Journal: Cell Death & Disease

Article Title: TGF- β 1 and hypoxia-dependent expression of MKP-1 leads tumor resistance to death receptor-mediated cell death

doi: 10.1038/cddis.2013.42

Figure Lengend Snippet: Knockdown of Smad2 or MKP-1 abolishes crosstalk between TGF- β 1 and TNF- α . Knockdown of Smad2 and MKP-1 was achieved by siRNA transfection as described in the Materials and methods . Each cytokine was applied 48 h after siRNA transfection. ( a ) 24 h post-transfection, MC38 cells were immunized with SIINFEKL peptide. Six hours after coculture of target (MC38 cells) and effector (OT-1 mouse-derived CD8 + T cells) cells, cytotoxicity was evaluated by LDH release assay as in (mean±S.E.M., n =4; Bonferroni's post hoc test was applied for multiple comparisons in two-way ANOVA, ***P <0.001, N.S, not significant). ( b ) 24 h after treatment with each cytokine in Huh-7 cells, viability was evaluated by WST-1 assay under conditions of siRNA knockdown (mean±S.E.M., n =4; *P <0.05, ***P <0.001 by Student's t -test). ( c ) Huh-7 cells were incubated in the presence or absence of TGF- β 1 (10 ng/ml) for 1 h, and were then treated with TNF- α (10 ng/ml) for up to 30 min. Cell lysates were subjected to immunoblot analysis as in

Article Snippet: Antibodies specific for IKK- β , phospho-IKK, phospho-I κ B- α , JNK, phospho-JNK, p38, phospho-p38, p42/44, phospho-p42/44, Smad2, phospho-Smad2, MKK3, phospho-MKK3/6, MKK4, phospho-MKK4, MKK7, phospho-MKK7, TAK1, and phospho-TAK1 were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, Transfection, Derivative Assay, Lactate Dehydrogenase Assay, WST-1 Assay, Incubation, Western Blot

Journal: Cell Death & Disease

Article Title: TGF- β 1 and hypoxia-dependent expression of MKP-1 leads tumor resistance to death receptor-mediated cell death

doi: 10.1038/cddis.2013.42

Figure Lengend Snippet: Tumor-specific expression of MKP-1. ( a ) MKP-1 expression was measured in colorectal and prostate cancer patients from the GEO database using a complementary DNA microarray (mean±S.E.M.; Tukey's post hoc test was applied to significant group effects in ANOVA, P <0.0001; ***P <0.001, compared with normal tissues). ( b ) The correlation between TGF- β pathway activity and MKP-1 expression in prostate tissue was evaluated as described in the Materials and methods . ( c ) Immunofluorescence staining with FITC (green) and Hoechst (blue) of a human liver tissue array. Of the 50 HCC samples, 39 were MKP-1 positive (78%); nine normal samples did not show MKP-1 expression (0%). ( d ) Immunoperoxidase staining of tissue samples from HCC patients using DAB and hematoxilin. T, tumor region; N, adjacent normal region. Of 23 HCC patients, 16 showed MKP-1 expression (69.6%) ( e ) Cell lysates were subjected to immunoblot analysis of HIF-1 α , Smad2, and MKP-1 in the presence or absence of siHIF-1 α and hypoxia (oxygen concentration: 1%). GAPDH was used as the loading control

Article Snippet: Antibodies specific for IKK- β , phospho-IKK, phospho-I κ B- α , JNK, phospho-JNK, p38, phospho-p38, p42/44, phospho-p42/44, Smad2, phospho-Smad2, MKK3, phospho-MKK3/6, MKK4, phospho-MKK4, MKK7, phospho-MKK7, TAK1, and phospho-TAK1 were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Microarray, Activity Assay, Immunofluorescence, Staining, Immunoperoxidase Staining, Western Blot, Concentration Assay, Control

Journal:

Article Title: Recombinant rabies virus vaccine strain SAD-L16 inoculated intracerebrally in young mice produces a severe encephalitis with extensive neuronal apoptosis

doi:

Figure Lengend Snippet: Transferase-mediated dUTP-biotin nick end labelling (TUNEL) and staining (A to D) and immunoperoxidase staining for activated caspase-3 (E to H) in L16-infected mouse brains. The TUNEL staining is present in neurons in the cerebellar external and internal granular layers (A) 6 d postinoculation (pi), in many neurons in the cerebral cortex (B) 4 d pi, and in hippocampal pyramidal neurons and neurons in the dentate gyrus of the hippocampus (C) 5 d pi, while there is low background staining in the hippocampus of a mock-infected mouse (D). Activated caspase-3 staining is present in many neurons in the internal granular layer of the cerebellum (E) 5 d pi, in some Purkinje cells (F) 4 d pi, in both hippocampal pyramidal neurons and in neurons in the dentate gyrus of the hippocampus (G) 4 d pi, and there is low background staining in the hippocampus of a mock-infected mouse (H). A to D, TUNEL staining — methyl green; E to H, Immunoperoxidase — hematoxylin. Magnification: A × 150; B × 95; C, D × 25; E × 130; F × 390; G, H × 20.

Article Snippet: Sections were successively treated with 5% normal goat serum, rabbit polyclonal antibody directed against cleaved (activated) caspase-3 (Asp175) (Cell Signaling Technology, Beverley, Massachusetts, USA) diluted 1:400 in 2% normal goat serum, biotinylated goat anti-rabbit IgG (Vector Laboratories) diluted 1:100 in 2% normal goat serum, 1% hydrogen peroxide in methanol, avidin-biotinylated horseradish peroxidase complex (Vector Laboratories), 3,3-diaminobenzidine tetrachloride (Polysciences) with 0.01% hydrogen peroxide, and 0.5% cupric sulfate in 0.15 M sodium chloride.

Techniques: TUNEL Assay, Staining, Immunoperoxidase Staining, Infection