Journal: Aging Cell

Article Title: Ghrelin Alleviates Aging‐Related Cognitive Impairment by Regulating Autophagy‐Related Signaling via the SHBG / JNK1 /Beclin1 Axis in Microglia

doi: 10.1111/acel.70732

Figure Lengend Snippet: CI in older and D‐gal‐induced mice is accompanied by microglial senescence and elevated ghrelin levels. (A) Experimental design of the D‐gal‐induced aging mouse model. (B) Percentage change in body weight of mice in the Young, Old, Control, and D‐gal groups, normalized to baseline body weight at the start of the experiment. (C) The NOL test comparing the mice from the Young and Old groups and from the Control and D‐gal groups. The recognition index was calculated as the ratio of time spent exploring in the novel location to the total time spent exploring both locations. (D) NOR test comparing the mice from the Young and Old groups and from the Control and D‐gal groups. The recognition index was calculated as the ratio of time spent exploring the novel object relative to the total exploration time for both objects. (E) MWM test comparing the mice from the Young and Old groups and from the Control and D‐gal groups, including swimming trajectories and escape latency. (F) Western blot analysis of senescence markers p16 Ink4a , p21 Cip1 , LaminB1, γH2AX, and HMGB1 in the mice brain tissue from the Young, Old, Control, and D‐gal groups. (G) Immunofluorescence co‐localization of Iba‐1 + microglia with LaminB1, γH2AX, and HMGB1 in the hippocampal CA1 and CA3 regions of the mice from Young, Old, Control, and D‐gal groups (magnification: ×400, scale bar = 25 μm). (H) ELISA‐based quantification of hippocampal cytokines (IL‐1α, IL‐1β, G‐CSF, IL‐4, IL‐6, IL‐8, CXCL10, LIF, MCP1, and M‐CSF) in the Young, Old, Control, and D‐gal groups. (I) ELISA measurement of plasma total ghrelin and acylated ghrelin in the Young, Old, Control, and D‐gal groups. ** p < 0.01, *** p < 0.001.

Article Snippet: Following antibody stripping and re‐blocking, sections were incubated overnight at 4°C with a second primary antibody: γH2AX (1:400; ab81299, Abcam), high‐mobility group box 1 (HMGB1) (1:400; AWA10032 , Abiowell), p21 Cip1 (1:200; ab188224, Abcam), p16 Ink4a (1:200; bs‐23797R, Bioss, China), or Lamin B1 (1:400; ab8982, Abcam).

Techniques: Control, Western Blot, Analysis, Tissue, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Aging Cell

Article Title: Ghrelin Alleviates Aging‐Related Cognitive Impairment by Regulating Autophagy‐Related Signaling via the SHBG / JNK1 /Beclin1 Axis in Microglia

doi: 10.1111/acel.70732

Figure Lengend Snippet: Ghrelin administration alleviates D‐gal‐induced microglial senescence and CI. (A) Experimental design of ghrelin treatment in the D‐gal‐induced aging mouse model. (B) Percentage change in body weight of mice in the Control, Control + Ghrelin, D‐gal, and D‐gal + Ghrelin groups, normalized to baseline body weight at the start of the experiment. (C) The NOL test comparing the mice from the Control, Control + Ghrelin, D‐gal, and D‐gal + Ghrelin groups. The recognition index was calculated as the ratio of time spent exploring in the novel location to the total time spent exploring both locations. (D) NOR test comparing the mice from the Control, Control + Ghrelin, D‐gal, and D‐gal+Ghrelin groups. The recognition index was calculated as the ratio of time spent exploring the novel object relative to the total exploration time for both objects. (E) MWM test comparing the mice from the Control, Control + Ghrelin, D‐gal, and D‐gal + Ghrelin groups, including swimming trajectories and escape latency. (F) Western blot analysis of senescence markers p16 Ink4a , p21 Cip1 , LaminB1, γH2AX, and HMGB1 in the mice brain tissue from the Control, Control+Ghrelin, D‐gal, and D‐gal+Ghrelin groups. (G) Immunofluorescence co‐localization of Iba‐1 + microglia with LaminB1, γH2AX, and HMGB1 in the hippocampal CA1 region of the mice from Control, Control+Ghrelin, D‐gal, and D‐gal + Ghrelin groups (magnification: ×400, scale bar = 25 μm). (H) ELISA‐based quantification of hippocampal cytokines (IL‐1α, IL‐1β, G‐CSF, IL‐4, IL‐6, IL‐8, IP10/CXCL10, LIF, MCP1, and M‐CSF) in the Control, Control + Ghrelin, D‐gal, and D‐gal + Ghrelin groups. *** p < 0.001.

