sa β gal staining solution  (Cell Signaling Technology Inc)

Journal: Biochemistry and Biophysics Reports

Article Title: Screening of metabolic-related biomarkers linking intervertebral disc degeneration and type 2 diabetes based on comprehensive bioinformatics analysis and machine learning

doi: 10.1016/j.bbrep.2026.102593

Figure Lengend Snippet: BCAA metabolism and immune changes in degenerated NP cells. (A) qRT-PCR was conducted to analysis the mRNA levels of BCAA metabolism-related enzymes and BCAA transporter in grade I/II and grade III/IV NP tissues; n = 20; P < 0.05. (B, C) Expression levels of mRNA for pro-inflammatory factors, BCAA metabolism-related enzymes, and BCAA transporters in control group and TNF-α-treated HNPC cells. (D) Expression of SA-β-gal in HNPC cells stimulated with different concentrations of TNF-α. (E, F) p16, p21, p53 mRNA level in TNF-α-stimulated HNPC cells,and correlation analysis with TNF-α concentration.

Article Snippet: Cellular senescence was assessed using Senescence-Associated β-Galactosidase (SA-β-gal) staining (Senescence β-Galactosidase Staining Kit, #C0602, Beyotime, China), performed according to the manufacturer's protocol.

Techniques: Quantitative RT-PCR, Expressing, Control, Concentration Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS attenuates full-blown bone marrow senescence during GC-induced skeletal degeneration. ( A ) Schematic illustration of the experimental design for assessing bone marrow senescence at 4 weeks after combined SCS and MPS treatment. ( B ) Representative images of SA-β-Gal–positive cells (green) in femur after MPS treatment. BM indicates bone marrow; TBM indicates trabecular bone matrix. (Scale bars, 100 μm and 25 μm) ( C – E ) Representative immunofluorescence images at week 4 showing Emcn + sinusoidal ECs, ALP + osteoblasts, and p16 + senescent cells (C), with corresponding quantification of Emcn + p16 + (D) and ALP + p16 + cells (E). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) ( F – H ) Flow cytometry analysis of CD45 − Ter119 − CD31 + arteriolar ECs in the femur after PBS or SCS treatment (F). Ki-67 + proliferative status was further analyzed within this population (G), and corresponding double-positive cell quantification is shown in (H). n = 6 biological replicates. ( I – K ) Representative flow cytometry plots of CD45 − Ter119 − CD31 − leptin receptor + (LepR + ) mesenchymal stem cells (MSCs) in the bone marrow at 4 weeks (I), with analysis of the proportion of SA-β-Gal–positive cells (J) and corresponding quantification (K). n = 6 biological replicates. ( L ) Representative flow cytometry plots of CD45 − Ter119 − CD144 + cells (including endothelial cells and endothelial progenitors) in the bone marrow at week 4 post-MPS treatment. ( M and N ) Gating and analysis of CD45 − Ter119 − CD144 + HMGB1 + ECs by flow cytometry (M), and corresponding quantification (N). n = 6 biological replicates. ( O and P ) Representative immunofluorescence images showing OPN + osteoblasts and γ-H2A.X + DNA damage marker–positive cells in the femur at 4 weeks (O), with quantification of senescent osteoblasts (P). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined using an unpaired two-tailed Student's t -test ( D, E, H, K, N and P ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Immunofluorescence, Flow Cytometry, Marker, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Comparative analysis of SCS and D + Q drugs on glucocorticoid-induced bone marrow senescence inhibition. ( A ) Schematic diagram showing the treatment of SCS and D + Q after glucocorticoid-induced senescence. ( B and C ) Representative flow cytometry images of bone marrow SA-β-Gal for senescence detection on day 42 (B), with corresponding quantification analysis (C). n = 6 biological replicates. ( D and E ) ELISA detection of TNF-α and IL-1β levels in bone marrow supernatant. n = 6 biological replicates. ( F ) Schematic diagram of SCS and D + Q treatment in the early stage of glucocorticoid-induced senescence. ( G and H ) Representative flow cytometry images of p16-positive senescent cells in bone marrow on day 42 (G), with corresponding quantification analysis (H). n = 6 biological replicates. ( I and J ) ELISA detection of TNF-α and IL-1β levels in bone marrow supernatant. n = 6 biological replicates. ( K-M ) Representative images of HE staining of the distal femur with macro and high-magnification images (K), and quantification of trabecular and cortical bone empty lacunae (L and M). n = 6 biological replicates. (Scale bars, 550 μm and 25 μm) ( N and O ) Representative ALP staining images of in vitro osteogenic differentiation of bone marrow LepR + MSCs after 14 days (N), with corresponding quantification analysis (O). n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C , D , E , H , I , J , L , M and O ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Inhibition, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, In Vitro

