Journal: Molecular and Cellular Biology

Article Title: Differences and Similarities in TRAIL- and Tumor Necrosis Factor-Mediated Necroptotic Signaling in Cancer Cells

doi: 10.1128/MCB.00941-15

Figure Lengend Snippet: TRAIL- and TNF-induced necroptosis are mediated by ceramide that is generated by A-SMase and N-SMase. (A to D) Cells were pretreated for 2 h with the indicated concentrations of inhibitors of A-SMase (ARC39, zoledronic acid, TP064/14e, D609, desipramine, and imipramine), N-SMase (3-OMS, spiroepoxide, and GW4869), and ceramide synthase (fumonisin B1), with the subsequent addition of 30 ng/ml killerTRAIL and 20 μM zVAD for 14 h (L929Ts) (A), 100 ng/ml hrTNF in combination with 20 μM zVAD for 5 h (L929Ts) (B), 100 ng/ml killerTRAIL and 20 μM zVAD for 16 h (NIH 3T3) (C), and 100 ng/ml hrTNF and 20 μM zVAD for 16 h (NIH 3T3) (D). (E) Inhibitors of A-SMase and N-SMase protect human Jurkat I.42 cells (FADD deficient and TNF-R2 positive) from TNF-mediated necroptosis. Cells were treated with the indicated concentrations of inhibitors and stimulated afterwards with 100 ng/ml hrTNF in combination with 50 μM zVAD for 6 h. (F) Inhibitors of A-SMase, HtrA2/Omi, UCH-L1, and vacuolar H+-ATPase protect the human pancreas adenocarcinoma cell line A818-6 from TRAIL-mediated necroptosis. Cells were pretreated for 2 h (or 3 h for LDN57444) with the indicated concentrations of inhibitors with the subsequent addition of 100 ng/ml killerTRAIL in combination with 50 μM zVAD for 24 h. Morphological changes of human A818-6 pancreas adenocarcinoma cells after induction of TRAIL-mediated necroptosis in combination with inhibitors of A-SMase, HtrA2/Omi, and vacuolar H+-ATPase were observed. Cells were pretreated for 2 h with 10 μM ARC39, 25 μM Ucf-101, or 10 μM BafA1 with the subsequent addition of 100 ng/ml killerTRAIL in combination with 50 μM zVAD for 24 h. Arrowheads in micrographs show typical necroptotic morphologies. Bar, 100 μm. Shown are means ± standard deviations (n = 3 [A to D] and n = 9 [E and F]), with differences being considered significant at P values of <0.05 (*), <0.01 (**), and <0.001 (***) (as determined by a t test).

Article Snippet: Highly purified human recombinant TNF (hrTNF) was provided by BASF Bioresearch (Ludwigshafen, Germany). zVAD, N -benzyloxycarbonyl-Asp-Glu-Val-Asp- O -methyl-fluoromethylketone (zDEVD), and N -benzyloxycarbonyl-Ile-Glu-Thr-Asp- O -methyl-fluoromethylketone (zIETD) were purchased from Bachem (Bubendorf, Switzerland).

Techniques: Generated

Journal: Molecular and Cellular Biology

Article Title: Differences and Similarities in TRAIL- and Tumor Necrosis Factor-Mediated Necroptotic Signaling in Cancer Cells

