Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5-Hydroxymethylcytosine-Mediated Sp1 Transcription Factor Expression.

doi: 10.1002/mco2.70261

Figure Lengend Snippet: FIGURE 1 5-hmC levels in senescent HUVECs and PQ-induced aging mice. (A) β-galactosidase staining and senescent cell ratio in replicative senescent HUVECs (n = 4 for each group, unpaired two-sided t-test, scale bars = 100 µm). (B) Protein levels of p16, p21, and p53 in replicative senescent HUVECs (n = 3 for each group, unpaired two-sided t-test). (C) 5-hmC levels in replicative senescent HUVECs assayed by using a dot blot; methylene blue staining used as an internal control of the DNA content of samples (n = 3 for each group, unpaired two-sided t-test). (D) Levels of p-γH2AX and 5-hmC in replicative senescent HUVECs (n = 6 for each group, unpaired two-sided t-test, scale bars = 100 µm). Levels of p-γH2AX and 5-hmC in the heart (E, scale bars = 100 µm) and aorta tissue (F, scale bars = 400 µm) of PQ-treated mice (n = 3 for each group, unpaired two-sided t-test). (G) 5-mC and 5-hmC landscape changes of the p21 in the senescent cells compared with the young cells. (H) 5-mC and 5-hmC landscape changes in the p53 in the senescent cells compared with the young cells. All data are expressed as the mean ± standard error of mean (SEM). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Before staining, the samples were treated with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.5% triton X-100 for 15 min. Then the tissue sections or cells were incubated with primary antibodies overnight at 4◦C, followed by incubation with secondary antibodies for 1 h. The primary antibodies used were against 5-hmC antibody (1:200, 39769, Active Motif), p-γH2AX antibody (1:200, ab26350, Abcam), SP1 (1:100, A19649, ABclonal), TET3 (1:200, GTX-121453, GENETEX), β-galactosidase (1:100, 27198, CST), p16 (1:100, sc-1661, Santa Cruz), p21 (1:100, sc6246, Santa Cruz), and p53 (1:100, 2524, CST).

Techniques: Staining, Dot Blot, Control

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5-Hydroxymethylcytosine-Mediated Sp1 Transcription Factor Expression.

doi: 10.1002/mco2.70261

Figure Lengend Snippet: FIGURE 2 Role of TET3 in regulating cellular senescence. (A) Protein levels of TET2 and TET3 in replicative senescent HUVECs (n = 3 for each group, unpaired two-sided t-test). (B) Protein levels of TET2 and TET3 in HUVECs treated with PQ (n = 4 for each group, unpaired two-sided t-test). (C) Protein levels of p16, p21, and p53, and (D) Immunofluorescent imaging of p-γH2AX after TET2 or TET3 silencing in HUVECs treated with PQ (n = 3 for each group, one-way analysis of variance followed by Dunnett’s multiple comparisons test, scale bars = 100 µm). (E, F) Protein and mRNA levels of p16, p21, and p53 after TET2 or TET3 overexpression (n = 3 for each group, unpaired two-sided t-test). (G) Immunofluorescent imaging of p-γH2AX in HUVECs after TET2 or TET3 overexpression (n = 3 for each group, unpaired two-sided t-test, scale bars = 50 µm). (H) Cell proliferation after TET3 overexpression in HUVECs (n = 9 for each group, unpaired two-sided t-test). All the data are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Before staining, the samples were treated with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.5% triton X-100 for 15 min. Then the tissue sections or cells were incubated with primary antibodies overnight at 4◦C, followed by incubation with secondary antibodies for 1 h. The primary antibodies used were against 5-hmC antibody (1:200, 39769, Active Motif), p-γH2AX antibody (1:200, ab26350, Abcam), SP1 (1:100, A19649, ABclonal), TET3 (1:200, GTX-121453, GENETEX), β-galactosidase (1:100, 27198, CST), p16 (1:100, sc-1661, Santa Cruz), p21 (1:100, sc6246, Santa Cruz), and p53 (1:100, 2524, CST).

