Journal: Nutrients

Article Title: Dihydromyricetin May Attenuate Skin Aging as a RAGE Inhibitor

doi: 10.3390/nu17111862

Figure Lengend Snippet: DHM delayed cellular senescence induced by AGEs in HFF-1 cells. ( a ) Representative confocal images of SAHF treated with various concentrations of DHM and 200 μg/mL AGEs, scale = 40 μm; ( b ) Quantification of fluorescence intensity of SAHF ( n = 4); ( c ) Representative image of senescence-associated β-galactosidase (SA-β-Gal) staining in cells (100×) treated with 50 μM DHM and/or 200 μg/mL AGEs, with blue-stained cells (indicated by dark arrows) representing SA-β-Gal-positive cells; ( d ) Quantification of the percentage of SA-β-Gal-positive cells ( n = 6); mRNA expression levels of ( e ) CDKN1A and ( f ) CDKN2A following treatment with 50 μM DHM and 200 μg/mL AGEs ( n = 3); ( g ) Representative Western blot showing p21 expression in treated cells. Data are presented as mean ± SEM. $ p < 0.05 vs. AGEs; & p < 0.05 vs. SC. Abbreviation for groups: SC, cells cultured with complete medium containing 0.025% DMSO; AGEs group, cells treated with 200 μg/mL AGEs; DHM, cells treated with 50 μM DHM; DHM + AGEs, cells treated with 50 μM DHM and 200 μg/mL AGEs.

Article Snippet: Then, the blocked PVDF membranes were incubated overnight at 4 °C with rabbit monoclonal antibodies against p21 Waf1/Cip1 (1:1000, #2947, Cell Signaling Technology, Danvers, MA, USA) or β-actin (1:1000, #4970, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Fluorescence, Staining, Expressing, Western Blot, Cell Culture

Journal: Stem cell research & therapy

Article Title: Chemical reprogramming culture for the expansion of salivary gland epithelial basal progenitor cells.

doi: 10.1186/s13287-025-04295-5

Figure Lengend Snippet: Fig. 4 Small molecules inhibit activation of oncogene-induced senescence and apoptosis in SG-BPCs. A Representative SA-β-galactosidase staining images of SG epithelial cells cultured under KEM and KEM + YAL conditions at PD40. Scale bar: 100 μm. B The β-gal + cells are quantified in conditions without and with the small-molecule compounds. An unpaired Two-tailed t-test is used to calculate significance. Quantification is conducted from a single experiment with three technical repeats and expressed as mean ± SD. ** = p < 0.01. C The gene expression of cellular senescence marker (CDKN1A, CDKN2A, MMP10, and TENM2) in SG epithelial cells untreated and treated with small molecules. D Representative TUNEL staining images of SG epithelial cells in the KEM and KEM + YAL groups at PD5 and PD40. Scale bar: 50 μm. E TUNEL + cells are quantified in conditions without and with the small-molecule compounds. An unpaired Two-tailed t-test is used to calculate significance. Quantification is conducted from a single experiment with three technical repeats and expressed as mean ± SD. * = p < 0.05. F The gene expression of cellular apoptosis markers (BMF and BCL-xL) in SG epithelial cells untreated and treated with small molecules. Two-way ANOVA (alpha = 0.05) is conducted on data presented in (C) and (F) followed by Tukey’s multiple comparisons. The data are representative of three independent experiments performed in triplicate and are expressed as mean ± SEM. * = p < 0.05, *** = p < 0.001

Article Snippet: Membranes were blocked with 5% skim milk and incubated with primary antibodies against p21 (1:000; #2947; CST), p65 (1:1000; #6956; CST), p-p65 (1:500; #3033; CST), SOX2 (1:1000; #9795; Abcam), SOX9 (1:500; #82630; CST), ID3 (1:500; #9837; CST) and β-actin (1:3000; #4967; CST).