Journal: Neoplasia (New York, N.Y.)

Article Title: The Cyclin C-CDK8/19 Mediator kinase module controls PRCC-TFE3 driven senescence in renal epithelium and tumorigenesis in TFE3-RCC

doi: 10.1016/j.neo.2026.101296

Figure Lengend Snippet: PRCC-TFE3 expression induces oncogene-induced senescence (OIS) . (A) Growth curves of doxycycline (Dox) inducible PRCC-TFE3 expressing HK-2 and HEK293 cells cultured in the presence or absence of Dox. Induction of PRCC-TFE3 markedly suppressed cell proliferation compared with non-induced controls (n = 3). (B) Senescence associated β-galactosidase (SA-β-gal) staining of HK-2 cells cultured in the absence or presence of Dox for 5 days. SA-β-gal positive cells were observed only upon PRCC-TFE3 induction (Dox+), whereas no positive cells were detected under non-induced conditions (Dox−). Black arrows indicate SA-β-gal positive cells. Representative images from three independent experiments are shown. Scale bar, 200 μm. (C) RT–qPCR analysis of lamin B1 mRNA levels in non-induced and PRCC-TFE3 induced HK-2 cells, showing reduced lamin B1 expression upon PRCC-TFE3 induction, a hallmark of cellular senescence (n = 3). (D) RT–qPCR analysis of senescence associated secretory phenotype (SASP) factor mRNAs (IL1A, IL6, and VEGF) in non-induced and PRCC-TFE3 induced HK-2 cells. PRCC-TFE3 induction significantly increased SASP gene expression (n = 3). (E, F) Cell cycle distribution of PRCC-TFE3 Dox-inducible HK-2 cells analyzed by BrdU incorporation and propidium iodide (PI) staining followed by flow cytometry at the indicated time points after doxycycline addition (Day 0, 1, 3, and 5). Cells were labeled with BrdU for 90 min prior to fixation. Representative flow cytometry plots are shown in (E). Quantitative analysis in (F) demonstrates a progressive accumulation of cells in the G0/G1 phase accompanied by a concomitant reduction in S phase entry upon PRCC-TFE3 induction (n = 3). (G) Western blot analysis of PRCC-TFE3, Rb, phosphorylated Rb (Ser780 and Ser807/811), p16, p21, p27, p53, CDK2, and CDK4 in PRCC-TFE3 Dox-inducible HK-2 cells at the indicated time points after induction. β-actin was used as a loading control. (H) Validation of p21 or p53 knockdown efficiency by RT-qPCR in PRCC-TFE3 Dox-inducible HK-2 cells stably expressing shRNA targeting luciferase (Control), p21 (p21 KD), or p53 (p53 KD) (left). Growth curves of the corresponding cell lines cultured with or without Dox, showing that PRCC-TFE3 induced growth arrest is partially relieved by p21 or p53 knockdown (right) (n = 3). Data are presented as means ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t-test for two-group comparisons, or two-way ANOVA followed by Sidak’s post hoc test for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: Cells were fixed and stained for senescence-associated β-galactosidase (SA-β-gal) activity using a commercially available SA-β-gal assay kit (Cell Signaling Technology), according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Staining, Quantitative RT-PCR, Gene Expression, BrdU Incorporation Assay, Flow Cytometry, Labeling, Western Blot, Control, Biomarker Discovery, Knockdown, Stable Transfection, shRNA, Luciferase, Two Tailed Test

Journal: Neoplasia (New York, N.Y.)

