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: bioRxiv

Article Title: The renal capsule: a vibrant and adaptive cell environment of the kidney in homeostasis, disease and aging

doi: 10.1101/2023.05.11.540033

Figure Lengend Snippet: ( a ) Schematic description of the experimental design for single cell based analyses. Created with BioRender.com. ( b ) opt-SNE plot of mass cytometry data depicting cell populations in the renal capsule (n=10 pooled mice). ( c ) Flow cytometry data of main immune cell types in the renal capsule (n=8 mice). ( d ) Iba-1 immunofluorescence imaging of macrophages in a whole-mounted mouse renal capsule. ( e ) Schematic description of the experimental design for RCAM and kidney macrophages characterization using bulk RNA-Seq. Created with BioRender.com. Volcano plot and heat maps of bulk RNA-Seq data of renal capsule macrophages and kidney macrophages with highlighted selected genes, representative image of two experiments (n=4,4). ( f ) UMAP of scRNA-Seq analysis of renal capsule cells, and horizontal slice chart with the proportions of 3 main interstitial macrophage subsets according to the scRNA-Seq data (2 samples of n=4 pooled mice each). Representative image of two experiments. ( g ) Pie charts with the proportion of 3 main interstitial macrophage subsets in mouse (n=6) and human (25 year-old donor) renal capsules and kidneys, according to flow cytometry analysis. ( h ) Inflammatory and phagocytic path scores of monocytes and macrophages, as previously described.

Article Snippet: Tissues were then stained with the following fluorophore-conjugated anti-mouse antibodies for (1:100, 1.5 hr, room temperature, gentle agitation) in blocking buffer: Iba-1 (78060S, Cell Signaling Technology), F4/80 (123122, Biolegend), CD206 (141710, Biolegend), CD45 (103172, Biolegend), and Lyve-1 (FAB2125G, R&D).

Techniques: Mass Cytometry, Flow Cytometry, Immunofluorescence, Imaging, RNA Sequencing Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Astragaloside IV Alleviates Ulcerative Colitis Progression by Inhibiting WDR5 ‐Mediated ENO1 H3K4me3 Modification

doi: 10.1002/kjm2.70064

Figure Lengend Snippet: WDR5, a target of ASI, is overexpressed in UC models. (A) The chemical structural formula of ASI was obtained from PubChem Substance. (B) Transcriptional differences between colonic biopsies from patients with UC and non‐inflammatory controls in the GSE38713 dataset. (C) The intersection of differentially expressed genes in the GSE38713 dataset, targets of ASI, and human TF and TF cofactors. (D) Six intersecting interactions were subjected to protein–protein interaction plotting (connections between two protein nodes represent evidence of interactions, and more connecting lines between the two indicate a closer relationship between the predicted interactions). (E) mRNA expression of WDR5 in NCM460 cells treated with ASI or not was examined using RT‐qPCR. (F) The protein expression of WDR5 in NCM460 cells treated with ASI was examined using western blot analysis. (G) Positive rate of WDR5 in the colonic tissues of mice detected by immunohistochemistry. The data are expressed as mean ± SEM, n = 3 (E, F) or 7 (G). ANOVA was applied for each comparison. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: After mixing with Protein A + G beads for 30 min at 4°C and centrifugation at 1000 g at 4°C, the supernatant was immunoprecipitated with antibodies against H3K4me3 (1:100, MBS8527387, MyBioSource) and WDR5 (1:50, 13,105, Cell Signaling Technologies, Beverly, MA, USA) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Comparison

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Astragaloside IV Alleviates Ulcerative Colitis Progression by Inhibiting WDR5 ‐Mediated ENO1 H3K4me3 Modification

doi: 10.1002/kjm2.70064

Figure Lengend Snippet: Overexpression of WDR5 weakens the effects of ASI on DSS‐induced NCM460 cells. (A) Detection of mRNA expression of WDR5 in NCM460 cells treated with ASI + OE‐Vector/OE‐WDR5 was examined using RT‐qPCR. (B) The viability of NCM460 cells was examined using the CCK‐8 assay. (C) The apoptosis of NCM460 cells was examined using the TUNEL assay. (D) The protein expression of Cleaved‐Caspase‐3 in NCM460 cells was examined using Western blot assays. (E) The protein expression of ZO‐1 and claudin‐3 in NCM460 cells was examined using Western blot assays. (F) TNF‐α, IL‐6, IL‐1β, and IL‐10 contents in NCM460 cell culture supernatant were examined using ELISA. The data are expressed as the means ± SEM, n = 3. An unpaired t‐test (A) or ANOVA (B–F) was applied for each comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After mixing with Protein A + G beads for 30 min at 4°C and centrifugation at 1000 g at 4°C, the supernatant was immunoprecipitated with antibodies against H3K4me3 (1:100, MBS8527387, MyBioSource) and WDR5 (1:50, 13,105, Cell Signaling Technologies, Beverly, MA, USA) at 4°C overnight.