Techniques: Activation Assay, Staining, Cell Culture, Two Tailed Test, Gene Expression, Marker, TUNEL Assay

Journal: Cell Death Discovery

Article Title: Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies

doi: 10.1038/s41420-025-02379-y

Figure Lengend Snippet: A Top . Representative images ( n = 3) of SA-β-gal staining in A549 lung cancer cells treated with senescence-inducing concentrations of bleomycin, alisertib, doxorubicin or palbociclib for 7 days (scale bar: 500 μm for proliferative/untreated cells; 200 μm for TIS cells). Bottom left . Expression levels of phospho-RB Ser807/Ser811 and p21 WAF1/Cip1 were detected by immunoblotting in whole cell lysates of proliferative (untreated) and TIS A549 cancer cells (7-day) using specific antibodies and β-actin/GAPDH as loading controls. The figure shows a representative immunoblot from multiple ( n = 3) independent experiments (PLB Palbociclib, DOX Doxorubicin, ALI Alisertib, BLEO Bleomycin). Bottom right . Representative flow cytometry plots showing the gating of the cell cycle distribution of proliferative (untreated) and TIS phenotypes. The histogram shows the percentage (mean ± S.D., n = 3) of cells in the four cell cycle phases as a function of the treatment condition. B Top . Representative microphotographs of proliferative (untreated) and TIS cancer cells labeled with IncuCyte ® Annexin V Green reagent 3 days after harvest and reseeding. Bottom . TIS cancer cells and proliferative (untreated) cells were reseeded into 96-well plates (6000 cells/well and 2000 cells/well, respectively) and cultured with increasing concentrations of ABT-263/navitoclax. After 5 days, cell viability was measured using the alarmarBlue™ assay. Shown are representative images of alamarBlue™-based cell viability assays showing changes in the number of metabolically active cells in response to serial dilutions of BH3 mimetics and dose-response curves for ABT-263/navitoclax in proliferative (untreated) and TIS A549 lung cancer cells (untreated = 100%). Bar graphs of the IC 50 values determined as the μmol/L concentrations of ABT-263/navitoclax required to decrease cell viability by 50%, and the senolytic indexes obtained by dividing the IC 50 values of ABT-263/navitoclax in proliferative A549 cells by those obtained in TIS A549 cells. Data represent the mean ± S.D. of ≥3 independent experiments performed in triplicate. Statistically significant differences (ANOVA analysis) between proliferative and TIS phenotypes means are shown. n.s. not statistically significant. C TIS cancer cells and proliferative (untreated) cells were replated into 96-well plates (6000 cells/well and 2000 cells/well, respectively) and cultured with increasing concentrations of ABT-199/venetoclax, A1331852 or S63845. After 5 days, cell viability was measured using the alarmarBlue™ assay. Shown are representative images of alamarBlue™-based cell viability assays showing changes in the number of metabolically active cells in response to serial dilutions of BH3 mimetics and dose-response curves for ABT-199/venetoclax, A1331852 or S63845 in proliferative (untreated) and TIS A549 lung cancer cells (untreated = 100%). See also Table .

Article Snippet: Antibody against p21 WAF1/CIP1 (12D1; Cat. #2947) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Staining, Expressing, Western Blot, Flow Cytometry, Labeling, Cell Culture, Metabolic Labelling

