Journal: Nature Communications

Article Title: Resistance to anti-LAG-3 plus anti-PD-1 therapy in head and neck cancer is mediated by Sox9+ tumor cells interaction with Fpr1+ neutrophils

doi: 10.1038/s41467-025-59050-4

Figure Lengend Snippet: A The UMAP plot shows five epithelial subtypes in distinct colors, with bar graphs indicating their quantities. B The UMAP plot (left) shows the distributions of control, resistant, and sensitive groups, with cell proportions (center) and subtype compositions (right). C Heatmap of top 10 signature genes for three groups. D CellChat circle plot shows differences in intercellular communication number and strength between two groups. E The UMAP plot shows 11 immune cell subtypes, with bar graphs indicating their numbers. F The UMAP plot shows 11 immune subtypes across two groups. G CellChat analysis compares epithelial-immune information flow in Resi and Sens groups, with relative (left) and absolute (right) flow shown. H Scatter plot of ANNEXIN pathway signaling in cell clusters from CellChat analysis. Bubble plots from CellChat ( I ) and CellPhoneDB (J) show epithelial subtype communication with Neu1 receptors and ligands. P values were calculated using a permutation test, assessing the significance of cell-cell communication by comparing observed mean expression with a null distribution generated by random permutations ( I ). P values were calculated using a permutation test, which generates a null distribution by randomly shuffling cell labels to determine the specificity of interactions ( J ). P values are one-sided and exact. K Violin plot (left) and FeaturePlot (right) show Anxa1 expression in epithelial cells across groups. The box plot elements are defined as described in the Statistics and Reproducibility section. Each group includes cells from 3 mice. P values were calculated one-way ANOVA. L Violin plot showing Anxa1 expression across epithelial cell subtypes. The box plot elements are defined as described in the Statistics and Reproducibility section. Each group includes cells from 9 mice. P values were calculated one-way ANOVA. M Representative IHC staining and score of Anxa1 in HNSCC. n = 6 control, n = 6 resistant, n = 8 sensitive mice. Scale bar = 65 μm. Data shown as as mean ± SD. P values were calculated one-way ANOVA with Tukey’s multiple comparisons test. Source data and exact p values are provided as a file.

Article Snippet: Following a previously published method, HL60 cells were incubated with 500 nM ANXA1 (MCE, HY-P7512) for 6 h, or sorted Fpr1+ neutrophils were incubated with 500 nM Anxa1 (MCE, HY-P72078) for 6 h . Following the manufacturer’s instructions, Fpr1+ neutrophils treated with or without Anxa1 were stained with MitoTracker Red (Thermo Fisher, A66443) and Hoechst 33342 (Beyotime, R0305S-6) under light-protected conditions at 37 °C for 20 min. After staining, images were captured using a super-resolution microscope (Nikon, Tokyo, Japan) in SIM mode.

Techniques: Control, Expressing, Generated, Immunohistochemistry

Journal: Nature Communications

Article Title: Resistance to anti-LAG-3 plus anti-PD-1 therapy in head and neck cancer is mediated by Sox9+ tumor cells interaction with Fpr1+ neutrophils

doi: 10.1038/s41467-025-59050-4

Figure Lengend Snippet: A The experimental design of the HNSCC tumorigenesis model and treatment strategy in the Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. B Representative image of tongue visible lesions in Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. n = 6 mice in each group. Scale bar, 2 mm. C Quantification of HNSCC lesion number and lesion area (mm 3 ) in Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. D Representative H&E staining of HNSCC and Quantification of HNSCC invasion grades in Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. Scale bar, 65 μm. n = 6 mice in each group. Statistical significance was assessed using the Pearson chi-square test. P value is exact and two-sided. Representative IHC staining and IHC Score of Ki67 ( E ), Anxa1 ( F ), and Fpr1 ( G ) in HNSCC of Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. The scale bar is 65 μm. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. Representative immunofluorescence staining of Gzma+γδT ( H ) or Gzma+Cd8 T ( I ) cells in mouse HNSCC tissues from Resi; Anxa1 cKO-Con and Resi; Anxa1 cKO groups. Epithelial tissues were stained with anti-Gzma antibody (green) and anti-Tcr g/d ( H ) or anti-Cd8a ( I ) antibody (red). Cell nuclei were stained with DAPI (blue). Scale bar, 30 μm. On the right, the corresponding percentages of Gzma+γδT ( H ) or Gzma+Cd8 T ( I ) cells in terms of area and number are provided. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. Source data and exact p values are provided as a file.

Article Snippet: Following a previously published method, HL60 cells were incubated with 500 nM ANXA1 (MCE, HY-P7512) for 6 h, or sorted Fpr1+ neutrophils were incubated with 500 nM Anxa1 (MCE, HY-P72078) for 6 h . Following the manufacturer’s instructions, Fpr1+ neutrophils treated with or without Anxa1 were stained with MitoTracker Red (Thermo Fisher, A66443) and Hoechst 33342 (Beyotime, R0305S-6) under light-protected conditions at 37 °C for 20 min. After staining, images were captured using a super-resolution microscope (Nikon, Tokyo, Japan) in SIM mode.

