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

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Gastrodin Promotes the Survival of Random-Pattern Skin Flaps via Autophagy Flux Stimulation

doi: 10.1155/2021/6611668

Figure Lengend Snippet: GAS reduces oxidative stress in skin flaps (random pattern). (a) IHC has been employed to determine the expression of SOD1 in the flaps of all groups (original magnification: 200x; scale bar: 50 μ m). (b) The histogram of the OD value of SOD1 in each group of IHC. (c–e) Immunoblotting detects the expression of eNOS, SOD1, and HO1 proteins in the flaps. Gel electrophoresis has been carried out under similar experimental conditions, and the sheared spots are indicated here. (f–h) Histograms of the OD values of VEGF, MMP9, and cadherin 5 in all groups as analyzed via immunoblotting. The obtained results are expressed as mean ± SE, n = 6 per group. ∗ P value < 0.05 and ∗∗ P value < 0.01 vs. the control group.

Article Snippet: VEGF, SOD1, MMP9, anti-HO1, and anti-CASP3 monoclonal antibodies were procured from the Proteintech Group (Chicago). eNOS, CYC, and anti-Bax (Bax) monoclonal antibodies were obtained from Cell Signaling Technologies (Beverly, Massachusetts).

Techniques: Expressing, Western Blot, Nucleic Acid Electrophoresis, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Gastrodin Promotes the Survival of Random-Pattern Skin Flaps via Autophagy Flux Stimulation

doi: 10.1155/2021/6611668

Figure Lengend Snippet: Attenuation of autophagy reverses the impact of GAS on oxidative stress, apoptotic process, angiogenesis, and the survival of the flap. (a) Immunofluorescence staining showed that there were autophagosomes (red) in the cells of the flaps in the control group, the GAS group, the GAS+3MA group, and the 3MA group via LC3II (scale bar: 20 μ m). p62-positive cells (green) (b, c). The percentages of LC3II-positive cells and p62-positive cells were quantitatively analyzed in the dermis of all groups. (d, f) Western blotting was used to detect autophagy-associated proteins, i.e., Beclin1, Vps34, SQSTM1/p62, CTSD, and LC3II; angiogenesis-associated proteins, i.e., VEGF, MMP9, and cadherin 5; apoptosis-associated proteins, i.e., CYC, Bax, and CASP3; and oxidative stress-associated protein, i.e., HO1, SOD1, and eNOS in all groups. Gel electrophoresis was carried out under the same conditions in the experiment, and the cropped spots are indicated here. (e, g) The OD values of Beclin1, Vps34, SQSTM1/p62, CTSD, MMP9, LC3II, cadherin 5, VEGF, CYC, CASP3, Bax, HO1, eNOS, and SOD1, in all groups. (h) Digital photos (scale bar: 1 cm) of the flaps in the control group, the GAS group, the GAS+3MA group, and the 3MA group on POD3 and POD7. (i) Quantitative analysis of the percentage of survival area in each group. (j) Digital photos have been taken from inside of the flaps in the control group, the GAS group, the GAS+3MA group, and the 3MA group on POD7 (scale bar: 1 cm). (k) Histogram of the percentage of water content in each group. (l) Full-field LDBF image of each group of skin flaps in POD7 (scale bar: 1 cm). (m) Quantitative analysis of the blood flow signal intensity of skin flaps in each group. (n) H&E staining shows the blood vessels in the flap zone II of the control group, the GAS group, the GAS+3MA group, and the 3MA group (original magnification: 200x; scale bar: 50 μ m). (o) Histogram of MVD percentage in each group. (p) CD34IHC in the control group, the GAS group, the GAS+3MA group, and the 3MA group showed the blood vessels in zone II (original magnification: 200x; scale bar: 50 μ m). (q) Histogram of the percentage of CD34-positive blood vessels in all groups. The obtained results are represented as mean ± SE, n = 6 for all groups. $ P value < 0.05 and $$ P value < 0.01, in comparison with the control group; @ P value < 0.05 and @@ P value < 0.01, relative to the GAS group.

Article Snippet: VEGF, SOD1, MMP9, anti-HO1, and anti-CASP3 monoclonal antibodies were procured from the Proteintech Group (Chicago). eNOS, CYC, and anti-Bax (Bax) monoclonal antibodies were obtained from Cell Signaling Technologies (Beverly, Massachusetts).

Techniques: Immunofluorescence, Staining, Control, Western Blot, Nucleic Acid Electrophoresis, Comparison