Journal: Cell Death Discovery

Article Title: Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies

doi: 10.1038/s41420-025-02379-y

Figure Lengend Snippet: A Left . Representative flow cytometry plots showing the gating of the cell cycle distribution of proliferative (untreated) and olaparib-treated IMEC BRCA1 +/+ and IMEC BRCA1 mut/+ cells. The histograms show the percentage (mean ± S.D., n = 3) of cells in the four cell cycle phases as a function of the treatment condition. Right . Expression levels of phospho-RB Ser807/Ser811 and p21 WAF1/Cip1 were detected by immunoblotting in whole cell lysates of proliferative (untreated) and olaparib-treated IMEC BRCA1 +/+ and IMEC BRCA1 mut/+ cells (7-day) using specific antibodies and β-actin/GAPDH as loading controls. The figure shows a representative immunoblot from multiple (n = 3) independent experiments. B . Left . Representative images of SA-β-gal staining in proliferative (untreated) and olaparib-treated IMEC BRCA1 +/+ and IMEC BRCA1 mut/+ cells (7-day) from three independent experiments (scale bar: 200 μm). Right top . Recovery of proliferative potential was evaluated by reseeding untreated and olaparib-treated IMEC BRCA1 +/+ and IMEC BRCA1 mut/+ cells (7-day) cultures (2000 cells/well) in drug-free conditions. After 10 days, cell growth was monitored by crystal violet staining. Shown are representative images from three technical replicates. Right bottom . Representative flow cytometry plots showing the gating of the cell cycle distribution olaparib-treated IMEC BRCA1 mut/+ cells reseeded in drug-free conditions for 3 days. C Top . The synthetic lethal interaction between the PARPi olaparib and DNA repair due to BRCA1 deficiency triggers breast epithelial cancer cell senescence. Bottom . BH3 profiling was performed to measure mitochondrial depolarization in proliferative (untreated) and olaparib-treated IMEC BRCA1 +/+ and IMEC BRCA1 mut/+ breast epithelial cells exposed to activator and sensitizer BH3 peptides. Figure shows a global heat map of % mitochondrial depolarization caused by increasing concentrations of the activator (BIM, BID, PUMA) and sensitizer (BMF, BAD, NOXA, HRK) peptides in proliferative (untreated) and olaparib-treated cells. Data shown are the mean of ≥3 independent experiments using three technical replicates for each peptide. D Proliferative and olaparib-treated IMEC BRCA1 +/+ and IMEC BRCA1 mut/+ cells (7-day) were incubated with serial dilutions of ABT-263/navitoclax, A1331852, and ABT-199/venetoclax. After 5 days, cell viability was measured using the alarmarBlue™ assay. Top . Graphs show the senolytic indexes obtained by dividing the IC 50 values of BH3 mimetics in proliferative IMEC ( BRCA1 +/+ and BRCA1 mut/+ ) cells by those obtained in corresponding olaparib-treated IMEC ( BRCA1 +/+ and BRCA1 mut/+ ) cells. Bottom . Graphs show the senolytic indexes obtained by dividing the IC 50 values of BH3 mimetics in proliferative IMEC ( BRCA1 +/+ and BRCA1 mut/+ ) cells by those obtained in olaparib-treated IMEC BRCA1 +/+ cells. Data represent the mean ( columns ) ± S.D. ( bars ) of 3 or more independent experiments. Statistically significant differences (ANOVA analysis) between the means for untreated and olaparib-treated cells are shown. n.s. not statistically significant. E Global heat map of % mitochondrial depolarization in proliferative (untreated) IMEC BRCA1 +/+ (normal-like breast epithelium, 0), IMEC BRCA1 mut/+ (breast cancer-prone breast epithelium, 1), A549 (lung adenocarcinoma, 2), and LoVo (colon cancer cells, 3).

Article Snippet: Antibody against p21 WAF1/CIP1 (12D1; Cat. #2947) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Flow Cytometry, Expressing, Western Blot, Staining, Incubation

Journal: In Vivo

Article Title: Secretome from HMGB1 Box A‐over‐expressing Adipose‐derived Stem Cells Shows Potential for Skin Rejuvenation by Senescence Reversal in PM2.5‐induced Senescence Cells via Stem Cell Induction

doi: 10.21873/invivo.13881

Figure Lengend Snippet: Effect of PM2.5 on cellular senescence evaluated based on p21/p16 level and reversal of senescence by BoxA-SC. DP and HWPc cells were treated with 50 μg/ml PM2.5 treatment for four days. Then, PM2.5-treated senescent cells were treated with BoxA-SC (1:20) for 48 h. (A) The mRNA expression levels of p21 and p16 were assayed using RT-qPCR in DP cells. The mRNA level was normalized to the mRNA of the housekeeping GAPDH gene. The relative mRNA expression was calculated by using comparative CT cycles. (C) The expression of p21 and p16 was assayed using immunofluorescence staining and the fluorescence intensity was determined using Image J software. (B, D) HWPc cells were assessed for the mRNA levels and protein expression level of p21 and p16 by RT-qPCR and immunofluorescence analysis, respectively. Data are presented as mean±SD (n=3). Significant compared to the control group, *p<0.05, **p<0.01, ***p<0.001 versus untreated control DP or HWPc cells and, # p<0.05, ### p<0.001 versus PM2.5-induced senescence in DP or HWPc cells.

Article Snippet: The rabbit monoclonal antibodies against p21 Waf1/Cip1 (cat no: 2947) and p16 INK4A (cat no: 92803) were acquired from Cell Signaling (Beverly, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Fluorescence, Software, Control