Techniques: Two Tailed Test, Staining, Immunohistochemistry, Immunofluorescence

Journal: Nature Communications

Article Title: Resistance to anti-LAG-3 plus anti-PD-1 therapy in head and neck cancer is mediated by Sox9+ tumor cells interaction with Fpr1+ neutrophils

doi: 10.1038/s41467-025-59050-4

Figure Lengend Snippet: A Venn diagram of genes identified by FindMarkers and SCENIC analyses. B Violin plot (left) shows Sox9 expression across groups, and FeaturePlot (right) depicts Sox9 expression in epithelial cells. The box plot elements are defined as described in the Statistics and Reproducibility section. Each group includes cells from 3 mice. P values were calculated one-way ANOVA. C Violin plot showing Sox9 expression across epithelial cell subtypes. The box plot elements are defined as described in the Statistics and Reproducibility section. Each group includes cells from 9 mice. P values were calculated one-way ANOVA. D Representative IHC staining and score of Sox9 in HNSCC. n = 6 control, n = 6 resistant, n = 8 sensitive mice. Scale bar = 65 μm. Data shown as mean ± SD. P values were calculated one-way ANOVA with Tukey’s multiple comparisons test. E ChIP of genomic DNA from resistant mouse HNSCC tissues was performed with a Sox9-specific antibody. Left: gel image, right: qPCR analysis of Anxa1 promoter ChIP signal relative to IgG group. Two primer pairs were used. The experiment was independently repeated three times. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. F Luciferase reporter assay assessed Anxa1 promoter activity in Moc1 cells. A schematic diagram shows cloning the Anxa1 promoter into the pZX001 vector to generate pGL3 luciferase plasmid. Binding motif information was retrieved from footprintDB database. G The relative activity of the Anxa1 promoter was detected by luciferase assay across the three groups. The experiment was independently repeated three times. Data shown as mean ± SD. P values were calculated one-way ANOVA with Tukey’s multiple comparisons test. H Representative immunofluorescence colocalization of Anxa1 (red) and Sox9 (green) in Resi mouse HNSCC tissues. Nuclei counterstained with DAPI (blue). Scale bar = 20 μm. Source data and exact p values are provided as a file.

Article Snippet: Following a previously published method, HL60 cells were incubated with 500 nM ANXA1 (MCE, HY-P7512) for 6 h, or sorted Fpr1+ neutrophils were incubated with 500 nM Anxa1 (MCE, HY-P72078) for 6 h . Following the manufacturer’s instructions, Fpr1+ neutrophils treated with or without Anxa1 were stained with MitoTracker Red (Thermo Fisher, A66443) and Hoechst 33342 (Beyotime, R0305S-6) under light-protected conditions at 37 °C for 20 min. After staining, images were captured using a super-resolution microscope (Nikon, Tokyo, Japan) in SIM mode.

Techniques: Expressing, Immunohistochemistry, Control, Two Tailed Test, Luciferase, Reporter Assay, Activity Assay, Cloning, Plasmid Preparation, Binding Assay, Immunofluorescence

Journal: Nature Communications

Article Title: Resistance to anti-LAG-3 plus anti-PD-1 therapy in head and neck cancer is mediated by Sox9+ tumor cells interaction with Fpr1+ neutrophils

doi: 10.1038/s41467-025-59050-4

Figure Lengend Snippet: A The experimental design of the HNSCC tumorigenesis model and treatment strategy in the Resi; DTR+ and Resi; DTR− groups. B Representative image of tongue visible lesions in Resi; DTR+ and Resi; DTR− groups. n = 6 mice in each group. Scale bar, 2 mm. C Quantification of HNSCC lesion number and lesion area (mm 3 ) in Resi; DTR+ and Resi; DTR− groups. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. D Representative H&E staining of HNSCC and Quantification of HNSCC invasion grades in Resi; DTR+ and Resi; DTR− groups. Scale bar, 65 μm. n = 6 mice in each group. Statistical significance was assessed using the Pearson chi-square test. P value is exact and two-sided. Representative IHC staining and IHC Score of Ki67 ( E ), Sox9 ( F ), Anxa1 ( G ), and Fpr1 ( H ) in HNSCC of Resi; DTR+ and Resi; DTR− groups. The scale bar is 65 μm. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. Representative immunofluorescence staining of Gzma+γδT ( I ) or Gzma+Cd8 T ( J ) cells in mouse HNSCC tissues from Resi; DTR+ and Resi; DTR− groups. Epithelial tissues were stained with anti-Gzma antibody (green) and anti-Tcr g/d ( I ) or anti-Cd8a ( J ) antibody (red). Cell nuclei were stained with DAPI (blue). Scale bar, 30 μm. On the right, the corresponding percentages of Gzma+γδT ( I ) or Gzma+Cd8 T ( J ) cells in terms of area and number are provided. n = 6 mice in each group. Data shown as mean ± SD. P values are presented by two-tailed unpaired Student’s t test. Source data and exact p values are provided as a file.

Article Snippet: Following a previously published method, HL60 cells were incubated with 500 nM ANXA1 (MCE, HY-P7512) for 6 h, or sorted Fpr1+ neutrophils were incubated with 500 nM Anxa1 (MCE, HY-P72078) for 6 h . Following the manufacturer’s instructions, Fpr1+ neutrophils treated with or without Anxa1 were stained with MitoTracker Red (Thermo Fisher, A66443) and Hoechst 33342 (Beyotime, R0305S-6) under light-protected conditions at 37 °C for 20 min. After staining, images were captured using a super-resolution microscope (Nikon, Tokyo, Japan) in SIM mode.

Techniques: Two Tailed Test, Staining, Immunohistochemistry, Immunofluorescence

Journal: Journal of Proteome Research

Article Title: Effect of Tamoxifen on Proteome Expression during In Vitro Myogenesis in Murine Skeletal Muscle C 2 C 12 Cells

doi: 10.1021/acs.jproteome.3c00340

Figure Lengend Snippet: Dilutions of Primary Antibody

Article Snippet: annexin A1 , Monoclonal IgG1 (mouse) , ProteinTech, 66344-1 , 1:2500.