Article Snippet: Following antibody stripping and re‐blocking, sections were incubated overnight at 4°C with a second primary antibody: γH2AX (1:400; ab81299, Abcam), high‐mobility group box 1 (HMGB1) (1:400; AWA10032 , Abiowell), p21 Cip1 (1:200; ab188224, Abcam), p16 Ink4a (1:200; bs‐23797R, Bioss, China), or Lamin B1 (1:400; ab8982, Abcam).

Techniques: Control, Western Blot, Analysis, Tissue, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: A biomimetic hybrid membrane vesicle nanoplatform attenuates tendinopathy through neuroinflammation modulation and tendon regeneration

doi: 10.1016/j.xcrm.2026.102937

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal anti-p16 , ABclonal , Cat# A11651; RRID: AB_2861619.

Techniques: Recombinant, Lysis, Hydroxyproline Assay, Enzyme-linked Immunosorbent Assay, Isolation, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Staining, Software

Journal: Cell Genomics

Article Title: Chromatin topology and distal elements underlie divergent cell-type-specific regulation of 9p21 locus cell cycle genes

doi: 10.1016/j.xgen.2026.101304

Figure Lengend Snippet: Tissue-specific expression of 9p21 transcripts (A) Left: approximate genomic organization of the 9p21 transcripts—p16 INK4A and p14 ARF ( CDKN2A ), p15 INK4B ( CDKN2B ), and the long noncoding RNA ANRIL ( CDKN2B-AS1 )—and expected results from various RNA-seq strategies. Right: reported functions of each protein encoded by the 9p21 cell cycle regulators. (B) Heatmap shows average transcript per million (TPM) per tissue site; RNA-seq data are from GTEx v.8 ( n ≥ 70 donors per tissue). Tissues are ordered left to right by a p14 ARF -to-p16 INK4A ratio. Rows 1–2: isoform-level expression estimates for p16 INK4A and p14 ARF . Rows 3–6: gene-level expression estimates of CDKN2A , p15 INK4B ( CDKN2B ), ANRIL ( CDKN2B-AS1 ), and MTAP . (C) Scatterplots show the correlation (Pearson R) between TPM values for p16 INK4A , p14 ARF , or the sum of the two, with CDKN2A from two GTEx tissues at the extremes of the` p14 ARF -to-p16 INK4A ratio in (B). Each dot is a single donor sample. (D and E) UpSet plots of tissues from GTEx (D) and FANTOM (E) expressing one or more of the 9p21 transcripts. Threshold is ≥1 TPM using the average across donors for a given tissue in each dataset. (F) Violin plots show log expression (natural logarithm of TPM +1) of 9p21 transcripts across GTEx tissues grouped by donor age for 4 representative tissues. Each dot is a single donor sample. The white dot represents the median value. Adipose - visceral (omentum): n [20–30] = 49, n (60–70] = 169. Esophagus-gastroesophageal junction: n [20–30] = 36, n (60–70] = 97. Thyroid: n [20–30] = 53, n (60–70] = 219. Whole blood: n [20–30] = 73, n (60–70] = 253. ∗∗∗Bonferroni adjusted p value (adj p ) < 0.001, ∗∗adj p < 0.01, and ∗adj p < 0.05.

Article Snippet: Briefly, pooled sample libraries were dried and mixed with Hybridization Mix (Twist Bioscience, 104178), Custom CDKN2A Panels (Twist Bioscience, 101001) and Universal Blockers (Twist Bioscience, 100578).

