Journal: Journal of Cellular and Molecular Medicine

Article Title: Kaempferol attenuates liver fibrosis by inhibiting activin receptor–like kinase 5

doi: 10.1111/jcmm.14528

Figure Lengend Snippet: Kaempferol reduces Smad2 and Smad3 phosphorylation. A, Levels of Smad2, p‐Smad2, Smad3, p‐Smad3 and Smad4 in HSCs pretreated with TGF‐β1 (2 ng/mL) and kaempferol at different concentrations (2‐10 μmol/L) were detected using Western blotting and thereafter quantified. n = 3. B, Immunofluorescence images exhibit a significant reduction in TGF‐β1‐induced Smad2/3 translocation into the nuclei. Smad2/3 is shown by red fluorescence. Nuclei were stained with DAPI and shown by blue fluorescence. Scale bars, 50 μm. n = 3. C, Levels of Smad6 and Smad7 after HSCs were incubated with kaempferol or DMSO. n = 3. D, Kaempferol has no significant impact on the expression of TGF‐β1 based on ELISA assay. n = 3. E, Levels of TGF‐β receptor I and II with kaempferol incubation at different concentrations. n = 3. All experiments were carried out 3 times to assess reproducibility. Data are demonstrated as mean ± SD. ** P < 0.01; *** P < 0.001

Article Snippet: The separated proteins were immunodetected with the primary antibodies including the following: Col1a1 (Abcam, 1:500, ab34710), α‐SMA (Abcam, 1:500, ab32575), Smad2 (Cell Signaling Technology, 1:1000, #5339), Smad3 (CST, 1:1000, #9523), p‐Smad2 (CST, 1:1000, #18338), p‐Smad3 (CST, 1:1000, #9520), Smad6 (CST, 1:1000, #9519), Smad7 (Abcam, 1:1000, ab90086), Smad4 (CST, 1:1000, #38454), TGF‐β receptor I (CST, 1:1000, #3712) and TGF‐β receptor II (CST, 1:1000, #79424).

Techniques: Phospho-proteomics, Western Blot, Immunofluorescence, Translocation Assay, Fluorescence, Staining, Incubation, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The chromatin landscape of healthy and injured cell types in the human kidney

doi: 10.1101/2023.06.07.543965

Figure Lengend Snippet: Alignment of epigenomic features in bulk and regional human kidney samples. (A) Epigenomic features of marker genes for glomerulus ( PODXL) , proximal tubule (PT-S12, PDZK1) , and thick ascending loop of Henle (C-TAL, CASR) , displaying: 1) DNA methylation (DNAm) in the tubulointerstitium (TI) and glomerulus (GLOM), 2) bulk CUT&RUN for four histone modifications: H3K27ac, H3K27me3, H3K4me1, H3K4me3, and 3) assay for transposase-accessible chromatin using sequencing (ATAC-seq) on bulk tissue (Encode ENCSR297VGU). Gray stripes indicate active promoters wherein ATAC-seq peaks at transcriptional start sites coincide with DNAm dips, and H3K4me3 peaks. Variable H3K27ac peaks reflect compartments’ proportion within bulk tissue. (B) Differential methylation between GLOM and TI kidney compartments for summative methylation of 30,024 gene bodies. (C) Best-fit regression model of methylation and mRNA expression in identical samples (N=12) for differentially expressed genes (N=5408) between the GLOM and TI. Each dot represents a gene. Y-axis is the Log 2 fold change of mRNA between the GLOM and TI. X-axis is the Log 2 fold change of methylation between the Glom and TI. The best fit annotated gene region (summative promoter, exon, intron, of CpG island methylation) with the most negative correlation was identified as the promoter for 1867 genes and CpG island for 1327 genes. The inset represents methylation fold change distribution in annotated gene regions. (D) Genomic region call rule based on combination of histone marks. (E) Landscape correlation agreement between datasets (Fisher’s exact test). (F) Histone markers and spearman correlation with snRNA-seq expression in the PT-S12 and C-TAL and in the regional mRNA expression of the micro-dissected TI. (G) Cell type deconvolution of CUT&RUN for H3K27ac, H3K4me1, H3K4me3 active histone modifications at promoters. The RNA signature was taken from the HuBMAP/KPMP atlas snRNAseq[48], using the 10% most DE marker genes. (H) Upset plot depicting overlap in peaks of H3K27ac, H3K4me1, H3K4me3, and H3K27me3 with DNAm dips across the genome. Active promoters, predicted enhancers, and repressed regions are annotated. (I) Heatmap of CUT&RUN marks across the genome by annotated region after filtering for open chromatin and DNA methylation dips.

Article Snippet: Antibodies used in this study for CUT&RUN reactions: H3K27ac (Cell Signaling, 8173), H3K27me3 (Cell Signaling, 9733), H3K4me1 (Cell Signaling, 5326), H3K4me3 (Cell Signaling, 9751), and IgG (Cell Signaling, 2729).