Techniques:

Journal: Journal of Natural Products

Article Title: Resin Glycosides from Ipomoea funis as Inhibitors of P‑Glycoprotein in Multidrug-Resistant Breast Carcinoma Cells

doi: 10.1021/acs.jnatprod.5c01273

Figure Lengend Snippet: Effect of intrapilosin I ( 2 ) on apoptosis: (A) flow cytometry analysis with double staining using annexin V/PE and 7AAD in MCF-7 and MCF-7/Vin cells, bars represent the percentage of early and late apoptosis in the different cell phenotypes, data are presented as mean ± SD from three independent experiments. *** p < 0.01; (B) Western blot analysis of apoptosis-associated proteins (caspase-3) in MCF-7 and MCF-7/Vin cells after the indicated treatments for 48 h. Abbreviations: N, necrosis; LA, late apoptosis; EA, early apoptosis; V, viable.

Article Snippet: The effect of compound 2 on cell death was evaluated using a double staining assay with the PE Annexin V kit (130–119–353, Miltenyi Biotec) and 7AAD (A9400, Sigma-Aldrich).

Techniques: Flow Cytometry, Double Staining, Western Blot

Journal: BioImpacts

Article Title: Annexin A1, calreticulin and high mobility group box 1 are elevated in secondary progressive multiple sclerosis: Does immunogenic cell death occur in multiple sclerosis?

doi: 10.34172/bi.30264

Figure Lengend Snippet: Fig. 1. ANXA1 level in CSF sample of control group and SPMS patient. Bar graph related to ANXA1 measurement using standard ELISA assay that represents means ± SD measured in each group. A comparison between the results from control group and SPMS patient was done using a t-test. ELISA experiments were repeated at least three times. ** indicates: P < 0.01.

Article Snippet: Human ANXA1 commercial ELISA Kit (Elabscience; E-EL-H5512) was used to determine the level of ANXA1.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 3. Hydrodynamic stress (HDS) induces upregulation of Annexin 1 (ANXA1) in cancer cell clusters. (A) Gene ontology (GO) analysis of gene-expression microarray data for C45 CTOS treated with single-set pulse HDS. At 6 h after HDS, 123 genes were upregulated by more than 1.5-fold; these genes were analyzed for GO enrichment. Orange bars: the number of genes included in each GO term. Blue bars: the log10(1/P- value) for each GO term. (B) Venn diagram shows overlap of 9 genes that were upregulated, both by mild HDS (single-set pulse HDS), which did not disrupt cell membranes, and by strong HDS, which caused architectural disruptions13. (C) Relative expression of Annexin 1 (ANXA1) mRNA before (pre) and 6 h after single-set pulse HDS, in multiple CTOS lines. Data are the average ± SD; N = 3 for each run. *P < 0.05. (D) Relative expression of ANXA1 mRNA, before and 6 h after SLO treatment in multiple CTOS lines. Data are the average ± SD; N = 3 for each run. *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Gene Expression, Microarray, Expressing

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 4. Knock down of Annexin 1 (ANXA1) suppresses growth stimulated by hydrodynamic stress (HDS). (A) Semi-quantitative PCR analysis confirms the knockdown of ANXA1 gene expression. Relative ANXA1 expression levels are shown in C45 CTOS that were either transduced with an ANXA1 shRNA construct (sh#1 or sh#2) or a non-targeting (NT) control construct. ACTB: β-actin, the internal control gene. Data are the average ± SD. *P < 0.05. (B) Immunoblots show proteins from C45 CTOS that were not transduced (NT) or transduced with 2 different ANXA1 shRNAs (sh#1 and sh#2). Proteins were extracted at 1, 3, and 7 days (d1, d3, and d7, respectively) after subculturing. (C) Relative growth (ATP assay) of C45 CTOS transduced with control (NT) or ANXA1 shRNAs (sh#1 and sh#2) and cultured for 7 days after single-set pulse HDS. Data are the average ± SD, normalized to control (ctrl) values; N = 20 for each condition. *P < 0.05. (D,E) Relative growth (ATP assay) of C45 CTOS treated with control (NT) or ANXA1 shRNAs (sh#1 and sh#2), and cultured for 14 days with (D) multiple-set pulse HDS or with (E) continuous HDS. Data are the average ± SD, normalized to ctrl; N = 9 in (D) and N = 6 in (E) for each condition. *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Transduction, shRNA, Construct, Control, Western Blot, Subculturing Assay, ATP Assay, Cell Culture

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 5. Forced overexpression of Annexin 1 (ANXA1) potentiates hydrodynamic stress (HDS)-induced growth stimulation in colorectal cancer cell clusters. (A) Semi-quantitative PCR analysis confirms ANXA1 expression levels in C132 CTOS, transduced with either the ANXA1 construct (overexpression; OE) or the yellow fluorescent protein (YFP) control construct. Data are the average ± SD. *P < 0.05. (B) Immunoblots show ANXA1 expression in C132 CTOS transduced with ANXA1 (OE) or YFP (ctrl). The transduced ANXA1 had a higher molecular weight, because it was tagged with 3x FLAG. (C) Relative growth (ATP assay) of C132 CTOS with ANXA1 OE and YFP as control. CTOSs were cultured for 14 days with multiple-set pulse HDS (mHDS) or without HDS (ctrl). Data are the average ± SD, normalized to YFP without HDS; N = 8 for each condition. *P < 0.05. (D) Relative growth (ATP assay) of C132 CTOS with ANXA1 OE and YFP as control. CTOSs were cultured for 7 days with continuous HDS (cHDS) or without HDS (ctrl). Data are the average ± SD, normalized to control; N = 6 for each condition *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Expressing, Transduction, Construct, Control, Western Blot, Molecular Weight, ATP Assay, Cell Culture

