cellular senescence activity assay kit enz-kit 129  (Enzo Biochem)

Journal: bioRxiv

Article Title: Myeloid-specific TFAM deficiency drives mitochondrial DNA stress and exacerbates allergic airway inflammation

doi: 10.1101/2025.05.27.654580

Figure Lengend Snippet: Pharmacological inhibition of senescence improves asthmatic inflammation from DRA-challenged mice. (A) qRT-PCR quantification of the levels of Irf4, Ccl17, Arg1 in BMDM from WT. BMDM were treated with indicated concentrations of ABT-263 (ABT), stimulated with IL-4 and TGFβ. (B) Schematic illustration of ABT-263 (ABT, 1 mg/kg) treatment in DRA-induced mouse asthma model. Total cells and eosinophils influx in BALF were counted based on total amount of BAL cells in WT mice, analyzed by flow cytometry. (C) Total number of cells in DRA-exposed WT and TFAM KO mice. (D) Level of cytokines in BALF of ABT-263 (ABT, 1 mg/kg) treated DRA-exposed WT and TFAM KO mice by ELISA. (E) Lung SA-β-gal activity was measured from WT and TFAM KO using SA-β-gal activity kit. Graphs are plotted as mean ± SD. p-Values were obtained using one-way ANOVA followed by Tukey’s multiple comparison tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Senescence associated-β-galactosidase (SA-β-gal) activity from cells and homogenized lung tissues [ , ] were measured quantitatively using cellular senescence activity assay kit (Cell Signaling Technology) according to the manufacturer’s protocol.

Techniques: Inhibition, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Comparison

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis

doi: 10.1038/s41419-024-07131-5

Figure Lengend Snippet: A Scheme depicting the Hexosamine Biosynthetic Pathway and O -GlcNAcylation processes. GFAT1/2: Glutamine Fructose-6-Phosphate Amido Transferase 1/2 (rate limiting enzyme of the HBP), OGT: O -GlcNAc Transferase, OGA: O -GlcNAcase. B HCT116 cells were treated with increasing concentrations of SN38 ranging from 1 to 25 nM for 96 hours. Cell apoptosis was investigated by Western Blot analyses of cleaved-caspase 7 and cleaved-PARP1. GAPDH was used as a loading control. Data shown are representative of three independent experiments. C–E HCT116 cells were treated with 1 nM SN38 for 96 or with DMSO as a negative control. C Top: Representative images of the microscopic analysis of cell morphology showing an increase in the size of senescent cells. Pictures were taken at the same magnification (x200). Bottom: SA-β-Galactosidase activity assay demonstrating blue staining of senescent cells. D Left: Western blot analysis of O -GlcNAcylation levels, OGT, OGA, and both isoforms of GFAT (GFAT1 and GFAT2), as well as the expression of p21, cyclin D1, and EZH2, three senescence markers. Right: Quantification of relative protein expression from four independent experiments (optical density measurement relative to GAPDH) ( n = 4) (individual values and mean +/- SEM, ns: non-significant, * P < 0.05, ** P < 0.01, multiple unpaired t -tests). E qRT-PCR analysis of p21, cyclin D1, EZH2, GFAT1, GFAT2, OGT, and OGA transcript expression. Results represent the individual values and mean +/- SEM of five independent experiments ( n = 5) (ns: non-significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001, multiple unpaired t -tests).

Article Snippet: SA-β-gal activity was assessed using the cellular senescence activity assay kit (Enzo, ENZ-KIT 129) following the instructions provided in the product manual.

Techniques: Western Blot, Control, Negative Control, Activity Assay, Staining, Expressing, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis

doi: 10.1038/s41419-024-07131-5

Figure Lengend Snippet: HCT116 cells were transfected with a siRNA targeting OGA (si OGA) or a non-target control siRNA (siCTRL). 24 h later, the cells were treated with 1 nM SN38 for 72 hours. A Western blot analysis was performed to assess the expression of the three senescence markers p21, cyclin D1, and EZH2. The efficiency of siRNA was also confirmed by evaluating OGA los of expression and O-GlcNAcylation levels upregulation. GAPDH was used as a loading control. Results shown are representative of three independent experiments. B Quantitative fluorimetric determination of the SA-β-Galactosidase activity in the different experimental conditions. Results represent the individual values and mean +/- SEM of four independent experiments ( n = 4) (ns: non-significant, * P < 0.05, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests). Quantification of relative protein expression analyzed by Western Blot (optical density measurement relative to GAPDH) of p21 ( C ), cyclin D1 ( D ) and EZH2 ( E ) from three independent experiments ( n = 3) (individual values and mean +/- SEM, ns: non-significant, * P < 0.05, *** P < 0.001, **** P < 0.0001, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests).