Techniques: Tissue, Expressing, RNA Sequencing

Journal: Cell Genomics

Article Title: Chromatin topology and distal elements underlie divergent cell-type-specific regulation of 9p21 locus cell cycle genes

doi: 10.1016/j.xgen.2026.101304

Figure Lengend Snippet: Cell-type-specific expression of 9p21 transcripts (A) Schematic of 9p21 cell cycle genes and their isoforms (GENCODE v.34). Major p14 ARF and p16 INK4A isoforms (CDKN2A-214 or ENST00000579755.1 and CDKN2A-201 or ENST00000304494.9, respectively) were used to design a custom gene transfer format (GTF) to annotate p16 INK4A and p14 ARF from 5′ single-cell gene expression profiles. A subset of isoforms of ANRIL and the protein-coding isoforms of CDKN2B are shown. (B) Heatmap shows percentages (%) of cells annotated in each cell type from human tissues expressing the 9p21 cell cycle genes p16 INK4A , p14 ARF , or p15 INK4B or ANRIL. 10× 5′ data from He et al. analyzed with the custom GTF from (A) (cutoff for expression: unique molecular identifier [UMI] > 0). Dotted-line boxes highlight (1) p15 INK4B and ANRIL expression in gut epithelial cells, (2) p14 ARF expression in immune cells, and (3) tissue fibroblasts expressing p16 INK4A , p14 ARF , and p15 INK4B . (C–E) Barplots show the percentage of each cell type expressing the 9p21 genes or ANRIL. 10× 5′ data analyzed with the custom GTF from (B). Expression cutoff UMI > 0. Error bars: standard error of the mean (SEM) between donor percentages. Number of donors are shown above the plot. (C) Adult gut tissues from Elmentaite et al. Top: all cell types; bottom: immune cells only. (D) Heart tissues from Koenig et al. Top: all cell types; bottom: immune cells only. (E) Immune cells. Top: 10× Genomics 5′ v.2 peripheral blood mononuclear cell (PBMC) example data from the manufacturer. Bottom: 10× 5′ data from CD4 + T cells from Hashimoto et al.

Article Snippet: Briefly, pooled sample libraries were dried and mixed with Hybridization Mix (Twist Bioscience, 104178), Custom CDKN2A Panels (Twist Bioscience, 101001) and Universal Blockers (Twist Bioscience, 100578).

Techniques: Expressing, Single Cell, Gene Expression, Tissue, Single Cell RNA-Seq

Journal: Cell Genomics

Article Title: Chromatin topology and distal elements underlie divergent cell-type-specific regulation of 9p21 locus cell cycle genes

doi: 10.1016/j.xgen.2026.101304

Figure Lengend Snippet: Enrichment of p16 INK4A and p14 ARF from 3′ single-cell libraries reveals cell-type-specific expression differences (A) Overview schematic of the protocol for targeted enrichment of p16 INK4A and p14 ARF (exon1α/exon1β) from scRNA-seq libraries; protocol was adapted from our previous work. CB, cell barcode; UMI, unique molecular identifier. (B) Uniform manifold approximation and projection (UMAP) shows 3′ 10× data from selected breast tissue samples from Gray et al. n = 10 donors from which we performed targeted exon1α/exon1β enrichment. Cluster annotations are as in Gray et al. . F1-3, fibroblasts; BAP1-3, basal/myoepithelial cells; AP, alveolar progenitor cells; BL, basal-luminal alveolar cells; HSa, hormone sensing alpha cells; HSb, hormone sensing beta cells; LE, lymphatic endothelial cells; M, myeloid cells; NK, NK cells; P, plasma cells; PE, pericytes/smooth muscle cells; T, T cells; and VE, vascular endothelial cells. (C) Barplot shows (bars 1–3): the percent (%) of cells from the samples in (B) expressing p16 INK4A (estimated from reads mapping to exon1α), p14 ARF (estimated from reads mapping to exon1β), or CDKN2A from the published data without enrichment, as well as (bars 4 and 5) p16 INK4A and p14 ARF after exon1α/exon1β enrichment using the protocol from (A). Error bars are standard error between the mean percentage of cells per sample/donor, SEM. (D) Dot plot shows cell types from (B) ordered left to right by p14 ARF to p16 INK4A ratio. p16 INK4A and p14 ARF expression determined by the exon1α/exon1β enrichment protocol from (A), normalized by the total number of counts per cell. CDKN2A , MTAP , ANRIL , and p15 INK4B ( CDKN2B ) expression from Gray et al.