Journal: Scientific reports

Article Title: Hydrodynamic stress stimulates growth of cell clusters via the ANXA1/PI3K/AKT axis in colorectal cancer.

doi: 10.1038/s41598-019-56739-7

Figure Lengend Snippet: Figure 6. Hydrodynamic stress (HDS) stimulates growth by activating the PI3K/AKT pathway downstream of Annexin 1 (ANXA1). (A) Immunoblot shows changes in the expression of proteins from the C45 line of cancer tissue-originated spheroids (CTOSs), treated with single-set pulse HDS. Proteins were identified by the indicated antibodies over the 6 h following HDS treatment. pAKT: AKT phosphorylated (at the indicated residue); tAKT: total AKT protein; ACTB: β-actin, the internal control gene. (B) Densitometry analysis of immunoblots shows the level of phosphorylated AKT (at S473), relative to the total AKT at each time point. The C45 CTOS line was treated with control (NT) or ANXA1 shRNAs (sh#1 or sh#2), then single-set pulse HDS were applied. Protein samples were extracted at the indicated time points following HDS treatment. Data show the quantification of a single representative result, selected from 3 independent experiments (see Supplementary Fig. S10 for raw immunoblots). (C) Relative growth (ATP assay) of C45 CTOS, treated with 2.5 μM GDC-0941 (GDC), and cultured for 14 days with multiple-set pulse HDS (mHDS). Data are the average ± SD, normalized to the DMSO control (crtl); N = 8 for each condition. *P < 0.05. (D) Relative growth (ATP assay) of C45 CTOS, treated with 1 μM GDC-0941 (GDC), and cultured for 14 days with continuous HDS (cHDS). Data are the average ± SD, normalized to the DMSO control; N = 6 for each condition. *P < 0.05.

Article Snippet: For making short hairpin (sh) RNAs targeting ANXA1, we transferred the target sequences of the ANXA1 human shRNA constructs carried in pRS plasmids (TR314775, OriGene, Rockville, MD) to the pPB vector.

Techniques: Western Blot, Expressing, Residue, Control, ATP Assay, Cell Culture

Journal: PLoS Genetics

Article Title: Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury

doi: 10.1371/journal.pgen.1007070

Figure Lengend Snippet: The sarcolemmal wounding assay was performed on flexor digitorus brevis muscles from WT muscle in the presence or absence of recombinant osteopontin (rOPN; 10μl at 1μg/μl). A) The extent of sarcolemmal damage, monitored as FM4-64 area, was greater in rOPN-treated myofibers compared to vehicle-treated control, both over time and at end-point (arrows). B) Repair cap formation, monitored by GFP-tagged annexin A1 (GFP-ANXA1) was delayed in rOPN-treated myofibers (arrowheads). Repair cap diameter at end-point was smaller in treated compared to control myofibers (arrowheads). Over time image series represents stacked consecutive images of the injured site at 10° orientation to reveal the extent of dye accumulation or repair cap formation . FM4-64 and ANXA1-GFP pictures were acquired simultaneously. Marked line plots, avg±sem; box plots, Tukey distribution; n = 50 myofibers (5 mice)/group; #, P<0.05 vs vehicle, 2way ANOVA + Bonferroni; *, P<0.05 vs vehicle, unpaired t-test with Welch’s correction.

Article Snippet: The plasmid encoding human annexin A1 with a carboxy-terminal GFP was obtained from Origene (Rockville, MD; Cat# RG201569).

Techniques: Muscles, Recombinant, Control

Journal: PLoS Genetics

Article Title: Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury

doi: 10.1371/journal.pgen.1007070

Figure Lengend Snippet: In addition to Ltbp4 , Anxa6 has also been shown to modify muscular dystrophy in mice [ , ]. The deleterious alleles of Ltbp4 (L4) or Anxa6 (A6) (severe) were compared to those from the protective 129 strain (mild) in the sarcolemma injury assay. A) Doubly homozygous A6 mild /L4 mild myofibers had the least injury while doubly homozygous A6 severe /L4 severe fibers had the greatest injury, marked by FM4-64. Myofibers with mixed homozygous genotypes were intermediate with respect to FM4-64 marked injury. B) A similar pattern was observed for Annexin A1 (ANXA1) repair caps, where doubly homozygous mild alleles of L4 and A6 assembled caps more rapidly and produced larger repair caps than doubly homozygous severe alleles. However, ANXA1 repair caps were smaller with the A6 homozygous severe allele, despite the presence of the mild L4 allele, suggesting that repair cap formation is dominated by the A6 genotype. FM4-64 and ANXA1-GFP images used for the analyses in A-B were acquired simultaneously. Marked line plots, avg±sem; box plots, Tukey distribution; n = 50 myofibers (5 mice)/group; marked line plots: #, P<0.05 vs control (A6 mild /L4 mild ), +, P<0.05 vs A6 severe /L4 mild and A6 mild /L4 severe groups, 2way ANOVA + Bonferroni; boxplots: *, P<0.05 vs control (A6 mild /L4 mild ), +, P<0.05 vs A6 severe /L4 mild and A6 mild /L4 severe groups, 1way ANOVA + Bonferroni.