Article Title: The Cyclin C-CDK8/19 Mediator kinase module controls PRCC-TFE3 driven senescence in renal epithelium and tumorigenesis in TFE3-RCC

doi: 10.1016/j.neo.2026.101296

Figure Lengend Snippet: Loss of CCNC alleviates PRCC-TFE3 induced oncogene-induced senescence (OIS) . (A) Growth curves of PRCC-TFE3 doxycycline (Dox)-inducible HK-2 cell lines stably established by lentiviral transduction with an empty vector (Control) or with CCNC targeting sgRNAs (CCNC-g1 and CCNC-g2), resulting in CCNC knockout (KO), and cultured in the presence or absence of Dox. Cell numbers were measured at the indicated time points (n = 3). Notably, CCNC KO alleviated the growth suppression induced by PRCC-TFE3 expression. (B) Senescence-associated β-galactosidase (SA-β-gal) staining of control and CCNC KO PRCC-TFE3 doxycycline (Dox) inducible HK-2 cells after 5 days of PRCC-TFE3 induction. Robust SA-β-gal-positive cells were observed upon PRCC-TFE3 induction in control cells, whereas CCNC KO markedly attenuated SA-β-gal staining. Black arrows indicate SA-β-gal positive cells. Representative images from three independent experiments are shown. Scale bar, 200 μm. (C) RT-qPCR analysis of lamin B1 mRNA levels in non-induced and PRCC-TFE3 induced control and CCNC KO HK-2 cells, showing restoration of lamin B1 expression upon CCNC deletion (n = 3). (D) RT-qPCR analysis of senescence associated secretory phenotype (SASP) factor mRNAs (IL1A, IL6, and VEGF) in non-induced and PRCC-TFE3 induced control and CCNC KO HK-2 cells (n = 3). Induction of PRCC-TFE3 triggered a senescence associated increase in SASP factor expression in control cells, whereas this response was markedly attenuated upon CCNC KO. (E, F) Cell-cycle analysis of PRCC-TFE3 doxycycline (Dox)-inducible control and CCNC KO HK-2 cells. Cells were cultured in the presence or absence of Dox for 3 days, labeled with BrdU for 90 min, fixed, stained with phycoerythrin (PE)-conjugated anti-BrdU antibodies, and counterstained with propidium iodide (PI). Representative flow cytometry plots are shown in (E), and quantification of the G0/G1, S, and G2/M populations is summarized in (F) (n = 3). PRCC-TFE3 induction increased the G0/G1 fraction and reduced S-phase entry in control cells, whereas CCNC KO markedly attenuated these PRCC-TFE3-induced cell cycle changes. Data are presented as means ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t-test for two-group comparisons, or two-way ANOVA followed by Sidak’s post hoc test for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: Cells were fixed and stained for senescence-associated β-galactosidase (SA-β-gal) activity using a commercially available SA-β-gal assay kit (Cell Signaling Technology), according to the manufacturer’s instructions.

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Control, Knock-Out, Cell Culture, Expressing, Staining, Quantitative RT-PCR, Cell Cycle Assay, Labeling, Flow Cytometry, Two Tailed Test

Journal: Neoplasia (New York, N.Y.)

Article Title: The Cyclin C-CDK8/19 Mediator kinase module controls PRCC-TFE3 driven senescence in renal epithelium and tumorigenesis in TFE3-RCC