Article Snippet: SA-β-gal activity was assessed using the cellular senescence activity assay kit (Enzo, ENZ-KIT 129) following the instructions provided in the product manual.

Techniques: Transfection, Control, Western Blot, Expressing, Activity Assay, Comparison

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis

doi: 10.1038/s41419-024-07131-5

Figure Lengend Snippet: HCT116 cells were transfected with a siRNA targeting GFAT1 and GFAT2 (si GFAT1/2) or a non-target control siRNA (siCTRL). 24 h later, the cells were treated with 1 nM SN38 for 72 h. A Western blot analysis was performed to assess the expression of the senescence markers p21 and cyclin D1, the DNA damage marker γH2AX and the apoptosis marker cleaved-caspase 7. The efficiency of siRNA was also confirmed by evaluating GFAT1 and GFAT2 loss of expression. GAPDH was used as a loading control. Results shown are representative of three independent experiments. B Quantitative fluorimetric determination of the SA-β-Galactosidase activity in the different experimental conditions. Results represent the individual values and mean +/- SEM of four independent experiments ( n = 4) (ns: non-significant, ** P < 0.01, *** P < 0.001, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests). Quantification of relative protein expression analyzed by Western Blot (optical density measurement relative to GAPDH) of γH2AX ( C ) and cleaved-caspase 7 ( D ) from three independent experiments ( n = 3) (individual values and mean +/- SEM, ns: non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests). E Quantification of the ratio of viable cells through MTT assay from three independent experiments ( n = 3) (individual values and mean +/- SEM, **** P < 0.0001, *** P < 0.001, * P < 0.05, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests).

Article Snippet: SA-β-gal activity was assessed using the cellular senescence activity assay kit (Enzo, ENZ-KIT 129) following the instructions provided in the product manual.

Techniques: Transfection, Control, Western Blot, Expressing, Marker, Activity Assay, Comparison, MTT Assay

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis

doi: 10.1038/s41419-024-07131-5

Figure Lengend Snippet: HCT116 cells were transfected with a siRNA targeting OGT (si OGT) or a non-target control siRNA (siCTRL). 24 h later, the cells were treated with 1 nM SN38 for 72 h. A Western blot analysis was performed to assess the expression of the senescence markers p21 and cyclin D1, the DNA damage marker γH2AX and the apoptosis marker cleaved-caspase 7. The efficiency of siRNA was also confirmed by evaluating GFAT1 and GFAT2 loss of expression. GAPDH was used as a loading control. Results shown are representative of four independent experiments. B Quantitative fluorimetric determination of the SA-β-Galactosidase activity in the different experimental conditions. Results represent the individual values and mean +/- SEM of four independent experiments ( n = 4) (ns: non-significant, ** P < 0.01, *** P < 0.001, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests). Quantification of relative protein expression analyzed by Western Blot (optical density measurement relative to GAPDH) of γH2AX (C) and cleaved-caspase 7 (D) from three independent experiments ( n = 3) (individual values and mean +/- SEM, ns: non-significant, * P < 0.05, ** P < 0.01, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests). E Quantification of the ratio of viable cells through MTT assay from four independent experiments (n = 4) (mean +/- SEM, ** P < 0.01, **** P < 0.0001, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests).

Article Snippet: SA-β-gal activity was assessed using the cellular senescence activity assay kit (Enzo, ENZ-KIT 129) following the instructions provided in the product manual.

Techniques: Transfection, Control, Western Blot, Expressing, Marker, Activity Assay, Comparison, MTT Assay

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis

doi: 10.1038/s41419-024-07131-5

Figure Lengend Snippet: HCT116 cells were treated with increasing concentrations of the OGT inhibitor OSMI-4 ranging from 5 to 20 µM. 24 h later, the cells were treated with 1 nM SN38 for 72 h. A Western blot analysis was performed to assess the expression of the senescence markers p21 and cyclin D1, the DNA damage marker γH2AX and the apoptosis markers cleaved-caspase 7 and cleaved-PARP1. The treatment efficiency was also confirmed by evaluating O -GlcNAcylation levels. GAPDH was used as a loading control. Results shown are representative of three independent experiments. B Quantitative fluorimetric determination of the SA-β-Galactosidase activity in the different experimental conditions. Results represent the individual values and mean +/- SEM of three independent experiments ( n = 3) (ns: non-significant, * P < 0.05, ** P < 0.01, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests). Quantification of relative protein expression analyzed by Western Blot (optical density measurement relative to GAPDH) of γH2AX ( C ) cleaved-caspase 7 ( D ) and cleaved-PARP1 ( E ) from three independent experiments ( n = 3) (individual values and mean +/- SEM, ns: non-significant, * P < 0.05, ** P < 0.01, **** P < 0.0001, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests). F Quantification of the ratio of viable cells through MTT assay from three independent experiments ( n = 3) (mean +/- SEM, *** P < 0.001, **** P < 0.0001, ordinary one-way ANOVA with Fisher’s LSD multiple comparison tests).

Article Snippet: SA-β-gal activity was assessed using the cellular senescence activity assay kit (Enzo, ENZ-KIT 129) following the instructions provided in the product manual.

Techniques: Western Blot, Expressing, Marker, Control, Activity Assay, Comparison, MTT Assay

Journal: Aging Cell

Article Title: Lens capsule advanced glycation end products induce senescence in epithelial cells: Implications for secondary cataracts

doi: 10.1111/acel.14249

Figure Lengend Snippet: Senescent cells are present in the posterior lens capsule of pseudophakic eyes from patients and in vitro human capsular bag cultures. Representative image of an IOL‐containing capsule isolated from a 73‐year‐old pseudophakic human eye (a). Senescent cells were detected by senescence‐associated beta‐galactosidase (SA‐β‐gal)‐staining. A representative image of a posterior capsule lacking senescent cells in a pseudophakic eye (65‐year‐old) (b). A representative image showing senescent cells (indicated by arrows) in the posterior capsule of a 73‐year‐old (c). The enlarged image of C shows senescent cells at matrix contraction sites (d). Representative images show phase‐contrast and SA‐β‐gal positive senescent cells cultured under serum‐free conditions in 43‐ and 78‐year‐old capsular bags for 28 days (e–h). LECs cultured in six capsular bags from 43‐ to 82‐year‐old donors were quantified for the percentage of SA‐β‐gal‐stained cells (i) and p21 levels (j). Scale bars = 100 μm.

Article Snippet: After 96 h of culturing on ECM‐AGEs, cells were labeled for SA‐β‐gal using Cellular Senescence Live Cell Activity Assay Kit (ENZ‐KIT130, ENZO, Farmingdale, NY) and sorted into a senescent and non‐senescent population using flow cytometry according to manufacturer's protocol.

Techniques: In Vitro, Isolation, Staining, Cell Culture

Journal: Aging Cell

Article Title: Lens capsule advanced glycation end products induce senescence in epithelial cells: Implications for secondary cataracts

doi: 10.1111/acel.14249

Figure Lengend Snippet: Aged human lens capsules and ECM‐AGEs promote the senescence of LECs. Human primary LECs (from a 16‐year‐old lens) were seeded on acellular young (19‐year‐old) and aged (72‐year‐old) posterior capsules, and after 3 days, cells were screened for senescence markers. Representative microscopic images show SA‐β‐gal positive cells (blue) (a), and the bar graph shows the percentage of SA‐β‐gal positive cells (b). Cells were cultured on young (17‐ to 22‐year‐old, n = 9) and aged (71‐ to 73‐year‐old, n = 9) capsules for which CDKN2A (p16) and CDKN1A (p21) mRNA levels were measured at 48 h and normalized to GAPDH (c, d, n = 5). FHL124 cells were cultured on ECM or ECM‐AGEs for 96 h. The expressions of p16 (e), p21 (f), and p38 (g) in the cell lysates were measured by western blotting (h) and normalized to the β‐actin loading control ( n = 3). Data are mean ± SD of the indicated number of independent experiments. * p < 0.05; ** p < 0.01; **** p < 0.0001. Scale bar = 50 μm.

Article Snippet: After 96 h of culturing on ECM‐AGEs, cells were labeled for SA‐β‐gal using Cellular Senescence Live Cell Activity Assay Kit (ENZ‐KIT130, ENZO, Farmingdale, NY) and sorted into a senescent and non‐senescent population using flow cytometry according to manufacturer's protocol.

Techniques: Capsules, Cell Culture, Western Blot, Control

Journal: Aging Cell

Article Title: Lens capsule advanced glycation end products induce senescence in epithelial cells: Implications for secondary cataracts

doi: 10.1111/acel.14249

Figure Lengend Snippet: LEC senescence is AGE‐level and ROS‐dependent. ECM was coated in tissue culture wells for 24 h and incubated with or without a glycation mixture (0.25, 0.5, 1, 2×) for 7 days. FHL124 cells were cultured on ECM‐AGEs for 48 h to assess the mRNA levels of CDKN2A (p16) and CDKN1A (p21) (a, b), for 96 h to assess the expression of p21 and p16 protein (c–e) and for 24 h to measure ROS production (f). FHL124 cells on ECM or ECM‐AGEs were treated with or without 250 μM GKT136901 (GKT) for 72 h, and p16 and p21 expressions were measured by western blotting (g–i). Data are mean ± SD of three independent experiments. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After 96 h of culturing on ECM‐AGEs, cells were labeled for SA‐β‐gal using Cellular Senescence Live Cell Activity Assay Kit (ENZ‐KIT130, ENZO, Farmingdale, NY) and sorted into a senescent and non‐senescent population using flow cytometry according to manufacturer's protocol.

Techniques: Incubation, Cell Culture, Expressing, Western Blot

Journal: Aging Cell

Article Title: Lens capsule advanced glycation end products induce senescence in epithelial cells: Implications for secondary cataracts

doi: 10.1111/acel.14249

Figure Lengend Snippet: A RAGE antagonist inhibits senescence and EMT of LECs cultured on ECM‐AGEs. FHL124 cells were cultured on ECM or ECM‐AGEs. After 24 h, cells were treated with or without 20 μM RAGE antagonist FPSZM1 for an additional 72 h, then IL‐6 was measured from the collected media (a), and p16 was measured in cell lysates (b). Senescent cells were detected by senescence‐associated beta‐galactosidase (SA‐β‐gal)‐staining. Representative images of cells grown on ECM, ECM and FPSZM1 treatment, ECM‐AGEs, and ECM‐AGEs with FPSZM1 treatment (c) are shown. The expression of α‐SMA (d), fibronectin (e), and collagen I (f) were measured in cell lysates by western blotting (g). Wild‐type (WT) and RAGE knock‐out (RAGE KO) mouse LECs (mLECs) were cultured on ECM or ECM‐AGEs for 96 h. Senescent cells in WT and RAGE KO mLECs were detected by SA‐β‐gal‐staining and representative images of WT mLECs cultured on ECM and ECM‐AGEs, and RAGE KO mLECs cultured on ECM and ECM‐AGEs are shown in (h). p16 (i) and p21 (j) levels in cell lysates were measured by western blotting (k). Data are mean ± SD of three independent experiments. ns, not significant, * p < 0.05, *** p < 0.001, **** p < 0.0001. Scale bars = 10 μm.

Article Snippet: After 96 h of culturing on ECM‐AGEs, cells were labeled for SA‐β‐gal using Cellular Senescence Live Cell Activity Assay Kit (ENZ‐KIT130, ENZO, Farmingdale, NY) and sorted into a senescent and non‐senescent population using flow cytometry according to manufacturer's protocol.

Techniques: Cell Culture, Staining, Expressing, Western Blot, Knock-Out