Article Snippet: The plasmid encoding human annexin A1 with a carboxy-terminal GFP was obtained from Origene (Rockville, MD; Cat# RG201569).

Techniques: Produced, Control

Journal: bioRxiv

Article Title: Transplanting ANXA1⁻ CD8⁺ Naïve T cells Delay Aging Through Senolysis

doi: 10.64898/2026.02.21.707223

Figure Lengend Snippet: (A) Gene Set Enrichment Analysis (GSEA) comparing CD8⁺ naïve T cells from Elderly versus other groups of donors. Plots show significant enrichment of the Cellular Senescence (GO:0090398) and Senescence-Associated Secretory Phenotype (SASP; R-HSA-2559582) gene sets in the Elderly group. NES, normalized enrichment score. (B) Heatmap displaying the expression of senescence-associated signature genes in CD8⁺ naïve T cells across all donors. Each row is scaled using a Z-score. (C) Violin plot showing the normalized expression level of ANXA1 in CD8⁺ naïve T cells across the five age groups from scRNA-seq data. (D) Quantification of ANXA1 surface expression in CD8⁺ naïve T cells across the five age groups, as measured by flow cytometry. Each point represents an individual donor (Kid, n = 31; Young, n = 98; Middle-aged, n = 93; Pre-Elderly, n = 52; Elderly, n = 63). (E) Representative flowcytometry plots and summary quantification of total ANXA1 expression levels in CD8⁺ naïve T cells from human PBMCs (n = 5 per group). (F) Pathway enrichment analysis comparing human ANXA1⁺ versus ANXA1⁻ CD8⁺ naïve T cells from RNA-seq. The bubble plot shows key pathways significantly altered between the two subsets. Bubble size corresponds to the number of genes, and color indicates enrichment significance. (G) Representative immunofluorescence images for ANXA1 and p16 INK4a expression in human CD8⁺ naïve T cells. Scale bar, 10 µm. (H) Representative flow cytometry histogram and quantification p16 INK4a expression in human ANXA1⁺ versus ANXA1⁻ CD8⁺ naïve T cells (n = 5 per group). (I) Representative histogram of Cell Trace Violet (CTV) dye dilution in human ANXA1⁻ and ANXA1⁺ CD8⁺ naïve T cells after 72 hours of stimulation. Data are presented as mean ± SEM. P -value was determined by one-way ANOVA and two-tailed unpaired t-test.

Article Snippet: C57BL/6 CD45.2 mice, C57BL/6 CD45.1 mice, Anxa1 KO mice were purchased from Suzhou Cyagen Biotechnology Co., Ltd. affiliated to Cyagen US Inc.

Techniques: Expressing, Flow Cytometry, RNA Sequencing, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Transplanting ANXA1⁻ CD8⁺ Naïve T cells Delay Aging Through Senolysis

doi: 10.64898/2026.02.21.707223

Figure Lengend Snippet: (A) Representative flow cytometric analysis of CCL5 expression on CD8 + naïve T cells in samples of young (19-35 y) and old (>65 y) group (n= 5 per group). (B) Sub-clustering of CD8⁺ naïve T cells from the scRNA-seq dataset. UMAP projection showing distinct subclusters (C1-C6) identified within the CD8⁺ naïve T cell population. (C) Feature plot overlaying normalized ANXA1 expression onto the UMAP projection from ( B ), highlighting its specific enrichment in the C4 subcluster. (D) Scatter plots showing the correlation between the relative abundance of each subcluster (as a percentage of total CD8⁺ naïve T cells) and donor age. Lines represent linear regression fits; Pearson correlation coefficient (R) and P -values are indicated. ( E and F ) Violin plots ( E ) and heatmap ( F ) validating the naïve identity of both ANXA1 − and ANXA1⁺ CD8⁺ naïve T cells defined in human scRNA-seq data. Average expression of canonical lineage markers for naïve, memory, effector, and exhausted T cells is compared across three populations with CD8⁺ effector memory (Tem) cells as a non-naïve control. Expression values are scaled per row. (G) Representative immunofluorescence images and quantification of ANXA1 surface expression on mouse CD8⁺ naïve T cells from Young (8-week-old, n = 5) and Old (13-month-old, n = 5) mice. Scale bar, 5 µm. (H) Representative flow cytometry results and quantifications on the percentage of CD8 + naïve T cells and surface ANXA1 expression in young and old mice (n = 3 per group). Data in quantification plots are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA and two-tailed unpaired t-test.

Article Snippet: C57BL/6 CD45.2 mice, C57BL/6 CD45.1 mice, Anxa1 KO mice were purchased from Suzhou Cyagen Biotechnology Co., Ltd. affiliated to Cyagen US Inc.

Techniques: Expressing, Control, Immunofluorescence, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Transplanting ANXA1⁻ CD8⁺ Naïve T cells Delay Aging Through Senolysis

doi: 10.64898/2026.02.21.707223

Figure Lengend Snippet: (A) Heatmap of DEGs from RNA-seq of resting WT and Anxa1 KO CD8⁺ naïve T cells. (B) GO enrichment bubble plot of pathways upregulated in Anxa1 KO versus WT CD8⁺ naïve T cells at basal state.

Article Snippet: C57BL/6 CD45.2 mice, C57BL/6 CD45.1 mice, Anxa1 KO mice were purchased from Suzhou Cyagen Biotechnology Co., Ltd. affiliated to Cyagen US Inc.

