CDKN2A/p16INK4a Antibody - #AF5484

Fig. 6: Knockout of cdkn2a (p16) in Lyz2+ cells abrogates LSI-induced endplate sclerosis. a Representative immunofluorescent images of p16+ (red), F4/80+ (green) cells and DAPI (blue) staining of nuclei in mouse caudal endplates of L4/5 in Cdkn2aΔLyz2 or Cdkn2af/f mice at 8 weeks after LSI or sham surgery. Scale bars, 50 μm. b Representative immunofluorescent images of CD31 (green), Emcn (red) and DAPI (blue) staining of nuclei in the endplates of Cdkn2aΔLyz2 or Cdkn2af/f mice after LSI or sham surgery. Scale bars, 50 μm. c Permillage of CD31+Emcn+ area in the endplates of b. d Representative µCT images of the caudal endplates of L4/5 (coronal view) in Cdkn2aΔLyz2 or Cdkn2af/f mice at 8 weeks after LSI or sham surgery. Scale bars, 500 μm. e Quantitative analysis of the total porosity of d. f Representative images of safranin O and fast green staining of endplates in Cdkn2aΔLyz2 or Cdkn2af/f mice at 8 weeks after LSI or sham surgery. Scale bars, 50 μm. g Endplate scores of the endplates of f. h Representative immunohistochemical images of Osterix (Osx) in the endplates of Cdkn2aΔLyz2 or Cdkn2af/f mice at 8 weeks after LSI or sham surgery. Scale bars, 50 μm. i Quantitative analysis of the number of Osx+ cells in the endplates of h. j Representative images of immunofluorescent analysis of CGRP+ sensory nerves (red) and DAPI (blue) staining of nuclei in the endplates of Cdkn2aΔLyz2 or Cdkn2af/f mice after LSI or sham surgery. Scale bars, 50 μm. k Permillage of CGRP+ area in the endplates of l. n = 6 per group. Data are represented as means ± standard deviations, as determined by One-way ANOVA. Source data are provided as a Source Data file.

Figure 1 Cellular senescence is associated with aortic valve calcification. A) GSEA plot showing differential expression of signature genes in the cellular senescence pathway in calcific valve and normal valvular tissue based on the RNA-seq data sets from the GEO database. B) DEG plot showing the differential expression genes between the calcific valve and normal valvular tissue. C) Violin plots representing the expression of P21, P16, and P53 in the aortic valve from CAVD patients, elder and young individuals. D) Representative Vonkossa staining, Alizarin red staining, and immunohistochemical staining of P16, P21, and P53 in aortic valves from CAVD patients and controls. Scale bar 200 µm. E) Immunofluorescent staining of NQO-1 (red), CCND1 (red), and DAPI (blue) in the aortic valve from CAVD patients and controls. Scale bar 100 µm. F) Protein expression of ALP, Runx2, NQO1, and P21 in the aortic valve from CAVD patients (n = 10) and controls (n = 10). Bar plots representing the fold change of specific protein expression over control. Data are means ± SD. **p < 0.01; ***p < 0.001 (unpaired two-tailed Student's t-test).

Fig. 3. The effects of TCPP@PPPy-PPy/PVA gel on NPCs’ viability and anabolic/catabolic metabolism. (A) The TCPP@PPy-PPy/PVA gel’s impact on NPC viability within a 3D culture system was evaluated through live/dead fluorescence staining on days 1, 3, and 7. (B) The MTT assay was used to determine the impact of varying US intensity on cell viability, with the culture time plotted as a line graph. Data shown are means ± SD of n = 3 biological replicates. (C) NPCs from Control, TBHP, TBHP + US, TBHP + Gel, and TBHP + Gel + US groups were performed live/dead and phalloidin fluorescence staining. (D) The PCR assay results from the Control-, TBHP-, TBHP + US–, TBHP + Gel–, and TBHP + Gel + US–treated NPCs were normalized and subsequently displayed using a heatmap. Data shown are means ± SD of n = 3 biological replicates. (E) WB analysis of inflammatory factor expression in NPCs following treatment with Control, TBHP, TBHP + US, TBHP + Gel, and TBHP + Gel + US experimental conditions. (F) The WB assay band images of ECM metabolism–related protein in NPCs following treatment with Control, TBHP, TBHP + US, TBHP + Gel, and TBHP + Gel + US. (G) Control-, TBHP-, TBHP + US–, TBHP + Gel–, and TBHP + Gel + US–treated NPCs were stained by Alcian. The darker the blue staining represents the better the catabolism of proteoglycan in NPCs.

Fig. 1. Effects of AGEs in different concentrations on the senescence of BMSCs. The BMSCs were treated with AGEs (50–200 μg/mL) or BSA for 24–72 h. (A) SA-β-gal assay for detection of BMSCs senescence. Scale bar: 100 μm. (B) Detection of H3K9me3 by immunofluorescence in BMSCs. Scale bar: 100 μm. (C) Detection of γ-H2AX by immunofluorescence in BMSCs. Scale bar: 100 μm. (D–I) Representative Western blotting assay and quantitation of the level of P16, P21, P53. **p < 0.01 versus BSA.