Techniques: Marker, DNA Methylation Assay, Sequencing, Methylation, Expressing

Journal: bioRxiv

Article Title: The chromatin landscape of healthy and injured cell types in the human kidney

doi: 10.1101/2023.06.07.543965

Figure Lengend Snippet: Single cell epigenomics in health. The multiome reduction by uniform manifold approximation and projection (UMAP) of 47,217 nuclei in 72 clusters aligning with the HuBMAP/KPMP atlas is depicted for (A) snRNA-seq and (B) snATAC-seq assays. Highlighted clusters include podocytes (POD), proximal tubule (PT) S1, S2, S3 and adaptive PT (aPT), cortical thick ascending loop of Henle (C-TAL), and adaptive TAL (aTAL1 and aTAL2) cells. (C) Gene markers for POD, PT S1 merged with S2 (PT-S12) and C-TAL reveal cell type specificity of expression and chromatin accessibility. Dot plot (top) reveals transcript expression. Bar graph (bottom) represents the area under the curve (AUC) for open chromatin coverage summed across the entire gene. Genes (D) PDZK1 , (E) ESRRG , and (F) PODXL align across the snATAC-seq, WGBS, and CUT&RUN technologies. snATAC-seq peaks are displayed in tracks 1 to 3 with respective RNA expression in adjacent violin plots. DNAm levels (track 4) and differential DNAm (track 5) are depicted for the GLOM (blue) and TI (red). Histone marks for CUT&RUN are found in track 6-9 (H3K27ac, H3K27me3, H3K4me1 and H3K4me3 respectively). Aggregate open chromatin regions (track 10), and chromosome coordinates are below. Differentially accessible open chromatin peaks are annotated as red stripes (coincides with CpG island) or gray (no CpG island). Cohen’s Kappa (CK) agreement between snATAC-seq peaks with DNAm dips (G) or H3K4me3 peaks (H) in the 2194 differentially expressed genes of POD, PT-S12, and C-TAL. Perfect agreement (disagreement) is 1 (−1), ranging from G CK [0,0.2) no agreement to G CK [0.8,1] perfect agreement. Average CK across all genes: G T . Most peaks positively correlated between technologies. Smaller genes have stronger correlation. (I) Association of DNAm & histone marks with open chromatin. The correlation of open chromatin peaks with DNAm dips, and histone mark peaks is given for differentially expressed genes of the POD, PT-S12, and C-TAL (Fisher’s Exact test). (J) Upset plot shows the intersection of CUT&RUN peaks and DNAm dips across snATAC-seq PT-S12/aPT peaks, identifying regulatory regions for PT. (K) Heatmap of CUT&RUN marks in PT-S12/aPT in each annotated region after filtering for open chromatin and DNAm dips.

Article Snippet: Antibodies used in this study for CUT&RUN reactions: H3K27ac (Cell Signaling, 8173), H3K27me3 (Cell Signaling, 9733), H3K4me1 (Cell Signaling, 5326), H3K4me3 (Cell Signaling, 9751), and IgG (Cell Signaling, 2729).

Techniques: Expressing, RNA Expression

Journal: bioRxiv

Article Title: The chromatin landscape of healthy and injured cell types in the human kidney

doi: 10.1101/2023.06.07.543965

Figure Lengend Snippet: Adaptive cell state in the proximal tubule. (A) Differentially expressed genes (DEGs) between the PT-S12 and aPT cell types within the multiome atlas. (B) Diffusion map of PT-S1, PT-S2 and aPT nuclei. The inset shows the pseudotime trajectory from PT to aPT. (C) Gene expression localization in aPT cells ( ITGB3 , PROM1 , TPM1 ) for aPT marker genes. Canonical PT markers ( PDZK1 , SLC5A12 and RXRA ) localize to the PT-S12. (D) Differentially accessible (DA) peaks (N=137,993) between the PT-S12 and aPT (from multiome TRIPOD-seurat-aPTxPT-S12 object) that coincide with TI DNA methylation (DNAm) dips. Red = promoter dip, blue = dip outside promoter. (E) DA multiome peaks with DNAm dips and a CUT&RUN histone mark peak. Active promoter (Act pro ) = green, predicted enhancer (Pred enh ) = blue, repressed promoter (Rep pro ) = red, (N=27,174). (F) Top 15 clusters from GO-All pathway enrichment analysis based on DA. Key pathways of the adaptive process include platelet-derived growth factor binding, mesodermal cell differentiation, and integrin complex and adhesion. (G) DA peaks (N=2557) in upregulated DEGs of the PT-S12, targeted by a transcription factor (TF) in TRIPOD analyses. Orange dots represent new peaks (NP) in the PT-S12, where fewer than 2% of aPT nuclei had open chromatin (adjusted p <0.05). Gray dots display DA peaks present in both PT-S12 and aPT. All DA NP were found in PT-S12 cells for DEGs upregulated in the PT-S12. (H) DA peaks (N=4573) in upregulated DEGs of the aPT. Green = aPT NP. Gray = DA peak present in both PT-S12 and aPT. (I-J) Gene alignment of SLC5A12 , a PT-S12 marker, and PROM1 , an aPT marker, across snATAC-seq peaks, DNAm TI dips, and CUT&RUN histone marks (H3K27ac, H3K27me3, H3K4me1 and H3K4me3). Red stripe = NP with TF binding. (K) Differentially expressed TFs of the aPT and PT-S12 which target NP of PROM1 and SLC5A12 . (L-M) Chord diagram of SLC5A12 and PROM1 , respectively, with TF (TRUE = positive binding or DNAm Dip and FALSE = no binding or no DNAm Dip).

Article Snippet: Antibodies used in this study for CUT&RUN reactions: H3K27ac (Cell Signaling, 8173), H3K27me3 (Cell Signaling, 9733), H3K4me1 (Cell Signaling, 5326), H3K4me3 (Cell Signaling, 9751), and IgG (Cell Signaling, 2729).

Techniques: Diffusion-based Assay, Gene Expression, Marker, DNA Methylation Assay, Derivative Assay, Binding Assay, Cell Differentiation