Article Snippet: Briefly, pooled sample libraries were dried and mixed with Hybridization Mix (Twist Bioscience, 104178), Custom CDKN2A Panels (Twist Bioscience, 101001) and Universal Blockers (Twist Bioscience, 100578).

Techniques: Single Cell, Expressing, Tissue, Clinical Proteomics, cDNA Library Assay, Single Cell RNA-Seq, RNA Sequencing, Polymerase Chain Reaction, Reverse Transcription

Journal: Cell Genomics

Article Title: Chromatin topology and distal elements underlie divergent cell-type-specific regulation of 9p21 locus cell cycle genes

doi: 10.1016/j.xgen.2026.101304

Figure Lengend Snippet: A switch from p14 ARF to p16 INK4A and p15 INK4B across fibroblast replicative age (A) Experimental design for the WI-38 fibroblast replicative senescence (RS) experiment from Chan et al. (B) UMAP projection shows WI-38 cells from the RS time course from Chan et al. Increasing population doubling levels (PDLs) are colored from early (red) to late (blue) time points as in (A). (C) The UMAP projection from (B) colored by Senmayo score. (D) The same UMAP projection from (B), colored by expression of each of the 9p21 cell cycle genes and ANRIL . CDKN2A , p15 INK4B , and ANRIL expression determined from the scRNA-seq data from Chan et al. p16 INK4A and p14 ARF expression determined by exon1α/exon1β enrichment protocol, normalised as in D. Maximum value capped at 99th percentile. (E) Barplot shows the ratio of p14 ARF to p16 INK4A , as determined by the exon1α/exon1β enrichment protocol, across the RS time course. (F) Enriched ontologies among the top 200 genes most correlated (by PearsonR) with p14 ARF , p16 INK4A , or p15 INK4B from EnrichR. (G) Heatmap shows expression of marker genes and expression signatures from cells in the main cluster in (B)–(D). Cells arranged on pseudotime from cycling to senescent states as in Chan et al. (H) SenMayo, SASP, and scores from genes most correlated with p14 ARF , p16 INK4A (from targeted enrichment), or p15 INK4B (from original expression profiles) in cells as in (G).

Article Snippet: Briefly, pooled sample libraries were dried and mixed with Hybridization Mix (Twist Bioscience, 104178), Custom CDKN2A Panels (Twist Bioscience, 101001) and Universal Blockers (Twist Bioscience, 100578).

Techniques: Expressing, Marker, Single Cell RNA-Seq, ATAC-seq

Journal: Cell Genomics

Article Title: Chromatin topology and distal elements underlie divergent cell-type-specific regulation of 9p21 locus cell cycle genes

doi: 10.1016/j.xgen.2026.101304

Figure Lengend Snippet: Genomic and epigenomic landscape of 9p21 (A) Region Capture Micro-C (RCMC) data of chr9:20,300,000–22,600,000 from WI-38 fibroblasts. Data were combined across all PDLs . Contacts are shown at a 5 kb resolution. (B) RCMC data of chr9:21,750,000–22,500,000, zoomed in from (A). Contacts are shown at a 1 kb resolution. The remaining tracks from top to bottom: (1) eQTLs for CDKN2A , CDKN2B , ANRIL , and MTAP from selected GTEx v.10 tissues. Dot size represents posterior inclusion probability (PIP); (2 and 3) CTCF ChIP-seq, (4 and 5) H3K27ac ChIP-seq, and (6 and 7) ATAC-seq from WI-38 across replicative age (tracks are counts per million [CPM] normalized) (ATAC-seq data from Chan et al., PDL20 and PDL50 WI-38, H3K27ac, and CTCF ChIP-seq were generated for this study from PDL33 and PDL54 WI-38); (8) candidate cis -regulatory elements (cCREs) from ENCODE v.3 ; (9) regions selected for CRISPRa screening; and (10) gene track, modified by annotating the CDKN2A isoform ENST00000304494.9 as p16 INK4A and the ENST00000579755.1 isoform as p14 ARF . All tracks were visualized in IGV. , Putative TAD boundary insulator sites marked by strong CTCF ChIP-seq peaks are annotated in red boxes. (C) Summary of the regions selected for CRISPRa screening. TSS, transcription start site.

