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: Biomolecules and Biomedicine

Article Title: Glioblastoma induces CAF-like astrocyte activation via the AKT/mTOR–SERPINH1/COL5A1 axis

doi: 10.17305/bb.2025.11898

Figure Lengend Snippet: Localization of SERPINH1 and COL5A1. (A–C) Multicolor IHC staining of astrocytes (A, gfap), microglia (B, Iba1), and oligodendrocytes (C, MBP) in the GBM mouse model. Green indicates G422-GFP and Red indicates glial cells. Nuclei were stained using DAPI. The Merge panel shows the combined image. Red arrows indicate the localized recruitment of glial cells. The statistical results are shown in Figure S7 . (D–G) Co-localization labeling of SERPINH1 (D, 10×; E, 63×) and COL5A1 (F, 10×; G, 63×) with astrocytes in the GBM mouse model. Green indicates G422-GFP, and Red indicates astrocytes. White indicates SERPINH1/COL5A1 expression. Nuclei were stained using DAPI. The Merge panel shows the combined image. White arrows indicate areas with high levels of co-localization and the cells that co-localized. (H–K) Co-localization labeling of FAP (H, 10×; I, 63×) and S100A4 (J, 10×; K, 63×) with astrocytes in the GBM mouse model. Green indicates G422-GFP, and Red indicates astrocytes. White indicates FAP/S100A4 expression. Nuclei were stained using DAPI. The Merge panel shows the combined image. White arrows indicate areas with high levels of co-localization and the cells that co-localized. Red arrows indicate FAP/S100A4-positive and GFAP-negative cells. (L) Statistical chart of tissue immunofluorescence localization (E, G, I, and K) results SERPINH1,0.557 ± 0.011, n ═ 3; COL5A1, 0.590 ± 0.006, n ═ 3; FAP, 0.505 ± 0.013, n ═ 3; S100A4, 0.481 ± 0.003, n ═ 3. The experiment was repeated three times, and the results are representative of three independent experiments. In the statistical figures, P values are indicated with asterisks, where *represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001. GBM: Glioblastoma multiforme.

Article Snippet: Slides were then incubated overnight at 4 ∘ C with the following primary antibodies: SERPINH1 (sc-5293, Santa Cruz, 1:100), COL5A1 (WLH4136, Wanleibio, 1:100), GFAP ( HA600094 , HUABIO, 1:500), MBP (WL03919, Wanleibio, 1:50), IBA1 (sc-32725, Santa Cruz, 1:100), FAP (AF5344, Affinity, 1:50), and S100A4 (CY5799, Abways, 1:100).

Techniques: Immunohistochemistry, Staining, Labeling, Expressing, Immunofluorescence