doi: 10.1128/MCB.00941-15

Figure Lengend Snippet: (A and B) ROS are not uniformly the executioners of TRAIL-mediated necroptosis. (A) All tested cell lines were pretreated or not with 150 μM BHA or BHT for 1 h, with the subsequent addition of 30 ng/ml (14 h for L929Ts cells), 100 ng/ml (16 h for NIH 3T3 cells), or 50 ng/ml (20 h for Jurkat cells) killerTRAIL; 20 μM zVAD (L929Ts and NIH 3T3); or 50 μM zVAD and 2 μg/ml CHX (Jurkat). Each cell line was treated with 1 mM BuOOH for 24 h as a positive control for ROS production. (B) Cells were treated as described above for panel A, and loss of membrane integrity was measured. (C) Lack of p38α increases execution of TRAIL- and TNF-mediated necroptosis. p38α-deficient immortalized MEFs and their wild-type counterparts were prestimulated for 30 min with or without 20 μM zVAD and with or without 1 μg/ml CHX with the subsequent addition of 100 ng/ml killerTRAIL or 100 ng/ml hrTNF for 24 h. (D and E) Deficiency in p38α increases phosphorylation of IκBα and phosphorylation of p65 during TRAIL- and TNF-induced cell death. Cells were stimulated as described above for panel C for 1 h, followed by total lysis and Western blot analyses. Shown are Western blots for total IκBα, phosphorylated (Ser32) IκBα (p-IκBα), total p65 (*, unspecific band), and phosphorylated (Ser536) p65 (p-p65); p38α served as a control for deficiency, and β-actin served as a loading control. (F and G) Overexpression of Bcl-XL and Bcl-2 protects cells from TRAIL-mediated necroptosis. (F) Wild-type and Bcl-2-overexpressing Jurkat cells were stimulated with 50 ng/ml killerTRAIL or 100 ng/ml hrTNF, 50 μM zVAD, and 5 μg/ml CHX for 20 h. (G) Colo357 cells stably overexpressing Bcl-XL or an empty vector were stimulated with 100 ng/ml killerTRAIL or 100 ng/ml hrTNF alone or in combination with 5 μg/ml CHX, 50 μM zVAD, and 1 μM the Smac mimetic birinapant (B) for 24 h. Shown are means ± standard deviations (panels A and B show data from one representative experiment for one repetition out of four) (n = 9 [C, F, and G]), with differences being considered significant at a P value of <0.001 (***) (as determined by a t test). n.s., nonsignificant. Insets show control Western blots for endogenous p38α, Bcl-2, Bcl-XL, and β-actin, which served as a loading control.

Article Snippet: Highly purified human recombinant TNF (hrTNF) was provided by BASF Bioresearch (Ludwigshafen, Germany). zVAD, N -benzyloxycarbonyl-Asp-Glu-Val-Asp- O -methyl-fluoromethylketone (zDEVD), and N -benzyloxycarbonyl-Ile-Glu-Thr-Asp- O -methyl-fluoromethylketone (zIETD) were purchased from Bachem (Bubendorf, Switzerland).

Techniques: Positive Control, Lysis, Western Blot, Over Expression, Stable Transfection, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Differences and Similarities in TRAIL- and Tumor Necrosis Factor-Mediated Necroptotic Signaling in Cancer Cells