Article Snippet: Briefly, pooled sample libraries were dried and mixed with Hybridization Mix (Twist Bioscience, 104178), Custom CDKN2A Panels (Twist Bioscience, 101001) and Universal Blockers (Twist Bioscience, 100578).

Techniques: ChIP-sequencing, ATAC-seq, Generated, Screening, Modification, Hi-C, CRISPR

Journal: Cell Genomics

Article Title: Chromatin topology and distal elements underlie divergent cell-type-specific regulation of 9p21 locus cell cycle genes

doi: 10.1016/j.xgen.2026.101304

Figure Lengend Snippet: CRISPRa functionally links 9p21 CREs to target genes (A) Schematic of the CRISPRa screening workflow. (B) CRISPRa screen results overview, contextualized with CTCF and H3K27ac tracks. From top to bottom: (1) CTCF ChIP-seq track and (2) H3K27ac ChIP-seq, both from WI-38 at PDL54 (as in B) (CPM normalized). (3–6) CRISPRa screen results from SCEPTRE: each track represents the −log 10 ( p ) for each gRNA-gene pair with a significance of p < 0.05, plotted at the gRNA binding site. Results for each target gene are shown as separate tracks. Expression values for CDKN2B and ANRIL are derived from scRNA-seq libraries and for p16 INK4A and p14 ARF from the exon1α/exon1β enrichment protocol. (7) Regions targeted by CRISPRa. (8) gRNAs validated from arrayed single gRNA transfection experiments and RT-qPCR. (9) Gene track, modified by annotating the CDKN2A isoform ENST00000304494.9 as p16 INK4A and ENST00000579755.1 as p14 ARF . All tracks were visualized with IGV. , (C) Zoomed-in views on the gRNAs that were taken forward into validation experiments, corresponding to 3 promoter regions and 9 distal CREs (E1–E9). Each section represents a 2–4 kb zoomed-in region of the data in (B). (D) RT-qPCR results for validation experiments. Each dot represents the average of 4 replicates: two separate time-series experiments, with cells collected on days 4 and 8 post-transfection. Data were normalized relative to expression of GAPDH and non-targeting control gRNAs. Color of the dot: average fold change. Size of the dot: combined Z score measuring the strength and significance of the effect of the guide on each transcript. (E) Dot plot shows ChromBPNet sequence attribution scores and −log 10 ( q ) of motif annotation strength for selected candidate TF family binding site (TFBS) within the promoter and CRE regions from the ChromBPNet PDL50 model. Top 4 rows: transcript(s) regulated by each CRE. (F) Zoomed-in views of E1 and E9. From top to bottom: CTCF and H3K27ac tracks from PDL54, ChromBPNet attribution score from model trained on PDL50 ATAC-seq, FOS ChIP-seq data from IMR-90 (ENCODE: ENCSR124AIG and ENCFF906BPJ), common SNPs (dbSNP155 common SNP track), and PhastCons100 conservation score.

Article Snippet: Briefly, pooled sample libraries were dried and mixed with Hybridization Mix (Twist Bioscience, 104178), Custom CDKN2A Panels (Twist Bioscience, 101001) and Universal Blockers (Twist Bioscience, 100578).