doi: 10.1016/j.neo.2026.101296

Figure Lengend Snippet: Pharmacological inhibition of CDK8/19 alleviates PRCC-TFE3 induced oncogene-induced senescence (OIS) . (A) Growth curves of PRCC-TFE3 doxycycline (Dox)-inducible HK-2 cells treated with the CDK8/19 inhibitor MSC2530818 (100 nM) in the presence or absence of Dox. Cell numbers were measured at the indicated time points (n = 3). MSC2530818 reduced basal proliferation in Dox(–) cells; however, it markedly alleviated the growth suppression caused by PRCC-TFE3 induction upon Dox treatment. (B) Senescence-associated β-galactosidase (SA-β-gal) staining of PRCC-TFE3 Dox inducible HK-2 cells cultured for 5 days in the presence or absence of the CDK8/19 inhibitor MSC2530818. PRCC-TFE3 induction robustly increased SA-β-gal positive senescent cells, whereas MSC2530818 treatment markedly attenuated PRCC-TFE3 induced cellular senescence. Black arrows indicate SA-β-gal positive cells. Representative images from three independent experiments are shown. Scale bar, 200 μm. (C) RT-qPCR analysis of lamin B1 mRNA levels in non-induced and PRCC-TFE3 induced HK-2 cells treated with MSC2530818 (100 nM) (n = 3). MSC2530818 treatment reduced basal lamin B1 expression in non-induced cells; however, no additional decrease in lamin B1 levels was observed upon PRCC-TFE3 induction in the presence of MSC2530818, indicating that CDK8/19 inhibition prevents PRCC-TFE3 associated lamin B1 downregulation. (D) RT-qPCR analysis of senescence-associated secretory phenotype (SASP) factor mRNAs (IL1A, IL6, and VEGF) in non-induced and PRCC-TFE3 induced HK-2 cells treated with MSC2530818 (100 nM) (n = 3). PRCC-TFE3 induction was associated with robust upregulation of SASP factor expression, consistent with the induction of oncogene-induced senescence. Pharmacological inhibition of CDK8/19 by MSC2530818 markedly attenuated this SASP response, indicating suppression of PRCC-TFE3 induced senescence. (E) Cell-cycle analysis of PRCC-TFE3 doxycycline (Dox)-inducible HK-2 cells cultured for 3 days under the indicated combinations of Dox (−/+) and the CDK8/19 inhibitor MSC2530818 (100 nM). Cells were labeled with BrdU for 90 min, fixed, stained with phycoerythrin (PE)-conjugated anti-BrdU antibodies, and counterstained with propidium iodide (PI). Representative flow cytometry plots are shown (top), and quantitative analyses of the G0/G1, S, and G2/M populations are summarized (bottom) (n = 3). MSC2530818 treatment largely abrogated PRCC-TFE3 induced cell cycle arrest, restoring S phase entry. (F) Immunofluorescence staining of Cyclin C and HA-tagged PRCC-TFE3 in PRCC-TFE3 Dox-inducible HK-2 cells cultured in the absence (left) or presence of doxycycline (right). Upon PRCC-TFE3 induction, Cyclin C exhibits prominent punctate nuclear localization. Nuclei were counterstained with DAPI. Representative images are shown. Scale bars, 10 μm. Quantification of cells displaying Cyclin C nuclear puncta is shown on the right (n = 3). (G) Chromatin immunoprecipitation (ChIP)-qPCR analysis of HA-tagged PRCC-TFE3 and Cyclin C occupancy at the indicated gene regulatory regions in PRCC-TFE3 Dox-inducible HK-2 cells (n = 3). Induction of PRCC-TFE3 resulted in robust recruitment of PRCC-TFE3 to these genomic regions, accompanied by a concomitant increase in Cyclin C binding at the same loci, suggesting coordinated engagement of PRCC-TFE3 and Cyclin C at shared transcriptional regulatory sites. Data are presented as means ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t-test for two-group comparisons, or two-way ANOVA followed by Sidak’s post hoc test for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: Cells were fixed and stained for senescence-associated β-galactosidase (SA-β-gal) activity using a commercially available SA-β-gal assay kit (Cell Signaling Technology), according to the manufacturer’s instructions.

Techniques: Inhibition, Staining, Cell Culture, Quantitative RT-PCR, Expressing, Cell Cycle Assay, Labeling, Flow Cytometry, Immunofluorescence, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: OMICS Profiling Identifies Signatures of Senescence in Osteogenesis Imperfecta Osteoblasts Counteracted by 4‐PBA

doi: 10.1111/jcmm.71120

Figure Lengend Snippet: Senescence is attenuated by 4‐PBA in Col1a1 +/G349C and Col1a2 +/G610C OBs. (A) Scheme of the main genes involved in the regulation of cell cycle progression. Real time PCR analyses of P53 (B), P16 (C), Ki67 (D), Lmnb1 (E), and Foxo3 (F) expression in Col1a1 +/G349C and Col1a2 +/G610C OBs in absence or presence of 4‐PBA. (G, H) SA‐β‐gal staining and quantification of senescent cells in Col1a1 +/G349C and Col1a2 +/G610C OBs in absence or presence of 4‐PBA. Biological triplicates for each experiment were performed. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Senescence‐associated beta‐galactosidase (SA‐β‐Gal) staining of primary osteoblasts was performed in 24 well plates according to the manufacturers' instructions (Cell Signalling Technology), with a cell confluence at staining around 60%.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining

Journal: Journal of Pharmaceutical Analysis

Article Title: Novel bioactive peptides targeting Keap1-Nrf2 interaction for combating UVA-induced skin aging: Computational discovery and experimental validation

doi: 10.1016/j.jpha.2025.101446

Figure Lengend Snippet: Protective effects of Seq1 and Seq3 on ultraviolet A (UVA)-induced cellular aging in human keratinocytes (HaCaT) cells. (A) The effect of peptide treatment on the migration of HaCaT cells. (B) The extent of wound closure was quantified and depicted on a histogram. (C) Assessment of the effects of peptides on senescence associated β-galactosidase (SA-β-Gal) activity in HaCaT cells. (D) Quantitation of SA-β-gal positive cells in HaCaT cells. (E) Immunofluorescence (IF) analysis of phosphorylated γ-H2AX in response to UVA irradiation and peptide treatment at varying concentrations to determine their influence. (F) 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining reveals intracellular reactive oxygen species (ROS) levels in UVA-irradiated HaCaT cells following peptide or tert-butylhydroquinone (t-BHQ) treatment. (G) Fluorescence intensity of ROS. (H) Western blot analysis of Seq1 and Seq3 on the expression of matrix metalloproteinase (MMP)-1 and MMP-9 in UVA-induced HaCat cells. (I) Relative MMP-1 and MMP-9 messenger RNA (mRNA) levels in Seq1 and Seq3 treated UVA-induced HaCat cells. Gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). (J) Western blot analysis showing the change of inducible nitric oxide synthase (iNOS) and interleukin-1 beta (IL-1β) in HaCat cells. (K) Quantitation of IL-1β and tumor necrosis factor-alpha ( TNF-α ) released by HaCat cells by quantitative real-time polymerase chain reaction (qRT-PCR). (L) Glutathione peroxidase (GSH-Px) activity levels in HaCaT cells. (M) Measurement of superoxide dismutase (SOD) activity levels in HaCaT cells. (N) Nrf2-dependent antioxidant enzymes protein levels for reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), and glutamate-cysteine ligase modifier subunit (GCLM) in UVA-irradiated HaCaT cells treated with peptides. Protein expression normalized to GAPDH. (O) Quantitation of GCLM , HO-1 , and NQO1 released by HaCat cells by qRT-PCR. Unless otherwise indicated in the figure, the peptide concentration was 20 μM. All data are presented as means ± standard deviation (SD) ( n = 3). Statistical significance is denoted by ∗ P < 0.05 , ∗∗ P < 0.01 , ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 vs. UVA-irradiated group.

Article Snippet: To evaluate cellular senescence induced by peptides and UVA treatments, HaCaT cells were stained using the SA-β-Gal Staining Kit (C0602, Beyotime, Shanghai, China), following the manufacturer's guidelines.

Techniques: Migration, Activity Assay, Quantitation Assay, Immunofluorescence, Irradiation, Staining, Fluorescence, Western Blot, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Concentration Assay, Standard Deviation