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: Transplanting ANXA1⁻ CD8⁺ Naïve T cells Delay Aging Through Senolysis

doi: 10.64898/2026.02.21.707223

Figure Lengend Snippet: (A) Heatmap of DEGs in activated CD8⁺ naïve T cells from WT and Anxa1 KO mice. Cells were stimulated with anti-CD3/CD28 antibodies for 12 hours prior to RNA-seq. Each row is scaled using a Z-score. (B) GO enrichment of pathways upregulated in activated Anxa1 KO CD8⁺ naïve T cells compared with WT. ( C and D ) Representative flow cytometry histograms and quantification of activation markers CD69 ( C ) and CD25 ( D ) on WT and Anxa1 KO CD8⁺ naïve T cells 12 hours post-stimulation (n = 6 per group). (E) Representative flow cytometry plots and quantification of IFNγ-producing cells within WT and Anxa1 KO CD8⁺ T cells after stimulation (n = 6 per group). (F) Representative flow cytometry plots and quantification of the degranulation marker CD107a on WT and Anxa1 KO CD8⁺ T cells after stimulation (n = 6 per group). (G) Schematic of the B16-F10 inoculation model. C57BL/6 mice were inoculated subcutaneously with B16-F10 melanoma cells, followed by adoptive transfer of CD8⁺ T cells isolated from either WT or Anxa1 KO mice. (H) Representative image and quantification of excised tumors at day 20 post-inoculation. (n = 6 per group). Data in quantification plots are presented as mean ± SEM. P-values were determined by two-tailed unpaired t-test.

Article Snippet: C57BL/6 CD45.2 mice, C57BL/6 CD45.1 mice, Anxa1 KO mice were purchased from Suzhou Cyagen Biotechnology Co., Ltd. affiliated to Cyagen US Inc.

Techniques: RNA Sequencing, Flow Cytometry, Activation Assay, Marker, Adoptive Transfer Assay, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: Transplanting ANXA1⁻ CD8⁺ Naïve T cells Delay Aging Through Senolysis

doi: 10.64898/2026.02.21.707223

Figure Lengend Snippet: (A) Representative flow cytometry histograms and quantifications of CTV-staining, CD69 and CD25 expression of ANXA1 − CD8 + naïve T cells from young and aged mice (n = 3 per group). (B) Representative flow cytometry results and quantifications of Tc1 differentiation of ANXA1 − CD8 + naïve T cells from young and aged mice. (C) Representative images and quantification from a transwell invasion assay showing that both young and old mouse ANXA1⁻ T cells, but not ANXA1⁺ T cells (n = 5 per group), significantly inhibit the invasion of U-2 OS tumor cells, with arrows indicate tumor cells passed the transwell membrane. Data are presented as mean ± SEM. P -values were determined by one-way ANOVA and two-tailed unpaired t-test.

Article Snippet: C57BL/6 CD45.2 mice, C57BL/6 CD45.1 mice, Anxa1 KO mice were purchased from Suzhou Cyagen Biotechnology Co., Ltd. affiliated to Cyagen US Inc.

Techniques: Flow Cytometry, Staining, Expressing, Transwell Invasion Assay, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Transplanting ANXA1⁻ CD8⁺ Naïve T cells Delay Aging Through Senolysis

doi: 10.64898/2026.02.21.707223

Figure Lengend Snippet: (A) Schematic of the co-culture experiment using fibroblasts engineered to express p16 INK4a -GFP as a model of senescent cells. Human ANXA1 − or ANXA1 + CD8 + naïve T cells using FACS were co-cultured with fibroblasts for live imaging. (B) Representative stills from live-cell imaging showing the targeting and clearance of p16 INK4a -GFP⁺ senescent cells by ANXA1 − or ANXA1 + CD8 + naïve T cells 6 hours post-culture. (C) Representative immunofluorescence images and quantification of p53- and γH2AX- expressing fibroblasts after co-culture in UV-B induced senescence model (n = 6 per group), corresponding with . (D) Schematic of an Etoposide-induced senescence model. (E) Representative immunofluorescence images and quantification showing a significant reduction of p16 INK4a -positive senescent fibroblasts after co-culture with ANXA1⁻ T cells, but not with ANXA1⁺ T cells (n = 5 per group). Scale bar, 10 µm. (F) Representative immunofluorescence images and quantification of GZMB-secreting cells from different T cell groups after co-culture in (E) (n = 5 per group). Scale bar, 10 µm. Data are presented as mean ± SEM. P -values were determined by one-way ANOVA and two-tailed unpaired t-test.

Article Snippet: C57BL/6 CD45.2 mice, C57BL/6 CD45.1 mice, Anxa1 KO mice were purchased from Suzhou Cyagen Biotechnology Co., Ltd. affiliated to Cyagen US Inc.