Techniques: Over Expression, Expressing, Plasmid Preparation, Quantitative RT-PCR, CCK-8 Assay, TUNEL Assay, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Astragaloside IV Alleviates Ulcerative Colitis Progression by Inhibiting WDR5 ‐Mediated ENO1 H3K4me3 Modification

doi: 10.1002/kjm2.70064

Figure Lengend Snippet: WDR5 promotes ENO1 transcription through H3K4me3 modification. (A) The intersection of differentially expressed genes in the GSE38713 dataset and WDR5 targets in the hTFtarget website. (B) The intersection of differentially expressed genes in the GSE38713 dataset, WDR5 targets in the hTFtarget website, and UC‐related genes downloaded from the GeneCards dataset. (C) ChIP‐seq analysis of H3K4me3 binding peaks on the ENO1 promoter region. (D) ChIP‐seq analysis of WDR5 binding peaks on the ENO1 promoter region. (E) Correlation analysis of WDR5 and ENO1 expression in the colon in the GEPIA dataset. (F) The protein expression of ENO1 in the colonic tissues of mice was detected by western blot analysis. (G) ENO1 mRNA expression in DSS‐stimulated NCM460 cells infected with a lentiviral vector overexpressing WDR5 was assessed using RT‐qPCR. (H) Enrichment of the ENO1 promoter region by anti‐WDR5 and anti‐H3K4me3 in NCM460 cells overexpressing WDR5 was analyzed using a ChIP assay. (I) H3K4me3 protein expression in DSS‐stimulated NCM460 cells infected with a lentiviral vector overexpressing WDR5 was examined using Western blot assay. The data are expressed as the means ± SEM, n = 3 (G–I) or 7 (F). An unpaired t ‐test (G–I) or ANOVA (F) was applied for each comparison. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After mixing with Protein A + G beads for 30 min at 4°C and centrifugation at 1000 g at 4°C, the supernatant was immunoprecipitated with antibodies against H3K4me3 (1:100, MBS8527387, MyBioSource) and WDR5 (1:50, 13,105, Cell Signaling Technologies, Beverly, MA, USA) at 4°C overnight.

Techniques: Modification, ChIP-sequencing, Binding Assay, Expressing, Western Blot, Infection, Plasmid Preparation, Quantitative RT-PCR, Comparison

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Astragaloside IV Alleviates Ulcerative Colitis Progression by Inhibiting WDR5 ‐Mediated ENO1 H3K4me3 Modification

doi: 10.1002/kjm2.70064

Figure Lengend Snippet: Silencing of ENO1 alleviates DSS‐induced NCM460 cell injury in the presence of WDR5 overexpression. The mRNA and protein expression of ENO1 in NCM460 cells infected with OE‐WDR5 + sh‐NC/sh‐ENO1 was examined using RT‐qPCR (A) and western blot analysis (B). (C) The viability of NCM460 cells was examined using the CCK‐8 assay. (D) The apoptosis of NCM460 cells was examined using the TUNEL assay. (E) The protein expression of Cleaved‐Caspase‐3 in NCM460 cells was examined using Western blot assays. (F) TNF‐α, IL‐6, IL‐1β, and IL‐10 contents in NCM460 cells were examined using ELISA. The data are expressed as the means ± SEM, n = 3. An unpaired t ‐test was applied for each comparison. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After mixing with Protein A + G beads for 30 min at 4°C and centrifugation at 1000 g at 4°C, the supernatant was immunoprecipitated with antibodies against H3K4me3 (1:100, MBS8527387, MyBioSource) and WDR5 (1:50, 13,105, Cell Signaling Technologies, Beverly, MA, USA) at 4°C overnight.

Techniques: Over Expression, Expressing, Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Astragaloside IV Alleviates Ulcerative Colitis Progression by Inhibiting WDR5 ‐Mediated ENO1 H3K4me3 Modification

doi: 10.1002/kjm2.70064

Figure Lengend Snippet: Silencing of ENO1 mitigates the UC‐like symptom in mice in the presence of WDR5 overexpression. The mRNA and protein expression of ENO1 and WDR5 in colon tissues of mice treated with OE‐WDR5 + sh‐NC/sh‐ENO1 and ASI was examined using RT‐qPCR (A) and western blot analysis (B). (C) Positive rate of ENO1 and WDR5 in the colonic tissues of mice detected by immunohistochemistry. (D) Measurement of the length of the colon in mice at d 15 of modeling. (E) Analysis of colonic tissue pathology and tissue damage scores using HE staining. Detection of NO (F) and MPO content (G) in the colonic tissues of mice. The data are expressed as the means ± SEM, n = 7. ANOVA was applied for each comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After mixing with Protein A + G beads for 30 min at 4°C and centrifugation at 1000 g at 4°C, the supernatant was immunoprecipitated with antibodies against H3K4me3 (1:100, MBS8527387, MyBioSource) and WDR5 (1:50, 13,105, Cell Signaling Technologies, Beverly, MA, USA) at 4°C overnight.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining, Comparison

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Astragaloside IV Alleviates Ulcerative Colitis Progression by Inhibiting WDR5 ‐Mediated ENO1 H3K4me3 Modification

doi: 10.1002/kjm2.70064

Figure Lengend Snippet: Possible mechanism of action of ASI in UC. Inhibition of WDR5‐mediated modification of H3K4me3 on the ENO1 promoter by ASI hinders transcriptional activation of ENO1 to alleviate symptoms of UC.

Article Snippet: After mixing with Protein A + G beads for 30 min at 4°C and centrifugation at 1000 g at 4°C, the supernatant was immunoprecipitated with antibodies against H3K4me3 (1:100, MBS8527387, MyBioSource) and WDR5 (1:50, 13,105, Cell Signaling Technologies, Beverly, MA, USA) at 4°C overnight.

Techniques: Inhibition, Modification, Activation Assay

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