Techniques: Imaging, Over Expression

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5-Hydroxymethylcytosine-Mediated Sp1 Transcription Factor Expression.

doi: 10.1002/mco2.70261

Figure Lengend Snippet: FIGURE 3 Role of TET3 knockout in regulating cellular senescence. (A, B) Protein and mRNA levels of p16, p21, and p53 in HUVECs with TET3 knockout (n = 3 for each group, one-way analysis of variance followed by Dunnett’s multiple comparisons test). (C) β-galactosidase staining and senescent cell ratio in HUVECs with TET3 knockout (n = 3 for each group, one-way analysis of variance followed by Dunnett’s multiple comparisons test, scale bars = 100 µm). (D) Cell proliferation ratio in HUVECs with TET3 knockout (n = 12 for each group, unpaired two-sided t-test). (E, F) MRNA levels of SASP in HUVECs with TET3 knockout (n = 3 for each group, one-way analysis of variance followed by Dunnett’s multiple comparisons test). (G) Immunofluorescence imaging of p-γH2AX and 5-hmC in HUVECs with TET3 knockout (n = 9 for each group, one-way analysis of variance followed by Dunnett’s multiple comparisons test, scale bars = 100 µm). All data are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Before staining, the samples were treated with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.5% triton X-100 for 15 min. Then the tissue sections or cells were incubated with primary antibodies overnight at 4◦C, followed by incubation with secondary antibodies for 1 h. The primary antibodies used were against 5-hmC antibody (1:200, 39769, Active Motif), p-γH2AX antibody (1:200, ab26350, Abcam), SP1 (1:100, A19649, ABclonal), TET3 (1:200, GTX-121453, GENETEX), β-galactosidase (1:100, 27198, CST), p16 (1:100, sc-1661, Santa Cruz), p21 (1:100, sc6246, Santa Cruz), and p53 (1:100, 2524, CST).

Techniques: Knock-Out, Staining, Immunofluorescence, Imaging

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5-Hydroxymethylcytosine-Mediated Sp1 Transcription Factor Expression.

doi: 10.1002/mco2.70261

Figure Lengend Snippet: FIGURE 5 Regulation of TET3 on SP1-ETS-1 axis. (A) Changes in the total distribution of 5-hmC in the genome in TET3 knockout/PQ-treated HUVECs based on hMeDIP-seq. (B) Distribution of DhMSs. (C) Overlapping DhMGs associated with senescence are shown using a Venn diagram. (D) Levels of KAT5 and SP1 mRNA after TET3 knockdown (n = 3 for each group, unpaired two-sided t-test). (E) Level of SP1 protein after TET3 knockdown (n = 3 for each group, unpaired two-sided t-test). (F) Level of 5-hmC in SP1 protomer after TET3 knockdown. (G) The combination of SP1 and ETS-1 after TET3 knockdown. (H, I) Protein and mRNA levels of p16, p21, and p53 after ETS-1 knockdown (n = 3 for each group, one-way analysis of variance followed by Dunnett’s multiple comparisons test). All data are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Before staining, the samples were treated with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.5% triton X-100 for 15 min. Then the tissue sections or cells were incubated with primary antibodies overnight at 4◦C, followed by incubation with secondary antibodies for 1 h. The primary antibodies used were against 5-hmC antibody (1:200, 39769, Active Motif), p-γH2AX antibody (1:200, ab26350, Abcam), SP1 (1:100, A19649, ABclonal), TET3 (1:200, GTX-121453, GENETEX), β-galactosidase (1:100, 27198, CST), p16 (1:100, sc-1661, Santa Cruz), p21 (1:100, sc6246, Santa Cruz), and p53 (1:100, 2524, CST).

Techniques: Knock-Out, Knockdown

Journal: MedComm

Article Title: Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5-Hydroxymethylcytosine-Mediated Sp1 Transcription Factor Expression.

doi: 10.1002/mco2.70261

Figure Lengend Snippet: FIGURE 7 Role of p53 in regulating senescence in vivo and vitro. (A, B) Protein and mRNA levels of p16, p21, and p53 after p53 knockdown (n = 3 for each group, one-way analysis of variance followed by Dunnett’s multiple comparisons test). (C, D) Expression of p53, p16, and p21 mRNAs in the heart and aorta tissues of p53+/−- heterozygous mice treated with PQ (n = 5 for C, and 3 for D each group, one-way analysis of variance followed by Dunnett’s multiple comparisons test). (E) Protein levels of p53, p16, and p21 in the heart tissues of p53+/−-heterozygous mice treated with PQ (n = 3 for each group, one-way analysis of variance followed by Dunnett’s multiple comparisons test). (F, G) Levels of p16, p21, and β-galactosidase in the heart and aorta tissues of p53+/−-heterozygous mice treated with PQ (n = 3 for each group, one-way analysis of variance followed by Dunnett’s multiple comparisons test, scale bars = 100 µm). All data are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Before staining, the samples were treated with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.5% triton X-100 for 15 min. Then the tissue sections or cells were incubated with primary antibodies overnight at 4◦C, followed by incubation with secondary antibodies for 1 h. The primary antibodies used were against 5-hmC antibody (1:200, 39769, Active Motif), p-γH2AX antibody (1:200, ab26350, Abcam), SP1 (1:100, A19649, ABclonal), TET3 (1:200, GTX-121453, GENETEX), β-galactosidase (1:100, 27198, CST), p16 (1:100, sc-1661, Santa Cruz), p21 (1:100, sc6246, Santa Cruz), and p53 (1:100, 2524, CST).

Techniques: In Vivo, Knockdown, Expressing

Journal: Biomedicines

Article Title: Loss of LXRβ Drives CD4 + T Cell Senescence and Exacerbates the Progression of Colitis

doi: 10.3390/biomedicines14010152

Figure Lengend Snippet: Absence of LXRβ leads to CD4 + T cell senescence by upregulating cGAS/STING signaling. ( A ) Immunoblot analysis of the LXRβ, senescence markers (P16, P21, P53), and cGAS/STING abundance in WT and Lxrβ −/− CD4 + T cells ( n = 3 independent experiments). ( B ) SA-β-gal activity of control and GW3965-treated CD4 + T cells evaluated by flow cytometry using the fluorescent β-gal substrate C12FDG ( n = 4 independent experiments; representative flow cytometry results are provided). ( C – G ) qRT-PCR and Western blot analysis of changes in the senescence markers and cGAS/STING mRNA and protein levels in CD4 + T cells after GW3965 treatment ( n = 3 independent experiments). ( H ) SA-β-gal activity of control and H151-treated Lxrβ −/− CD4 + T cells evaluated by flow cytometry using the fluorescent β-gal substrate C12FDG ( n = 4 independent experiments; representative flow cytometry results are provided). ( I ) Immunoblot analysis of the senescence markers abundance in control and H151-treated Lxrβ −/− CD4 + T cells ( n = 3 independent experiments). Statistical analysis of the data was conducted using two-way ANOVA ( A , G , I ) and two-sided unpaired t -test ( B , C – F , H ). Data are presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Then, they were shifted to PVDF membranes and probed with antibodies against P16 (#23200, 1:1000), P53 (#2527, 1:1000) (all from Cell Signaling Technology, Danvers, MA, USA), P21 (sc-6246, 1:200) (Santa Cruz, CA, USA), LXRβ (60345-1-lg, 1:1000), cGAS (29958-1-AP, 1:2000), and STING (19851-1-AP, 1:2000) (all from Proteintech, Wuhan, China).

Techniques: Western Blot, Activity Assay, Control, Flow Cytometry, Quantitative RT-PCR

Journal: Materials Today Bio

Article Title: Increased substrate stiffness disrupts nuclear-cytoskeletal mechanical coupling in senescent cells

doi: 10.1016/j.mtbio.2025.102472

Figure Lengend Snippet: In vitro validation of cellular senescence in human dermal fibroblasts. (A) Schematic showing senescence induction mechanism in hdFs via three independent stimuli: (1) over-expression of p16 through a tetracycline-inducible construct enforcing a cell cycle arrest, (2) long-term cell culture leading to telomere shorting resulting in replicative senescence (RS), and (3) treatment with DNA crosslinking agent Mitomycin C (MMC) causing DNA damage. (B) Reduced proliferation of senescent hdFs (p16, RS, and MMC) expressed as population doublings. (C) Immunoblotting of cell cycle inhibitors p21Cip and p16Ink4a, and DNA damage marker phospho-H2A.X in hdFs and senescent hdFs (p16, RS, and MMC). (D) Representative phase-contrast images of hdFs (ctrl) and senescent hdFs (p16, RS and MMC) cultured for 14 days showing increased cellular morphology and increased senescence-associated-β-galactosidase activity (blue staining) in senescent hdFs. Scale bar 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Membranes were then blocked with TBS Blocking Buffer (927–50000; Li-Cor) and probed overnight with primary antibodies according to the manufacturer's instructions: Phospho-Histone H2A.X (Ser139) (2577; Cell Signaling), GAPDH, (2118; Cell Signaling), p16 INK4A (D7C1M) (80772; Cell Signaling), and P21 Waf1/Cip1 (12D1) (2947; Cell Signaling).

Techniques: In Vitro, Biomarker Discovery, Over Expression, Construct, Cell Culture, Western Blot, Marker, Activity Assay, Staining

Journal: Materials Today Bio

Article Title: Increased substrate stiffness disrupts nuclear-cytoskeletal mechanical coupling in senescent cells

doi: 10.1016/j.mtbio.2025.102472

Figure Lengend Snippet: Enhanced cell morphological alterations of senescent hdFs in response to stiffness-varying substrates. (A) Representative immunostained images of hdFs (ctrl) and senescent hdFs (p16, RS and MMC) cultured for one day on collagen-coated stiffness-varying substrates: soft (E = 1.7 kPa), intermediate-stiff (E = 48.3 kPa), and rigid TCP (E ∼ 1 GPa). Cells were stained for actin cytoskeleton (red) and cell nuclei (blue). (B) Quantification of changes in cell area and (C) cell aspect ratio in response to stiffness-varying substrates. Each data point represents an individual cell (N ≥ 60 cells) from at least 3 independent experiment. Significance levels indicate: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bar 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Membranes were then blocked with TBS Blocking Buffer (927–50000; Li-Cor) and probed overnight with primary antibodies according to the manufacturer's instructions: Phospho-Histone H2A.X (Ser139) (2577; Cell Signaling), GAPDH, (2118; Cell Signaling), p16 INK4A (D7C1M) (80772; Cell Signaling), and P21 Waf1/Cip1 (12D1) (2947; Cell Signaling).

Techniques: Cell Culture, Staining

Journal: Materials Today Bio

Article Title: Increased substrate stiffness disrupts nuclear-cytoskeletal mechanical coupling in senescent cells

doi: 10.1016/j.mtbio.2025.102472

Figure Lengend Snippet: Altered actin cytoskeletal re-arrangement of senescent hdFs in response to stiffness-varying substrates . (A) Representative orientation-colored images showing local orientation of actin fibers of hdFs (ctrl) and senescent hdFs (p16, RS and MMC) cultured for one day on collagen-coated stiffness-varying substrates: soft (E = 1.7 kPa), intermediate-stiff (E = 48.3 kPa), and rigid TCP (E ∼ 1 GPa). The different colors indicate different orientations of actin fibers according to the color scale with respect to the cell axis (0°). (B) Corresponding distribution plots of the actin orientation-colored images for the different stiffness within each cell group. (C) Box plots showing the circular variance of actin fiber distribution in individual cells, with lower values indicating highly aligned fibers and higher values indicating increased fiber isotropy and disorganization. Each data point represents an individual cell (N ≥ 60 cells) from at least 3 independent experiments. Significance levels indicate: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bar 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Membranes were then blocked with TBS Blocking Buffer (927–50000; Li-Cor) and probed overnight with primary antibodies according to the manufacturer's instructions: Phospho-Histone H2A.X (Ser139) (2577; Cell Signaling), GAPDH, (2118; Cell Signaling), p16 INK4A (D7C1M) (80772; Cell Signaling), and P21 Waf1/Cip1 (12D1) (2947; Cell Signaling).

Techniques: Cell Culture

Journal: Materials Today Bio

Article Title: Increased substrate stiffness disrupts nuclear-cytoskeletal mechanical coupling in senescent cells

doi: 10.1016/j.mtbio.2025.102472

Figure Lengend Snippet: Enhanced maturation and altered spatial distribution of focal adhesions of senescent hdFs in response to stiffness-varying substrates . (A) Representative immunostained images (left) and corresponding inverted binary images (right) showing focal adhesions (FAs) of hdFs (ctrl) and senescent hdFs (p16, RS and MMC) cultured for one day on collagen-coated stiffness-varying substrates: soft (E = 1.7 kPa), intermediate-stiff (E = 48.3 kPa), and rigid TCP (E ∼ 1 GPa). Cells were stained for actin cytoskeleton (red), cell nuclei (blue) and vinculin (green). (B) Quantification of mean FA size per cell and (C) percentage of FAs located at the periphery of the cell. Each data point represents an individual cell (N ≥ 50 cells) from 3 independent experiments. Significance levels indicate: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. For comparisons within same cell group, significance is indicated using asterisks accompanied by connecting lines. For comparisons between control cells (hdFs) and senescent hdFs under the same substrate condition, significance is indicated by asterisks placed directly above the box. Scale bar 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Membranes were then blocked with TBS Blocking Buffer (927–50000; Li-Cor) and probed overnight with primary antibodies according to the manufacturer's instructions: Phospho-Histone H2A.X (Ser139) (2577; Cell Signaling), GAPDH, (2118; Cell Signaling), p16 INK4A (D7C1M) (80772; Cell Signaling), and P21 Waf1/Cip1 (12D1) (2947; Cell Signaling).

Techniques: Cell Culture, Staining, Control

Journal: Journal of Ginseng Research

Article Title: Anti-skin aging effects of black ginseng extracts through regulating inflammatory factors and the CLOCK–ERK pathway in mice

doi: 10.1016/j.jgr.2025.11.014

Figure Lengend Snippet: Antiaging effect of Rg3 in vitro. (A&B) The effects of D-gal and Rg3 on the activity of MEFs were detected by the CCK-8 kit. (C) MEFs were stained with SA-β-Gal. A blue–green signal was observed in the cytoplasm of positive cells. (D) Analysis of the mRNA levels of p16, p21, TNF-α and IL-1β. The reversal effects of 30 μM Rg3 on antiaging and anti-inflammatory were more obvious. (E) Analysis of CAT, SOD and MDA levels.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 Scale bar = 50 μm.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against p16 INK4a , p21 Cip1/Waf1 (Proteintech, China), clock, ERK1/2, p-ERK1/2, K15, β-actin, and CD34 (Wanleibio, China).

Techniques: In Vitro, Activity Assay, CCK-8 Assay, Staining

Journal: Journal of Ginseng Research

Article Title: Anti-skin aging effects of black ginseng extracts through regulating inflammatory factors and the CLOCK–ERK pathway in mice

doi: 10.1016/j.jgr.2025.11.014

Figure Lengend Snippet: Effects of BGE and PTE on the markers of senescent cells in epidermis and antioxidant enzymes in serum. (A&B) The mRNA level of p16 and p21 in the epidermis. (C&D) Representative images of p16 and p21 immunohistochemical staining and quantitative analysis. Compared with the Model group, BGE or PTE reduced the gene and protein levels of p16 and p21. (E–G) CAT, SOD, and MDA content in the serum. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Scale bar = 20 μm.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against p16 INK4a , p21 Cip1/Waf1 (Proteintech, China), clock, ERK1/2, p-ERK1/2, K15, β-actin, and CD34 (Wanleibio, China).

Techniques: Immunohistochemical staining, Staining