Fig. 1 NPC senescence is accompanied by DNA damage and the activation of the cGAS-STING axis during IVDD. A: Overview of the overall workflow of the integrated single-cell analysis. B: UMAP visualizing human NPCs as five different subclusters after unsupervised clustering. Each point indicates a single cell, colored according to different cell subclusters. C: Relative proportion of each NPC cluster between the control group and IVDD group. D: Differentially expressed genes in each NPC cluster. E: Cell cycle analysis of all NPC subclusters or that between the control group and IVDD group. F: Senescence score based on single-cell sequencing of each NPC cluster between the control group and IVDD group. G: Representative T2-weighted MRI images and SOFG staining of NP tissues with varying degrees of degeneration. Scale bar = 20 μm. H and I: Representative images and quantitative results of immunohistochemistry for p16INK4a in NP tissues with different degrees of degeneration (n = 3). Scale bar = 20 μm. J and K: Representative images and quantitative results of IF staining for γH2AX in NP tissues with varying degrees of degeneration (n = 3). Scale bar = 20 μm. L and M: Representative images and quantitative results of IF staining for γH2AX and p16INK4a in primary rat NPCs treated with or without H2O2 for 24 h (n = 3). Scale bar = 20 μm. N and O: Representative images and quantitative results of IF staining for S-β-Gal in primary rat NPCs treated with or without H2O2 for 24 h (n = 3). P: Neutral comet assay in primary rat NPCs treated with or without H2O2 for 24 h. Q: Quantification of the percentage of DNA in the tail (Tail DNA) (n = 3). Significance was calculated by (I, K, M, O, and Q) one-way ANOVA. *p 

Fig. 1 NPC senescence is accompanied by DNA damage and the activation of the cGAS-STING axis during IVDD. A: Overview of the overall workflow of the integrated single-cell analysis. B: UMAP visualizing human NPCs as five different subclusters after unsupervised clustering. Each point indicates a single cell, colored according to different cell subclusters. C: Relative proportion of each NPC cluster between the control group and IVDD group. D: Differentially expressed genes in each NPC cluster. E: Cell cycle analysis of all NPC subclusters or that between the control group and IVDD group. F: Senescence score based on single-cell sequencing of each NPC cluster between the control group and IVDD group. G: Representative T2-weighted MRI images and SOFG staining of NP tissues with varying degrees of degeneration. Scale bar = 20 μm. H and I: Representative images and quantitative results of immunohistochemistry for p16INK4a in NP tissues with different degrees of degeneration (n = 3). Scale bar = 20 μm. J and K: Representative images and quantitative results of IF staining for γH2AX in NP tissues with varying degrees of degeneration (n = 3). Scale bar = 20 μm. L and M: Representative images and quantitative results of IF staining for γH2AX and p16INK4a in primary rat NPCs treated with or without H2O2 for 24 h (n = 3). Scale bar = 20 μm. N and O: Representative images and quantitative results of IF staining for S-β-Gal in primary rat NPCs treated with or without H2O2 for 24 h (n = 3). P: Neutral comet assay in primary rat NPCs treated with or without H2O2 for 24 h. Q: Quantification of the percentage of DNA in the tail (Tail DNA) (n = 3). Significance was calculated by (I, K, M, O, and Q) one-way ANOVA. *p 

FIGURE 5 Autophagy was excessively activated, and hair cell senescence was aggravated by DLK overexpression. (a, c–e) Western blot showing that DLK, p-JNK, and JNK3 were upregulated after DLK overexpression. (b, f–h) Western blot showing that the expression of ULK1, Beclin-1, and LC3B increased after DLK overexpression. (i–l) Western blot showing that the expression of p21, p16, and p53 increased after DLK overexpression. (m–p) Immunofluorescence showing that the expression of p21, p16, and p53 increased in HEI-OC1 cells following DLK overexpression. (q) Transmission electron microscopy image showing that autophagosomes (blue arrows) and autolysosomes (red arrows) increased in HEI-OC1 cells after DLK overexpression. (r) Number of autophagosomes and autolysosomes in a single cell in (p). Data are presented as mean ± SD

FIGURE 5 Autophagy was excessively activated, and hair cell senescence was aggravated by DLK overexpression. (a, c–e) Western blot showing that DLK, p-JNK, and JNK3 were upregulated after DLK overexpression. (b, f–h) Western blot showing that the expression of ULK1, Beclin-1, and LC3B increased after DLK overexpression. (i–l) Western blot showing that the expression of p21, p16, and p53 increased after DLK overexpression. (m–p) Immunofluorescence showing that the expression of p21, p16, and p53 increased in HEI-OC1 cells following DLK overexpression. (q) Transmission electron microscopy image showing that autophagosomes (blue arrows) and autolysosomes (red arrows) increased in HEI-OC1 cells after DLK overexpression. (r) Number of autophagosomes and autolysosomes in a single cell in (p). Data are presented as mean ± SD