Techniques: Screening, ChIP-sequencing, Binding Assay, Expressing, Derivative Assay, Transfection, Quantitative RT-PCR, Modification, Biomarker Discovery, Control, Sequencing, ATAC-seq, Chromatin Immunoprecipitation, CRISPR, Single Cell RNA-Seq

Journal: Cell Genomics

Article Title: Chromatin topology and distal elements underlie divergent cell-type-specific regulation of 9p21 locus cell cycle genes

doi: 10.1016/j.xgen.2026.101304

Figure Lengend Snippet: Characterization of 9p21 distal CREs (A) 3D genome interactions across 9p21 chr9:21,800,000–22,225,000. From top to bottom: (1) loop plot representing regions of high interactions, identified from RCMC data combined from all WI-38 time points in our RS time course. (2) Arrows represent the presence and orientation of CTCF motifs at the major CTCF peaks. (3–6) ChIP-seq for CTCF and H3K27ac from WI-38 PDL33 or PDL54; tracks are CPM normalized. (7–10) Virtual 4C visualizations of RCMC data from four viewpoints (E1 or p16 INK4A , p14 ARF /ANRIL, and p15 INK4B promoters) at 500 bp resolution. Anchor regions are indicated by the anchor symbol. (11) Track representing the location of validated CREs and promoter regions. (12) Gene track, modified by annotating the CDKN2A isoform ENST00000304494.9 as p16 INK4A and the ENST00000579755.1 isoform as p14 ARF . The orange-highlighted box marks a CTCF-bound region in MTAP that has strong interactions with E1 (gray-highlighted box). Blue highlight boxes mark the p16 INK4A , p14 ARF / ANRIL , and p15 INK4B promoter regions. (B) Change in 3D genome interactions with replicative age. RCMC data are shown at a 1 kb resolution as a subtraction plot, comparing PDL48 and PDL21 WI-38 fibroblasts. Each RCMC dataset was normalized to total reads in chr9. (C) Fine-mapped GWAS variants (UKBB) for phenotypes associated with common diseases or the diseases hemoglobin A1c, type 2 diabetes, apolipoprotein B, coronary artery disease, pulse pressure, lymphocyte count, albumin/globulin ratio, glaucoma, and IGF1 (insulin-like growth factor 1). Bottom: track representing the location of lead germline SNPs from cancer GWASs, grouped in two sets: one including breast and colorectal cancer, glioma, and leukemia and the other including lung cancer and melanoma. Dot size represents −log10( p ). eQTLs are the same as those in . Dot size represents posterior inclusion probability (PIP). Linkage disequilibrium (LD) is from UKBB ; LD block annotation was determined using R 2 > 0.5. (D) Heatmap shows the 9p21 WI-38 CREs and promoters annotated in this study as open (blue) or closed (gray) in published single-nucleus ATAC-seq data from human tissues that overlap with a validated 9p21 CRE or promoter region and are labeled by the region they overlap with. There are no regions that overlap E2, but there are 2 regions overlapping each of the promoters and E6, shown as separate rows. Transcripts regulated by each CRE are annotated to the right. Bottom 2 annotation rows represent cell-type groups and the organ where each cell type is mostly identified.

Article Snippet: Briefly, pooled sample libraries were dried and mixed with Hybridization Mix (Twist Bioscience, 104178), Custom CDKN2A Panels (Twist Bioscience, 101001) and Universal Blockers (Twist Bioscience, 100578).

Techniques: ChIP-sequencing, Modification, Blocking Assay, ATAC-seq, Labeling, GWAS

Journal: Cell Genomics

Article Title: Chromatin topology and distal elements underlie divergent cell-type-specific regulation of 9p21 locus cell cycle genes

doi: 10.1016/j.xgen.2026.101304

Figure Lengend Snippet:

Article Snippet: Briefly, pooled sample libraries were dried and mixed with Hybridization Mix (Twist Bioscience, 104178), Custom CDKN2A Panels (Twist Bioscience, 101001) and Universal Blockers (Twist Bioscience, 100578).

Techniques: Generated

Journal: Cell Genomics

Article Title: Chromatin topology and distal elements underlie divergent cell-type-specific regulation of 9p21 locus cell cycle genes

doi: 10.1016/j.xgen.2026.101304

Figure Lengend Snippet:

Article Snippet: Briefly, pooled sample libraries were dried and mixed with Hybridization Mix (Twist Bioscience, 104178), Custom CDKN2A Panels (Twist Bioscience, 101001) and Universal Blockers (Twist Bioscience, 100578).

Techniques: Single Cell RNA-Seq