Techniques: Co-Culture Assay, Cell Culture, Imaging, Live Cell Imaging, Immunofluorescence, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Transplanting ANXA1⁻ CD8⁺ Naïve T cells Delay Aging Through Senolysis

doi: 10.64898/2026.02.21.707223

Figure Lengend Snippet: (A) Representative stills from live-cell imaging of p16 INK4a -GFP⁺ senescent fibroblasts (green), normal fibroblasts (grey) co-cultured with sorted surface ANXA1⁻ CD8⁺ naïve T cells (magenta), with arrows indicate interaction spots. (B) Schematic diagram of the in vitro UV-B induced senescence co-culture system. Mouse fibroblasts (mFib) were induced into senescence by UV-B radiation and then co-cultured for 5 days with different subsets of sorted CD8⁺ naïve T cells. (C) Representative immunofluorescence images and quantification of p21-positive fibroblasts after co-culture with the indicated T cell subsets (n = 6 per group). Scale bar, 10 µm. (D) Western blot analysis of p16 INK4a protein levels in senescent fibroblasts following co-culture. (E) Schematic of the doxorubicin (Doxo)-induced systemic senescence model. Mice received a single intravenous ( i.v. ) injection of ANXA1⁻ CD8⁺ naïve T cells prior to treatment with Doxo. (F) Rotarod test quantification showing motor coordination and endurance in mice (n = 5 per group). (G) Weight loss of mice in different groups (n = 5 per group) in Doxo-induced senescence model. Initial body weight was normalized to 100%. (H) Representative images and quantifications of p16 INK4a and γH2AX in the heart and kidney from Doxo-treated mice (n = 5 per group). Scale bar, 5 µm. Data in quantification plots are presented as mean ± SEM. P-values were determined by one-way ANOVA and two-tailed unpaired t-test.

Article Snippet: C57BL/6 CD45.2 mice, C57BL/6 CD45.1 mice, Anxa1 KO mice were purchased from Suzhou Cyagen Biotechnology Co., Ltd. affiliated to Cyagen US Inc.

Techniques: Live Cell Imaging, Cell Culture, In Vitro, Co-Culture Assay, Immunofluorescence, Western Blot, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Transplanting ANXA1⁻ CD8⁺ Naïve T cells Delay Aging Through Senolysis

doi: 10.64898/2026.02.21.707223

Figure Lengend Snippet: (A) Schematic of the long-term therapeutic study design. Old (13-month-old) mice received monthly intravenous ( i.v. ) transfers of Anxa1⁻ CD8⁺ naïve T cells (Old-Treated group) or a vehicle control (Old-Ctrl group) for the duration of the study. (B) Kaplan-Meier survival curves for all mice with sexes combined. Statistical significance was determined by the Log-rank test. Median survival is indicated by dashed lines. (C) Representative photographs of mice at 30 months of age, highlighting the improved physical condition in the treated mouse. (D) Single-cell t-SNE projections of total CD45⁺ immune cells from the bone marrow of Old-Ctrl and Old-Treated mice (n = 2 per group). Left: Integrated UMAP colored by major cell lineages. Right: The same UMAP split by treatment group to visualize compositional shifts. (E) Box plots show the relative abundance of Inflammatory, Lymphoid, and Myeloid cell clusters as a percentage of total CD45⁺ cells between the two groups. (F) Violin plots comparing the senescence score, calculated per cell, between the Old-Ctrl and Old-Treated groups. The gene list for this score is provided in the Methods section. Statistical significance was determined by two-tailed unpaired t-test. (G) Density plot comparing the “Aging score” for all CD8⁺ T cells from the three experimental groups. The gene list for this score is provided in the Methods section. (H) GSEA enrichment analysis comparing Old-Treated versus Old-Ctrl CD8⁺ T cells. Bar plot shows NES for selected Hallmark gene sets. Purple bars indicate pathways enriched in Old-Ctrl, while orange bars indicate pathways enriched in Old-Treated cells.

Article Snippet: C57BL/6 CD45.2 mice, C57BL/6 CD45.1 mice, Anxa1 KO mice were purchased from Suzhou Cyagen Biotechnology Co., Ltd. affiliated to Cyagen US Inc.

Techniques: Control, Single Cell, Two Tailed Test

Journal: Scientific reports

Article Title: Annexins A2 and A5 are potential early biomarkers of hepatocarcinogenesis.

doi: 10.1038/s41598-023-34117-8

Figure Lengend Snippet: Figure 1. Expression of Anxa1, Anxa2, Anxa5, Anxa8, and Anxa10 genes during rat hepatocarcinogenesis. The relative mRNA levels of annexin genes in liver tissue were determined by RT-qPCR. Anxa1, Anxa2, Anxa5, Anxa8, and Anxa10 expression was normalized to ribosomal 18 s mRNA level. Bars show the relative expression (RE) compared to the NL group and represent the mean ± SD. p values were calculated by the ANOVA test. n = 5 animals/group. NL normal liver, W week. ***p < 0.001; **p < 0.01.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% skimmed milk and then incubated overnight at 4 °C with primary antibodies against ANXA1 (1:300), ANXA2 (1:500), ANXA5 (1:500), ANXA10 (1:500; NBP1-90,156; RRID: AB_11004664), and glutathione S-transferases P1, (GSTP1, 1:10,000; NBP2-16,756), from Novus Biologicals; CO, USA; ANXA8 (1:500; ab111693; RRID: AB_10858116), glyceraldehyde-3-phosphate dehydrogenase (GADPH, 1:10,000; ab181602; RRID: AB_2630358), and Lamin β1 (1:10,000; Ab16048; RRID: AB_443298), from Abcam; MA, USA; and prostaglandin reductase 1, (Ptgr1, 1:2600; GTX118527; RRID: AB_10618960) from GeneTex; CA, USA.

