Journal: Molecular and Cellular Biology

Article Title: Requirement of FADD, NEMO, and BAX/BAK for Aberrant Mitochondrial Function in Tumor Necrosis Factor Alpha-Induced Necrosis

doi: 10.1128/mcb.05303-11

Figure Lengend Snippet: FIG. 10. Overexpression of Bcl-xL attenuates TCZ-induced necroptosis. (A) Wild-type MEFs were transfected with Bcl-xL plasmid. The overexpression of Bcl-xL was assessed by Western blotting. (B) MEFs were cultured in glass-bottom petri dishes, and cells were challenged with TCZ for 20 h. After the treatment, live cells were simultaneously loaded with the m indicator TMRE and the plasma membrane integrity marker TOTO-3 to assess the mitochondrial function and cell viability, respectively. (C) Quantitation of m maintenance and cell viability after TCZ treatment. (D) Wild-type MEFs were treated with TCZ for 4 and 6 h. Cells were isolated and treated with 10 mM bis-maleimide cross-linker. The BAX oligomers were analyzed by anti-BAX immunoblotting. (E) Wild-type MEFs were treated with TCZ for 6 h, and cells were fixed and immunostained with anti-cytochrome c antibody (green), anti-Smac/Diablo antibody (green), anti-HtrA2/Omi (green), and anti-AIF antibody (green). Hoechst dye (blue) was used as a nuclear stain. (F) Quantitation of mitochondrial intermembrane space proteins (cytochrome c, Smac/Diablo, HtrA2/Omi, and AIF). Data are presented as means SEMs of at least three independent experiments.

Article Snippet: Cells were probed for anti-cytochrome c antibody (catalog no. 556433; BD Pharmingen), Smac/Diablo antibody (abcam8115), HtrA2/Omi antibody (ab64111), and apoptosis-inducing factor (AIF) antibody (catalog no. 4642; Cell Signaling Technology) and visualized using an Alexa Fluor-488 secondary antibody (Invitrogen).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Cell Culture, Clinical Proteomics, Membrane, Marker, Quantitation Assay, Isolation, Staining

Journal: Investigative ophthalmology & visual science

Article Title: Selective Serotonin Reuptake Inhibitors Aggravate Depression-Associated Dry Eye Via Activating the NF-κB Pathway.

doi: 10.1167/iovs.18-25572

Figure Lengend Snippet: FIGURE 1. SSRI treatment is closely related to depression-associated dry eye disease and causes increased tear serotonin levels with a serious inflammatory response and cell apoptosis at the ocular surface. (A) The clinical indicators based on the baseline data and data collected after 90 days of treatment in the SSRI and control groups. The P value means the difference between ‘‘change’’ in SSRI and control groups. (B) ELISA analysis showing the tear serotonin levels of the patients with SSRI treatment and the control group. (C, D) Protein chip analysis showing the inflammatory cytokine content following SSRI treatment, based on examination of 507 proteins, including TLR4, IL-6, and IL-10. (E) qRT-PCR analysis showing the conjunctival TLR2 and TLR4 and inflammatory cytokine IL1b, IL10, and TNFa mRNA levels between the patients with SSRI treatment and the control group. (F) qRT-PCR analysis showing the conjunctival mRNA levels of the proapoptotic genes AIF, BAD, and BAX and the antiapoptotic genes BCL2 and XIAP between the patients with SSRI treatment and the control group. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were incubated overnight at 48C with primary antibodies, including IL-1b (1:1000; Abcam), IL-6 (1:1000; Proteintech, Wuhan, China), IL-10 (1:500; Abcam), TNF-a (1:500; Abcam), TLR2 (1:1000; Abcam), TLR4 (1:200; Santa Cruz Biotechnology), AIF (1:1000; Proteintech), BAD (1:1000; Abcam), BAX (1:1000; Proteintech), P-p65 (1:2000; Abcam), p65 (1:2000; Abcam), IjBa (1:1000; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:12000; Proteintech).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Investigative ophthalmology & visual science

Article Title: Selective Serotonin Reuptake Inhibitors Aggravate Depression-Associated Dry Eye Via Activating the NF-κB Pathway.

doi: 10.1167/iovs.18-25572

Figure Lengend Snippet: FIGURE 4. Serotonin can induce corneal epithelial cell inflammation and apoptosis. (A) qRT-PCR analysis showing the mRNA levels of the inflammatory cytokines TLR4, TLR2, IL1b, IL6, IL10, and TNFa with the serotonin level. (B, C) Western blot analysis showing the protein levels in the growth plate of TLR4, TLR2, IL-1b, IL-6, IL-10, and TNF-a with the serotonin level. (D) ELISA analysis of the related IL-1b, IL-10, and TNF-a content in the cell supernatants in cells with increased serotonin levels. (E, F) Western blot analysis showing the protein levels in the growth plate of the proapoptotic proteins BAX, BAD, and AIF with the serotonin level. (E) Flow cytometry showing apoptosis in corneal epithelial cells with serotonin treatment. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were incubated overnight at 48C with primary antibodies, including IL-1b (1:1000; Abcam), IL-6 (1:1000; Proteintech, Wuhan, China), IL-10 (1:500; Abcam), TNF-a (1:500; Abcam), TLR2 (1:1000; Abcam), TLR4 (1:200; Santa Cruz Biotechnology), AIF (1:1000; Proteintech), BAD (1:1000; Abcam), BAX (1:1000; Proteintech), P-p65 (1:2000; Abcam), p65 (1:2000; Abcam), IjBa (1:1000; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:12000; Proteintech).

