Journal: The American Journal of Pathology

Article Title: Regulation of Apo2L/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Thyroid Carcinoma Cells

doi: 10.1016/s0002-9440(10)64220-4

Figure Lengend Snippet: Figure 7. A: Flow cytometric analysis for TRAIL receptors (DR4, DR5, DcR1, and DcR2, shaded peaks) in SW579 cells treated with or without IGF-1 (100 ng/ml) for 8 hours revealed no changes. Control antibody staining appears as unshaded peaks. B: Treatment of SW579 cells with IGF-1 (100 or 300 ng/ml) for 8 hours up-regulated the apoptosis inhibitors FLIP, cIAP2, XIAP, and survivin, but not cIAP1 or the anti-apoptotic members of the Bcl-2 family Bcl-2, Bcl-xL, A1, and Mcl-1. IGF-1 also down-regulated the proapoptotic Bax.

Article Snippet: Recombinant human TRAIL was obtained from Immunex Corporation (Seattle, WA), in a leucine zipper (LZ) form that promotes and stabilizes the formation of trimers, as previously reported;13 for comparison, several experiments were repeated using the recombinant Apo2L form from Genentech Inc. (South San Francisco, CA).20 The goat polyclonal antibodies for DR4, DR5, DcR1, and mouse monoclonal antibody for tubulin were from Santa Cruz Biotechnologies (Santa Cruz, CA); the anti-DcR2 rabbit polyclonal antibody was from Imgenex (San Diego, CA); cycloheximide, geldanamycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma Chemical Co. (St Louis, MO); IGF-1, bFGF, EGF, IFN- , and TNF- were from R&D Systems (Minneapolis, MN; bisindolylmaleimide (BIM) III and wortmannin were from Calbiochem (La Jolla, CA); rabbit polyclonal antibody for caspase-10 was from Research Diagnostics Inc. (Flanders, NJ); the IGF-1 receptor neutralizing antibody aIR3 was from Oncogene Research (Cambridge, MA); and the enhanced chemiluminescence (ECL) kit, which includes the peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies, was from Amersham (Arlington Heights, IL).

Techniques: Control, Staining

Journal: The American Journal of Pathology

Article Title: Regulation of Apo2L/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Thyroid Carcinoma Cells

doi: 10.1016/s0002-9440(10)64220-4

Figure Lengend Snippet: Figure 11. A and B: Flow cytometric analysis of TRAIL receptors DR4 (A) and DR5 (B) after a 48-hour treatment with or without IFN- (500 IU/ml) or TNF- (50 ng/ml) in SW579 cells. Control antibody staining is also shown. C: Evaluation of the protein levels of caspase-8, caspase-10, caspase-3, FLIP, and TRAIL in SW579 cells after a 48-hour treatment with or without IFN- (500 IU/ml) or TNF- (50 ng/ml). IFN- (500 IU/ml) up-regulated caspase-8 and TNF- (50 ng/ml) and up-regulated caspases-10 and -3. Additionally, TNF- induced the expression of TRAIL itself.

Article Snippet: Recombinant human TRAIL was obtained from Immunex Corporation (Seattle, WA), in a leucine zipper (LZ) form that promotes and stabilizes the formation of trimers, as previously reported;13 for comparison, several experiments were repeated using the recombinant Apo2L form from Genentech Inc. (South San Francisco, CA).20 The goat polyclonal antibodies for DR4, DR5, DcR1, and mouse monoclonal antibody for tubulin were from Santa Cruz Biotechnologies (Santa Cruz, CA); the anti-DcR2 rabbit polyclonal antibody was from Imgenex (San Diego, CA); cycloheximide, geldanamycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma Chemical Co. (St Louis, MO); IGF-1, bFGF, EGF, IFN- , and TNF- were from R&D Systems (Minneapolis, MN; bisindolylmaleimide (BIM) III and wortmannin were from Calbiochem (La Jolla, CA); rabbit polyclonal antibody for caspase-10 was from Research Diagnostics Inc. (Flanders, NJ); the IGF-1 receptor neutralizing antibody aIR3 was from Oncogene Research (Cambridge, MA); and the enhanced chemiluminescence (ECL) kit, which includes the peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies, was from Amersham (Arlington Heights, IL).

Techniques: Control, Staining, Expressing

Journal: The American Journal of Pathology

Article Title: Regulation of Apo2L/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Thyroid Carcinoma Cells

doi: 10.1016/s0002-9440(10)64220-4

Figure Lengend Snippet: Figure 2. Protective role of FLIP against TRAIL-induced apoptosis in thyroid carcinoma cells. A: TRAIL-resistant SW579-TR cells express higher levels of FLIP, but not Bcl-2, than the parental, TRAIL-sensitive cells. B: Pretreatment of SW579-TR cells with cycloheximide (CHX, 10 g/ml) or bisindolylmale- imide (BIM III, 20 mol/L) restored sensitivity to LZ-TRAIL (300 ng/ml) (no TRAIL, black bars; TRAIL treatment, white bars). Cell survival was quan- tified with the MTT assay. C: CHX and BIM III specifically down-regulated FLIP protein levels in SW579-TR cells after 8 hours of treatment, but not those of cIAP-1, cIAP-2, Bcl-2, or Bcl-xL. Tubulin levels are shown for comparison. D: Indirect confirmation of the anti-apoptotic role of FLIP, SW579-TR cells were sensitized to LZ-TRAIL (300 ng/ml) by FLIP anti-sense oligonucleotides (AS), but not by control oligonucleotides (CO). Cell survival was quantified with the MTT assay.

Article Snippet: Recombinant human TRAIL was obtained from Immunex Corporation (Seattle, WA), in a leucine zipper (LZ) form that promotes and stabilizes the formation of trimers, as previously reported;13 for comparison, several experiments were repeated using the recombinant Apo2L form from Genentech Inc. (South San Francisco, CA).20 The goat polyclonal antibodies for DR4, DR5, DcR1, and mouse monoclonal antibody for tubulin were from Santa Cruz Biotechnologies (Santa Cruz, CA); the anti-DcR2 rabbit polyclonal antibody was from Imgenex (San Diego, CA); cycloheximide, geldanamycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma Chemical Co. (St Louis, MO); IGF-1, bFGF, EGF, IFN- , and TNF- were from R&D Systems (Minneapolis, MN; bisindolylmaleimide (BIM) III and wortmannin were from Calbiochem (La Jolla, CA); rabbit polyclonal antibody for caspase-10 was from Research Diagnostics Inc. (Flanders, NJ); the IGF-1 receptor neutralizing antibody aIR3 was from Oncogene Research (Cambridge, MA); and the enhanced chemiluminescence (ECL) kit, which includes the peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies, was from Amersham (Arlington Heights, IL).

Techniques: MTT Assay, Comparison, Control