 µM) and paclitaxel (25 nM) and/or Caspase 8 inhibitor ZIETD (25 µM), birinapant (12.5 μM), and staurosporine (1 µM) for 18 h to induce caspase activity. Results of Caspase 8 cell-based assay are expressed as average luciferase units ± S.E.M., n = 4. Single asterisk (*) represents p < 0.05 based on t test. D Caspase 3 and 7 activity was measured in the described cell lines and exposed to similar conditions described in C . E Ovcar3 cells expressing either control (caspase 8 WT) or Caspase 8 shRNA (Caspase 8 KD) were exposed for 72 h to combined cisplatin (2.5 µM) and paclitaxel (25 nM), birinapant, 12.5 μM, and/or ZIETD (25 µM) or NEC1 (25 µM) as indicated. Viability was assessed by XTT assay. Results are expressed as average ± S.E.M., n = 3. Single asterisk (*) represents p < 0.01 based on t test comparing Caspase 8 WT versus KD; Double asterisks (**) represent p < 0.01 based on t test comparing ZIETD to Necrostatin.

Article Snippet: All viability studies were performed according to the above format, using 25 μM Caspase 8 inhibitor ZIETD (Z-IETD-FMK, FMK-007, R&D Systems), 20 μM NEC1 (Necrostatin-1, Tocris), or birinapant added to cells either alone or in combination, as described.

Techniques: Transduction, Control, shRNA, XTT Assay, Expressing, Activity Assay, Cell Based Assay, Luciferase

Journal: Oncotarget

Article Title: Sensitization of neuroblastoma for vincristine-induced apoptosis by Smac mimetic LCL161 is attended by G2 cell cycle arrest but is independent of NFκB, RIP1 and TNF-α

doi: 10.18632/oncotarget.21193

Figure Lengend Snippet: Neuroblastoma cell lines were treated with the indicated concentrations of vincristine, LCL161 and Necrostatin (Nec-1) and apoptosis was determined by flow cytometry (Annexin V and PI staining) after 48 h. Values represent the mean ± SD of three independent experiments. ‡p≤ 0.05 (VCR vs. VCR + LCL161).

Article Snippet: Necrostatin-1 (Nec-1) from StressMarq Biosciences Inc. was used to inhibit necroptosis and RIP1 kinase activity.

Techniques: Flow Cytometry, Staining