doi: 10.1128/MCB.00941-15

Figure Lengend Snippet: (A and B) Autophagy does not play a crucial role in TNF- or TRAIL-mediated necroptosis in L929Ts or Jurkat I.42 cells. (A) L929ATCC cells were prestimulated for 2 h with the indicated concentrations of inhibitors of autophagy (3-MA), lysosome formation (CQ), or vesicle acidification (BafA1) and stimulated afterwards for 24 h with 20 μM zVAD. L929Ts cells were pretreated for 2 h with 3-MA, CQ, or BafA1 and stimulated with 20 μM zVAD, 30 ng/ml killerTRAIL for 14 h, or 100 ng/ml hrTNF for 5 h. Jurkat I.42 (FADD-deficient and TNF-R2-positive) cells were pretreated for 2 h with the indicated concentrations of 3-MA, CQ, or BafA1 before the addition of 50 μM zVAD in combination with 100 ng/ml hrTNF for 6 h. (B) Morphological analyses of the influence of 3-MA, CQ, or BafA1 on zVAD-induced autophagy in L929ATCC cells (top) and TRAIL-mediated (middle) and TNF-mediated (bottom) necroptosis in L929Ts cells. Cells were stimulated as described above for panel A. Bar, 100 μm. (C) Atg5 is required for both TRAIL- and TNF-mediated RIPK1-dependent necroptosis. Atg5-deficient immortalized MEFs, their wild-type counterparts, and Atg5-deficient cells retransfected with wild-type Atg5 (Atg5RE) or mutated Atg5 (Atg5T75A) were prestimulated or not with 50 μM Nec-1s for 2 h, followed by the addition of 30 ng/ml killerTRAIL or 100 ng/ml hrTNF, 20 μM zVAD, and/or 1 μg/ml CHX to induce necroptosis or 100 ng/ml hrTNF or 30 ng/ml killerTRAIL together with 1 μg/ml CHX to induce apoptosis for 18 h. In order to verify their authenticity, all cell lines were left untreated in full medium or treated for 2 h with Earle's balanced salt solution (EB SS) to induce autophagy by starvation accompanied by the appearance of LC3-II as an indicator of autophagy. Insets show Western blots for LC3 under normal and starvation conditions (Earle's balanced salt solution), with β-actin serving as a loading control. (D) Morphological analyses of necroptosis in Atg5-deficient cells retransfected with wild-type Atg5 (Atg5RE), mutated Atg5 (Atg5T75A), and their wild-type counterparts. Cells were stimulated as described above for panel C. The bar in microphotographs is 100 μm. (E) Lack of Atg16L1 protects cells from TNF-mediated but not TRAIL-mediated necroptosis. Atg16L1-deficient immortalized MEFs, their wild-type counterparts, and Atg16L1-deficient cells transiently retransfected for 48 h with wild-type Atg16L1 (Atg16L1RE) were prestimulated or not with 50 μM Nec-1s for 2 h, followed by stimulation with 30 ng/ml killerTRAIL or 100 ng/ml hrTNF together with 20 μM zVAD and/or 1 μg/ml CHX to induce necroptosis or with 30 ng/ml killerTRAIL or 100 ng/ml hrTNF together with 1 μg/ml CHX to induce apoptosis for 24 h. The authenticity of all cell lines and efficiency of transfection with Atg16L1 were verified by Western blotting, with β-actin serving as a loading control. (F) Morphological analyses of necroptosis in Atg16L1-deficient and wild-type MEFs and MEFs retransfected with wild-type Atg16L1 (Atg16L1RE). Cells were stimulated as described above for panel E. Arrowheads indicate typical necroptotic morphology. Bar, 100 μm. (G) Receptor internalization is necessary for TNF-mediated but not for TRAIL-mediated necroptosis. L929Ts cells were prestimulated for 1 h with the indicated concentrations of PitStop2 or dynasore and stimulated with 30 ng/ml killerTRAIL and 20 μM zVAD or with 100 ng/ml hrTNF and 20 μM zVAD. Shown are means ± standard deviations (n = 6 for L929Ts and n = 4 for Jurkat I.42 cells [A], n = 3 [C], and n = 6 [A]), and data were considered significant at a P value of <0.001 (***) (as determined by a t test). (H) TNF but not TRAIL receptor is internalized during necroptosis in L929Ts cells. Cell surface-bound receptors were coupled to Fc-TNF or Fc-TRAIL ligands labeled with protein G-Alexa Fluor 488 alone or in combination with 20 μM zVAD at 4°C, followed by a temperature shift to 37°C for 30 min. A minimum of 10,000 cells were analyzed for each sample. Internalization Wizard was used to measure the ratio of the intensity inside the cell to the intensity of the entire cell (percent). (I) Representative images of cells (bright field) with a stained plasma membrane and TNF or TRAIL ligand-receptor complexes. Colocalization of ligand-receptor complexes with the plasma membrane (yellow) and internalization of TNF ligand-receptor complexes but not TRAIL ligand-receptor complexes were observed after 30 min of stimulation.

Article Snippet: Highly purified human recombinant TNF (hrTNF) was provided by BASF Bioresearch (Ludwigshafen, Germany). zVAD, N -benzyloxycarbonyl-Asp-Glu-Val-Asp- O -methyl-fluoromethylketone (zDEVD), and N -benzyloxycarbonyl-Ile-Glu-Thr-Asp- O -methyl-fluoromethylketone (zIETD) were purchased from Bachem (Bubendorf, Switzerland).

Techniques: Western Blot, Transfection, Labeling, Staining

Journal: Gels

Article Title: Polysaccharide-Based Drug Carriers—A Patent Analysis

doi: 10.3390/gels10120801

Figure Lengend Snippet: Selected relevant patents of fucoidan-based drug carriers.

Article Snippet: Highly purified and/or modified fucan compositions for the treatment of fibrous adhesions , ZA202100275B [ ] , ARC Medical Devices Inc. , 2 November 2018.