Techniques: Expressing, Quantitative RT-PCR

Journal: Scientific reports

Article Title: Annexins A2 and A5 are potential early biomarkers of hepatocarcinogenesis.

doi: 10.1038/s41598-023-34117-8

Figure Lengend Snippet: Figure 3. Increased annexins in cells of nodules during hepatocarcinogenesis. Representative images of ANXA1, ANXA2, and ANXA5 expression in NL tissue, preneoplastic lesions (W6 and W12), and HCC tumor cells (W18 and W22) were obtained by IF analysis. Annexin proteins were labeled with Alexa Fluor 488 (green), while nuclei were stained with DAPI (blue). Magnification: 400X. Scale bar = 50 μm. n = 5 animals/group. Images were captured with ZEISS Axio-A1 Microscopy.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% skimmed milk and then incubated overnight at 4 °C with primary antibodies against ANXA1 (1:300), ANXA2 (1:500), ANXA5 (1:500), ANXA10 (1:500; NBP1-90,156; RRID: AB_11004664), and glutathione S-transferases P1, (GSTP1, 1:10,000; NBP2-16,756), from Novus Biologicals; CO, USA; ANXA8 (1:500; ab111693; RRID: AB_10858116), glyceraldehyde-3-phosphate dehydrogenase (GADPH, 1:10,000; ab181602; RRID: AB_2630358), and Lamin β1 (1:10,000; Ab16048; RRID: AB_443298), from Abcam; MA, USA; and prostaglandin reductase 1, (Ptgr1, 1:2600; GTX118527; RRID: AB_10618960) from GeneTex; CA, USA.

Techniques: Expressing, Labeling, Staining, Microscopy

Journal: Scientific reports

Article Title: Annexins A2 and A5 are potential early biomarkers of hepatocarcinogenesis.

doi: 10.1038/s41598-023-34117-8

Figure Lengend Snippet: Figure 4. Subcellular expression levels of ANXA1, ANXA2, and ANXA5 proteins. The graphs show nuclear and cytosolic protein expression in liver samples from all experimental groups normalized to Lamin B1 and GAPDH, respectively. Bars show the relative levels (RL) compared to the NL group and represent the mean ± SD. p values were calculated by the ANOVA test. n = 3 animals/group. NL normal liver, W week. **p < 0.01; *p < 0.05. Images were captured with the Uvitec MINI HD6 photo-documentation system. Original blots of figure are presented in Supplementary Information S1.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% skimmed milk and then incubated overnight at 4 °C with primary antibodies against ANXA1 (1:300), ANXA2 (1:500), ANXA5 (1:500), ANXA10 (1:500; NBP1-90,156; RRID: AB_11004664), and glutathione S-transferases P1, (GSTP1, 1:10,000; NBP2-16,756), from Novus Biologicals; CO, USA; ANXA8 (1:500; ab111693; RRID: AB_10858116), glyceraldehyde-3-phosphate dehydrogenase (GADPH, 1:10,000; ab181602; RRID: AB_2630358), and Lamin β1 (1:10,000; Ab16048; RRID: AB_443298), from Abcam; MA, USA; and prostaglandin reductase 1, (Ptgr1, 1:2600; GTX118527; RRID: AB_10618960) from GeneTex; CA, USA.

Techniques: Expressing

Journal: Scientific reports

Article Title: Annexins A2 and A5 are potential early biomarkers of hepatocarcinogenesis.

doi: 10.1038/s41598-023-34117-8

Figure Lengend Snippet: Figure 6. Kaplan–Meier curves for overall survival (OS). Plots show the percent of 5-year overall survival associated with the expression levels of ANXA1 (a), ANXA2 (b), ANXA5 (c), AFP (d), PTGR1 (e), and GSTP1 (f) in HCC patients. Data were obtained from the liver hepatocellular carcinoma (LIHC) project, which comprises data from 364 HCC primary tumors, (https://portal.gdc.cancer.gov/projects/TCGA-LIHC).

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% skimmed milk and then incubated overnight at 4 °C with primary antibodies against ANXA1 (1:300), ANXA2 (1:500), ANXA5 (1:500), ANXA10 (1:500; NBP1-90,156; RRID: AB_11004664), and glutathione S-transferases P1, (GSTP1, 1:10,000; NBP2-16,756), from Novus Biologicals; CO, USA; ANXA8 (1:500; ab111693; RRID: AB_10858116), glyceraldehyde-3-phosphate dehydrogenase (GADPH, 1:10,000; ab181602; RRID: AB_2630358), and Lamin β1 (1:10,000; Ab16048; RRID: AB_443298), from Abcam; MA, USA; and prostaglandin reductase 1, (Ptgr1, 1:2600; GTX118527; RRID: AB_10618960) from GeneTex; CA, USA.

Techniques: Expressing

Journal: Scientific reports

Article Title: Annexins A2 and A5 are potential early biomarkers of hepatocarcinogenesis.

doi: 10.1038/s41598-023-34117-8

Figure Lengend Snippet: Figure 7. Graphic summary. The summary scheme shows or represents the elevation of ANXA1, ANXA2, and ANXA5 during experimental hepatocarcinogenesis and its association with survival. NL normal liver, W week. Created with BioRender.com.

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% skimmed milk and then incubated overnight at 4 °C with primary antibodies against ANXA1 (1:300), ANXA2 (1:500), ANXA5 (1:500), ANXA10 (1:500; NBP1-90,156; RRID: AB_11004664), and glutathione S-transferases P1, (GSTP1, 1:10,000; NBP2-16,756), from Novus Biologicals; CO, USA; ANXA8 (1:500; ab111693; RRID: AB_10858116), glyceraldehyde-3-phosphate dehydrogenase (GADPH, 1:10,000; ab181602; RRID: AB_2630358), and Lamin β1 (1:10,000; Ab16048; RRID: AB_443298), from Abcam; MA, USA; and prostaglandin reductase 1, (Ptgr1, 1:2600; GTX118527; RRID: AB_10618960) from GeneTex; CA, USA.

Techniques: