Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide-Activated Macrophages Suppress Cellular Senescence and Promote Rejuvenation in Human Dermal Fibroblasts

doi: 10.3390/ijms26157061

Figure Lengend Snippet: Expression of the p16 and p21 genes in Young cells. The schematic protocol (1)–(7) is shown at the top of the figure. The test conditions in figures ( A – D ) correspond to the schematic protocol (1)–(7). Young cells were cultured for 24 h after replacing half of the medium with EMEM, Old cell-conditioned medium (Old CM), or THP/LPS-conditioned medium (THP/LPS-CM; 0–1000 ng/mL). ( A , B ) Total RNA was extracted, and the relative mRNA expression levels of p16 ( A ) and p21 ( B ) were measured by Quantitative real-time PCR (RT-qPCR) using GAPDH as a housekeeping gene. ( C , D ) Young cells were treated with EMEM or THP/LPS-CM (0, 10, or 100 ng/mL) for 24 h. Cells were lysed, and 12.5 μg of total protein per lane was separated by SDS-PAGE, followed by western blot analysis using antibodies against P16 ( C ) or P21 ( D ). Representative western blot images are shown in the boxed regions of panels C and D. Band intensities were normalized to β-actin and quantified. Data are presented as mean ± SD (n = 3). Different letters (a–c) indicate statistically significant differences ( p < 0.05, ANOVA, Tukey–Kramer).

Article Snippet: Proteins were resolved via 12.5% SDS-PAGE at 30 mA for 90 min and transferred onto PVDF membranes at 300 mA for 1 h. Membranes were then blocked with PBS containing 0.1% Tween-20 and 5% skim milk for 1 h, followed by incubation overnight at 4 °C with primary antibodies against P21 Waf1/Cip1 (1:1000) and β-actin (1:2000; both from Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide-Activated Macrophages Suppress Cellular Senescence and Promote Rejuvenation in Human Dermal Fibroblasts

doi: 10.3390/ijms26157061

Figure Lengend Snippet: Changes in P16, P21, and Ki-67 expression in senescent NB1RGB cells treated with THP/LPS-CM. The schematic protocol (1)–(7) is shown at the top of the figure. The test conditions in figures ( A – D ) correspond to the schematic protocol (1)–(7). ( A ) Young and Old NB1RGB cells were treated with EMEM, THP/LPS (0 ng/mL)-CM, or THP/LPS (100 ng/mL)-CM for 24 h. Relative mRNA expression levels of p16 , p21 , and Ki-67 were analyzed by RT-qPCR and normalized to the levels in Young cells. ( B ) Old cells were treated with LPS (10, 100, or 1000 ng/mL) for 24 h, and the expression levels of p16 , p21 , and Ki-67 were assessed by RT-qPCR. In both ( A , B ), bars represent mean ± SD (n = 5). Different letters (a–c) indicate statistically significant differences ( p < 0.05). ( C ) Young and Old cells were treated with EMEM, THP/LPS (0 ng/mL)-CM, or THP/LPS (100 ng/mL)-CM for 24 h, then immunofluorescence staining was performed using antibodies against P21 and Ki-67 (n = 5). Representative fluorescence images were acquired using a 20× objective lens with 2× digital zoom. In P21 and Ki-67 single-staining panels green = P21 or Ki-67, red = actin, and blue = nuclei. In double-staining panels green = P21, red = Ki-67, and blue = nuclei. Scale bars = 50 μm. ( D ) Based on the immunofluorescence images, the percentages of P21-positive cells, Ki-67-positive cells, and total cell counts per field were quantified. Data are expressed as mean ± SD (n = 5). Different letters (a–c) indicate statistically significant differences ( p < 0.05, ANOVA, Tukey–Kramer).

Article Snippet: Proteins were resolved via 12.5% SDS-PAGE at 30 mA for 90 min and transferred onto PVDF membranes at 300 mA for 1 h. Membranes were then blocked with PBS containing 0.1% Tween-20 and 5% skim milk for 1 h, followed by incubation overnight at 4 °C with primary antibodies against P21 Waf1/Cip1 (1:1000) and β-actin (1:2000; both from Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Fluorescence, Double Staining

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI can effectively maintain vascular function in naturally aging mice. (A) Western blot experiments were conducted and quantitatively analyzed to determine the changes in protein levels of Ki-67, Lamin B, P21, P16, and γ-H2AX in the aortas of naturally aged mice after FOXO4-DRI or PBS treatment. n ≥ 4 per group. (B) RT-qPCR analysis was performed to examine the mRNA levels of Il-1β, Il-6, Cxcl15 , and Tnf-α in the aortas of naturally aged mice after FOXO4-DRI and PBS treatments. n = 3 per group. (C) Representative SA-β-Gal staining and quantitative analysis were performed to determine the number of positive cells in the aortas of naturally aged mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. (D) Representative HE staining and quantitative analysis were performed to evaluate the thickness of the aortas in naturally aged mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. (E) Color Doppler imaging and analysis were performed to detect and analyze the structural and blood flow conditions of the aortas in naturally aged mice after FOXO4-DRI and PBS treatments. n = 6 per group. (F) Representative DHE staining and quantitative analysis were performed to assess the ROS levels in the aortas of naturally aged mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: The primary antibodies employed were: P53 (Proteintech, 60283-2-Ig), pSer46-P53 (CST, 2521), FOXO4 (Proteintech, 21535-1-AP), P21 (CST, 2947), P16 (CST, 18769), BAX (CST, 2772), BCL2 (CST, 3498), Ki-67 (Proteintech, 84192-4-RR), Lamin B (CST, 13435), Cleaved Caspase 3 (CST, 9664), γ-H2AX (Abcam, ab81299), and GAPDH (Abcam, ab8245) as an internal reference for normalizing protein expression levels.

Techniques: Western Blot, Quantitative RT-PCR, Staining, Imaging, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI can effectively maintain vascular function in progeroid mice. (A) Western blot experiments were conducted and quantitatively analyzed to determine the changes in protein levels of Ki-67, Lamin B, P21, P16, and γ-H2AX in the aortas of progeroid mice after FOXO4-DRI or PBS treatment. n ≥ 4 per group. (B) RT-qPCR analysis was performed to examine the mRNA levels of Il-1β , Il-6 , Cxcl15 , and Tnf-α in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. n = 3 per group. (C) Representative SA-β-Gal staining and quantitative analysis were performed to determine the number of positive cells in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. Scale bar = 100 μm. n = 5 per group. (D) Representative HE staining and quantitative analysis were performed to evaluate the thickness in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. Scale bar = 100 μm. n = 5 per group. (E) Color Doppler imaging and analysis were performed to detect and analyze the structural and blood flow conditions in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. n = 5 per group. (F) Representative DHE staining and quantitative analysis were performed to assess the ROS levels in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: The primary antibodies employed were: P53 (Proteintech, 60283-2-Ig), pSer46-P53 (CST, 2521), FOXO4 (Proteintech, 21535-1-AP), P21 (CST, 2947), P16 (CST, 18769), BAX (CST, 2772), BCL2 (CST, 3498), Ki-67 (Proteintech, 84192-4-RR), Lamin B (CST, 13435), Cleaved Caspase 3 (CST, 9664), γ-H2AX (Abcam, ab81299), and GAPDH (Abcam, ab8245) as an internal reference for normalizing protein expression levels.

Techniques: Western Blot, Quantitative RT-PCR, Staining, Imaging, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: Glucose and oxygen deprivation can induce endothelial cell senescence. (A) Western blot analysis was performed to quantify the changes in protein levels of Ki-67, Lamin B, FOXO4, P53, P21, P16, and γ-H2AX in HUVECs following OGD treatment, with n ≥ 4 per group. (B) Representative immunofluorescence staining was conducted to analyze the content and localization changes of Ki-67, P21, γ-H2AX, and P53 different groups of HUVECs, with n = 5 per group. (C) Subcellular localization of FOXO4 in different groups of HUVECs was analyzed through representative immunofluorescence staining (FOXO4 in red, DAPI in blue), with n = 5 per group, scale bar = 40 μm. (D) Representative SA-β-GAL staining and quantification were performed to evaluate the number of positive cells in HUVECs after OGD treatment, with n = 5 per group, scale bar = 100 μm. (E) Tube formation assays analyzed relative tube lengths in different groups following OGD treatment, with n = 5 per group, scale bar = 400 μm. (F) Scratch assays were conducted to assess endothelial cell migration coverage at 0, 12, 24, and 36 h across different groups, with n = 5 per group, scale bar = 400 μm. Data are represented as mean ± SEM. Statistical analysis assessed by 2-way RM ANOVA (G) Representative DHE staining and quantification evaluated the number of DHE-positive cells in different groups, with n = 5 per group, scale bar = 100 μm. (H) RT-qPCR analysis was performed in HUVECs following OGD treatment to measure the mRNA levels of IL-6 , IL-8 , IL-1β , and TNF-α , with n = 3 per group. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: The primary antibodies employed were: P53 (Proteintech, 60283-2-Ig), pSer46-P53 (CST, 2521), FOXO4 (Proteintech, 21535-1-AP), P21 (CST, 2947), P16 (CST, 18769), BAX (CST, 2772), BCL2 (CST, 3498), Ki-67 (Proteintech, 84192-4-RR), Lamin B (CST, 13435), Cleaved Caspase 3 (CST, 9664), γ-H2AX (Abcam, ab81299), and GAPDH (Abcam, ab8245) as an internal reference for normalizing protein expression levels.

Techniques: Western Blot, Immunofluorescence, Staining, Migration, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI can alleviate endothelial cell senescence induced by OGD. (A) Western blot analysis was conducted to quantify the changes in protein levels of Ki-67, Lamin B, P21, P16, and γ-H2AX in senescent HUVECs following treatment with FOXO4-DRI or PBS, with n ≥ 4 per group. (B) Representative immunofluorescence analysis (green for Ki-67, blue for DAPI) was performed to assess the number of Ki-67 positive HUVECs in different groups, with n = 5 per group and scale bar = 40 μm. (C) Fluorescent staining was carried out to analyze the content and localization changes of P21 and γ-H2AX in different groups, with n = 5 per group and scale bar = 40 μm. (D) Representative SA-β-GAL staining and quantification were performed to evaluate the number of positive cells in different groups, with n = 5 per group and scale bar = 100 μm. (E) Tube formation assays were conducted to analyze relative tube lengths in different groups, with n = 5 per group and scale bar = 400 μm. (F) Representative DHE staining and quantification were carried out to evaluate the number of DHE-positive cells in different groups, with n = 5 per group and scale bar = 100 μm. (G) Scratch assays were performed to assess endothelial cell migration coverage at 0, 12, 24, and 36 h in different groups, with n = 5 per group and scale bar = 400 μm. Data are represented as mean ± SEM. Statistical analysis assessed by 2-way RM ANOVA. (H) RT-qPCR analysis was performed to detect mRNA levels of IL-6 , IL-8 , IL-1β , and TNF-α in different groups, with n = 3 per group. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: The primary antibodies employed were: P53 (Proteintech, 60283-2-Ig), pSer46-P53 (CST, 2521), FOXO4 (Proteintech, 21535-1-AP), P21 (CST, 2947), P16 (CST, 18769), BAX (CST, 2772), BCL2 (CST, 3498), Ki-67 (Proteintech, 84192-4-RR), Lamin B (CST, 13435), Cleaved Caspase 3 (CST, 9664), γ-H2AX (Abcam, ab81299), and GAPDH (Abcam, ab8245) as an internal reference for normalizing protein expression levels.

Techniques: Western Blot, Immunofluorescence, Staining, Migration, Quantitative RT-PCR, Two Tailed Test

Journal: Antioxidants

Article Title: Epicatechin Gallate Ameliorates UVB-Induced Photoaging by Inhibiting p38α-Mediated Autophagy and Oxidative Stress

doi: 10.3390/antiox15020180

Figure Lengend Snippet: Effects of ECG on UVB-induced senescence markers in HaCaT cells. ( A , B ) Cell cycle distribution analysis of HaCaT cells by flow cytometry. The histogram in ( A ) shows DNA content (propidium iodide staining intensity) on the x-axis and cell count on the y-axis. The stacked bar chart in ( B ) quantifies the percentage of cells in different cell cycle phases (G0/G1, S, and G2/M). ( C ) Relative mRNA expression levels of p53 , p16 , p21 , IL6 , and TNF-α determined by RT-qPCR. ( D , E ) Representative Western blots and densitometric analysis of p53, p16, and p21 proteins. ( F , I ) Representative immunofluorescence images of γH2AX and Lamin B1 (scale bar: 50 μm). γH2AX foci are shown in green, Lamin B1 is shown in red, and nuclei are counterstained with DAPI (blue). White arrows indicate nuclei with damage. ( G ) Representative images of SA-β-gal staining in HaCaT cells (scale bar: 200 μm). ( H ) Quantitative analysis of SA-β-gal positive cells. Data are presented as the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Rabbit anti-Beclin1 (BECN1) antibody (cat. #11306), mouse anti-tubulin antibody (cat. #66031-1-IG), rabbit anti-LaminB1 (LMNB1) antibody (cat. #12987-1-AP), rabbit anti-Cyclin-Dependent Kinase Inhibitor 1A (p21, CDKN1A) antibody (cat. #10355-1-AP) and rabbit anti-LC3 antibody (cat. #14600) were purchased from Proteintech group (Wuhan, China).

Techniques: Flow Cytometry, Staining, Cell Characterization, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Molecular Cancer

Article Title: Differential susceptibility and role for senescence in CART cells based on costimulatory domains

doi: 10.1186/s12943-025-02371-1

Figure Lengend Snippet: Recurrent activation/rest differentially induced senescence in CART cells with alternative costimulatory domains. A-E. The percentage of CD3 + T cells that are positive for ( A. ) p21, ( B. ) KLRG1, ( C. ) uPAR, ( D. ) CD27 and ( E. ) TIM-3 as measured by flow cytometry are plotted ( n = 6 donors) (A-D., two-way ANOVA, E., t-test). F-G. Expression of uPAR, TIM-3, and KLRG1 and loss of CD27 expression are considered senescence events. Simultaneous occurrence of two, three, and four of these events in ( F. ) D8 and ( G. ) D15 CART cells are plotted as percent of CD3 + cells (t-test). H. DNA is isolated from D0, D8 and D15 CART cells. The telomere lengths are measured by qPCR and plotted for each time point (two-way ANOVA). I-L. mRNA expression of ( I. ) p21, ( J. ) p53, ( K. ) PIM-1, and ( L. ) uPAR are measured by qPCR. Fold change is calculated by 2ˆ (–delta delta CT). The expression levels are normalized to BBζ D8 (two-way ANOVA). M. Recurrently activated CART cells were immunoblotted for DNA damage markers p-H2AX (Ser139), p-ATM (Ser1981), and p-Chk2 (Thr68). N. NSG mice received JeKo-1, were randomized, and received BBζ and 28ζ. The spleens from mice were collected and flowed for the indicated markers 21 days after CART injection ( n = 3, t-test). Error bars, SEM. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: p21 Waf1/Cip1 (cat# CST 8865) and p53 (cat# CST 5429) antibodies were purchased from Cell Signaling Technology (CST) (Danvers, MA, USA).

Techniques: Activation Assay, Flow Cytometry, Expressing, Isolation, Injection

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Euptox A Induces G1 Arrest and Autophagy via p38 MAPK- and PI3K/Akt/mTOR-Mediated Pathways in Mouse Splenocytes

doi: 10.1369/0022155417722118

Figure Lengend Snippet: Antibodies Used in the Study.

Article Snippet: p21 Waf1/Cip1 , Rabbit, polyclonal , BM4382 , Boster.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Mammary Epithelial Cell Polarity Is Regulated Differentially by p73 Isoforms via Epithelial-to-mesenchymal Transition

doi: 10.1074/jbc.m112.358143

Figure Lengend Snippet: FIGURE 4. p73-KD and TAp73-KD, but not Np73-KD induce EMT pheno- types in MCF10A cells. Western blots were prepared using extracts from MCF10A cells (lane 1), and MCF10A cells with p73-KD (lane 2), with Np73-KD (lane3),orwithTAp73-KD(lane4).MCF10AcellsweregrowninMatrigelfor20 days.TheblotswereprobedwithantibodiesagainstE-cadherin(A),-catenin (B), laminin V (B), Snail (B), Slug (B), Twist (B), p21 (C), PUMA (C), and actin (A–C), respectively. The basal levels of each gene were arbitrarily set at 1.0, and fold change is shown below each lane.

Article Snippet: Antibodies used were purchased from Calbiochem (anti-p73 (ab-1)-ER-13, anti-p73 (ab-2)-ER-15), Millipore (anti-laminin 2), Santa Cruz Biotechnology (anti- - catenin (E-5), anti-Snail-1, anti-Twist, p21, and anti-GAPDH), Cell Signaling (anti-Slug), BDTransduction Laboratories (antiE-cadherin), Prosci (PUMA), Sigma (anti-actin), and Bio-Rad (secondary antibodies against rabbit or mouse IgG conjugated with HRP).

Techniques: Western Blot