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Investigative ophthalmology & visual science

Article Title: Selective Serotonin Reuptake Inhibitors Aggravate Depression-Associated Dry Eye Via Activating the NF-κB Pathway.

doi: 10.1167/iovs.18-25572

Figure Lengend Snippet: FIGURE 5. Serotonin receptors are indispensable for serotonin-induced cell inflammation and apoptosis in corneal epithelial cells. (A) qRT-PCR analysis showing the HTR1A, 1B, 1D, 1E, 1F, 2A, 2B, 2C, 3A, 3B, 4, 5A, 6, and 7 mRNA levels in corneal epithelial cells. (B, C) Western blot assay showing the expression levels of the proinflammatory response element TLR4, inflammatory cytokines IL-6 and IL-10, and proapoptotic proteins BAX, BAD, and AIF in asenapine maleate–treated corneal epithelial cells. (D) ELISA analysis of the related IL-1b, IL-10, and TNF-a content in the supernatants of cells with accumulated serotonin treated with asenapine maleate. (G) Flow cytometry showing apoptosis in corneal epithelial cells with serotonin treated with asenapine maleate. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were incubated overnight at 48C with primary antibodies, including IL-1b (1:1000; Abcam), IL-6 (1:1000; Proteintech, Wuhan, China), IL-10 (1:500; Abcam), TNF-a (1:500; Abcam), TLR2 (1:1000; Abcam), TLR4 (1:200; Santa Cruz Biotechnology), AIF (1:1000; Proteintech), BAD (1:1000; Abcam), BAX (1:1000; Proteintech), P-p65 (1:2000; Abcam), p65 (1:2000; Abcam), IjBa (1:1000; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:12000; Proteintech).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Investigative ophthalmology & visual science

Article Title: Selective Serotonin Reuptake Inhibitors Aggravate Depression-Associated Dry Eye Via Activating the NF-κB Pathway.

doi: 10.1167/iovs.18-25572

Figure Lengend Snippet: FIGURE 6. Serotonin induces cell inflammation and apoptosis by activating the NF-jB pathway. (A, B) Western blot analysis showing the p65 phosphorylation level and the IjBa protein level with the serotonin level. (C, D) Western blot analysis showing the P-p65, IjBa, proinflammatory response element TLR4, inflammatory cytokine IL-1b, and proapoptotic protein BAX, BAD, and AIF protein levels in JSH-23–treated corneal epithelial cells. (E) ELISA analysis of the related IL-1b, IL-10, and TNF-a content in the supernatants of cells treated with the NF-jB signaling inhibitor. (F) Flow cytometry showing apoptosis in corneal epithelial cells with accumulated serotonin following JSH-23 treatment. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were incubated overnight at 48C with primary antibodies, including IL-1b (1:1000; Abcam), IL-6 (1:1000; Proteintech, Wuhan, China), IL-10 (1:500; Abcam), TNF-a (1:500; Abcam), TLR2 (1:1000; Abcam), TLR4 (1:200; Santa Cruz Biotechnology), AIF (1:1000; Proteintech), BAD (1:1000; Abcam), BAX (1:1000; Proteintech), P-p65 (1:2000; Abcam), p65 (1:2000; Abcam), IjBa (1:1000; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:12000; Proteintech).

Techniques: Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Oncotarget

Article Title: Molecular profiling of prostate cancer derived exosomes may reveal a predictive signature for response to docetaxel

doi:

Figure Lengend Snippet: (A) Heat map of the hierarchical clustering of the 100 most abundant protein identified. The values were mean centered and log-transformed. The relative protein abundance is colored-coded with red corresponding to a relatively high abundance, green to a relatively low abundance, and black indicating indifferent abundance values. Each exosome sample was analyzed in duplicates; (B) Western blot analysis of 10 μg of DU145 Tax-Sen and DU145 Tax-Res exosomes, probed for the indicated proteins. Rab5 and TSG101 were used as exosomal markers and AIF as a quality control of the exosomal isolation ( n = 2); (C) Quantification of the abundance values for MDR-1 and PABP4; (D) Western blot analysis of the sucrose gradient fractions from DU145 Tax-Sen and DU145 Tax-Res exosomes, probed for MDR-1/3 and TSG101 ( n = 2).

Article Snippet: The primary antibodies used in this study against Rab 5, CD81, CD82, Alix, Emmprin, Caveolin-1 were obtained from Cell Signaling Technology; TSG101, CD9 and CD82 from Abcam, MDR-1 and MDR-3 from Gene Tex; AIF, CD9, Endophilin A2 and CD63 from Santa Cruz Biotechnology; PACSIN2 from Abcam and Santa Cruz Biotechnology; Cyp17A from Novus Biologicals; IgG1-APC from Biolegend.

Techniques: Transformation Assay, Quantitative Proteomics, Western Blot, Control, Isolation