Techniques: Algae, Shear, Purification, Modification

Journal: Nucleic Acids Research

Article Title: A novel λ integrase-mediated seamless vector transgenesis platform for therapeutic protein expression

doi: 10.1093/nar/gky500

Figure Lengend Snippet: Anti-CD19 CAR expression from seamless vectors in hESCs. ( A ) A schematic drawing showing in vitro generation and ex vivo targeting of a two-reporter seamless vector ( attL4X-PGKssCAR-EF-Puro ) at genomic att H4X. Positions of relevant screening primers (PGK Rev 187, attP Rev and cs_attH4X_F1/F2), restriction sites and the probes used for Southern blotting are indicated. The purified 5.5 kb seamless vector resulting from in vitro recombination was analyzed by agarose gel electrophoresis shown at the left; supercoiled seamless vector: SC-SV; open circular seamless vector: OC-SV. ( B ) Sequence analysis of the targeted hESC clone #19. A chromatogram representation of the DNA sequence of the PCR product obtained with the cs_attH4X_F2 and attP Rev primer pair using genomic DNA from targeted hESC clone#19. The recombinant att L4X site (HOP’), resulting from λ-Int-mediated recombination between genomic att H4X (chr2:159357384-159357546) and att L4X (present on an attL4X-PGKssCAR-EF-Puro seamless vector), is boxed. ( C ) Flow cytometric analysis of anti-CD19 CAR expression in targeted hESC clone#19. A single parameter histogram overlay plots representation of stained non-transfected control hESCs onto the anti-CD19 CAR-stained population. An overlay obtained with cells from co-transfection of anti-CD19 CAR seamless expression vector with inactive Int expression plasmid and non-transfected control hESCs is shown in the left panel, indicating negligible expression. An overlay obtained with cells from co-transfection of anti-CD19 CAR seamless expression vector with Int-C3 expression plasmid and control hESCs clearly differentiate the anti-CD19 CAR-expressing population; clone#19 (after 8 weeks of culturing) is shown in middle panel. Long term and stable anti-CD19 CAR expression of clone#19 over a period of 6 months is shown in the right panel. ( D ) Western blot showing anti-CD19 CAR expression. Western analysis using anti-human CD3ζ primary antibody and a goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody shows anti-CD19 CAR expression in clone #19. (Top Panel). ß-actin was used as loading control (bottom panel). Lanes: ES, lysate from non-transfected hESCs; 19, lysate from clone #19 (a puromycin resistant clone obtained from targeting of seamless vector attL4X-PGKssCAR-EF-Puro and plasmid expressing Int C3); IN, lysate from an inactive clone (a puromycin resistant clone obtained from targeting of seamless vector attL4X-PGKssCAR-EF-Puro and plasmid expressing inactive integrase); JUR, a bulk culture of Jurkat T cells obtained from targeting attL4X-PGKss-CAR-EF-GFP seamless vector (expressing anti-CD19 CAR) along with plasmid expressing Int C3 was used as a positive control. Note: hESC clone #19 and Jurkat T cells show two bands at ∼55 kDa suggesting post translational modifications of anti-CD19 CAR whereas a ∼15kDa band in Jurkat T cells indicates the expression of native monomer of endogenous CD3ζ. ( E ) Southern blot analysis of the targeted hESC clone#19. A southern blot analysis showing the bands indicated by arrows obtained using PCR-derived digoxigenin-labeled probe complementary to CAR (left panel) or Puro (right Panel) with genomic DNA purified from targeted hESC clone#19 digested with BsrGI and/or NcoI . Lanes: pPCEP (10 8 , 10 7 ), copies of linearized vector pattP4X-PGKssCAR-EF-Puro-attH4X loaded as positive control; ES, genomic DNA from parental DNA; IN, genomic DNA obtained from inactive clone (a puromycin resistant clone obtained from co-transfection of attL4X-PGKssCAR-EF-Puro and plasmid expressing inactive integrase); 19, genomic DNA from targeted hESC clone. hESC clone#19 (carrying a single-copy transgene) is a puromycin resistant clone obtained through co-transfection of attL4X-PGKssCAR-EF-Puro and plasmid expressing active Int C3.

Article Snippet: Blots were washed with 1× PBST and incubated for 2 h at room temperature with goat anti-mouse IgG HRP conjugate secondary antibody (1:3000, R&D systems) in 4% skim milk.

Techniques: Expressing, In Vitro, Ex Vivo, Plasmid Preparation, Southern Blot, Purification, Agarose Gel Electrophoresis, Sequencing, Recombinant, Staining, Transfection, Control, Cotransfection, Western Blot, Positive Control, Derivative Assay, Labeling