rabbit anti sa β gal  (Cell Signaling Technology Inc)

Journal: Journal of Advanced Research

Article Title: Impaired stemness in aging periodontal ligament stem cells is mediated by the progerin/endoplasmic reticulum stress/p53 axis

doi: 10.1016/j.jare.2024.10.029

Figure Lengend Snippet: ATF6 regulated the stemness of OPDLSCs through p53/p21 pathway. (a) SA-β-gal staining in YPDLSCs and OPDLSCs. Scale bar, 200 μm. (b) The p53, p21, and p16 proteins levels in YPDLSCs and OPDLSCs. (c) SA-β-gal staining after transfection with the vector or ATF6 plasmid in YPDLSCs. Scale bar, 200 μm. (d) The p53, p21, and p16 proteins levels in YPDLSCs transfected with vector or ATF6 plasmid. (e) SA-β-gal staining in OPDLSCs transfected with scrambled or ATF6 siRNA. Scale bar, 200 μm. (f) The p53, p21, and p16 proteins levels in OPDLSCs transfected with scrambled or ATF6 siRNA. (g) The binding sites of ATF6 on the promoter of p53, and the design for luciferase reporters. (h) Dual luciferase assay was performed to detect the luciferase activity after co-transfection of vector or ATF6 plasmid together with WT-p53 or MUT-p53. Statistical significance was defined as * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Immunofluorescence staining was performed with antibodies against ATF6 (66563-1-lg, 1:200), SA-β-gal (15518-1-AP, 1:200), p53 (10442-1-AP, 1:200), and p21 (28248-1-AP, 1:200) (all from Proteintech).

Techniques: Staining, Transfection, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay, Cotransfection

Journal: Journal of Advanced Research

Article Title: Impaired stemness in aging periodontal ligament stem cells is mediated by the progerin/endoplasmic reticulum stress/p53 axis

doi: 10.1016/j.jare.2024.10.029

Figure Lengend Snippet: Inhibition of ATF6-mediated ER stress rescued the age-related alveolar bone loss via the p53/p21 pathway. (a) The SA-β-gal, p53, p21 expression in the alveolar bone of the young and old rats were explored by immunofluorescence. Scale bar, 50μm. (b) Micro-CT showed the alveolar bone loss in the SD rats. One site for each root of two molars were measured. (c) Ceapin-A7 (concentration, 0.06, 0.12, 0.6, 6, or 12 μM) was injected into the old SD rats, and immunofluorescence was performed to explore the expression of ATF6. Scale bar, 50 μm. (d) The SA-β-gal, p53, p21 expression in the alveolar bone of the 4-PBA (5 mM) and Ceapin-A7 (0.6 μM) groups compared to that in the saline (10 μL) group. Scale bar, 50 μm. (e) Micro-CT showed the alveolar bone loss in the SD rats of 4-PBA (5 mM) and Ceapin-A7 (0.6 μM) groups compared to that in the saline (10 μL) group. One site for each root of two molars were measured. (ab, alveolar bone. PDL, periodontal ligament). Statistical significance was defined as * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Immunofluorescence staining was performed with antibodies against ATF6 (66563-1-lg, 1:200), SA-β-gal (15518-1-AP, 1:200), p53 (10442-1-AP, 1:200), and p21 (28248-1-AP, 1:200) (all from Proteintech).

Techniques: Inhibition, Expressing, Immunofluorescence, Micro-CT, Concentration Assay, Injection, Saline

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Amelioration of experimental tendinopathy by lentiviral CD44 gene therapy targeting senescence-associated secretory phenotypes

doi: 10.1016/j.omtm.2022.06.006

Figure Lengend Snippet: Expression levels of p21, p53, and p16 in tendon tissues of normal hamstring (Ham) tendon during anterior cruciate ligament reconstruction and long head of bicep (LHB) with tendinopathic changes during surgical intervention (A and B) Representative figures of hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining for senescence-associated β-galactosidase (SA-β-gal), p53, p21, and p16 in tendon tissues of normal Ham (n = 10) and LHB (n = 12) tendons with tendinopathic changes (moderate to severe) during surgical intervention. Scale bars represent 20 μm in ×400 magnifications. Arrow heads indicate positive cells. (B) Harvested tissues from Ham, moderate, and severe LHB were subjected to IHC staining, and the p53-, p21-, p16-, and SA-β-gal-positive cells were counted and normalized with hematoxylin-counterstained total cells. Values are represented as the mean ± standard error of the mean (SEM). Spearman correlation rank test was used. (C) Representative figures of SA-β-gal activity in human tendon tissues from the normal Ham and LHB tendons. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) was used for nuclear staining. Arrow heads indicate SA-β-gal and DAPI double-positive cells. Scale bars represent 20 μm in ×400 magnifications. The blue-boxed areas are higher-magnification views of the red-boxed areas. (D) Representative figures of immunoblotting for p53, p21, and p16 expression levels (n = 2) and SA-β-gal activity (n = 1) in tenocytes from Ham and LHB tendons. Results are representative of at least two independent experiments. Scale bars represent 200 μm in ×40 magnifications.

Article Snippet: For immunohistochemical staining, the sections were deparaffinized in xylene, dehydrated in alcohol, epitope unmasked by heating, immersed in H 2 O 2 , and stained with antibodies against p53 (#sc-6243, Santa Cruz Biotechnology), p21 (#sc-6246, Santa Cruz Biotechnology), p16 (#sc-6243, Santa Cruz Biotechnology), SA-β-gal (#sc-377257, Santa Cruz Biotechnology), tnmd (#ab203676, Abcam), and BrdU (#GTX128091, GeneTex) in combination with chromogen 3-amino-9-ethylcarbazole (Zymed Laboratories).

Techniques: Expressing, Immunohistochemistry, Activity Assay, Staining, Western Blot

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Amelioration of experimental tendinopathy by lentiviral CD44 gene therapy targeting senescence-associated secretory phenotypes

doi: 10.1016/j.omtm.2022.06.006

Figure Lengend Snippet: Expression levels of CD44, P21, p53, p16, and SA-β-gal activity in rat primary tenocytes Tenocytes were isolated from the Achilles tendons of Sprague-Dawley rats treated (T) or untreated (Un) with intratendinous injection of collagenase I (10 lambda/rat, 1.5 mg/lambda) for 1 week. (A) Primary tenocytes were subjected to SA-β-gal activity assay. The black arrows indicate SA-β-gal-positive cells. Scale bars represent 50 μm in ×100 magnifications. SA-β-gal-positive cells were counted and quantitated (n = 3). (B) Expression levels of p53, p21, and p16 were determined via immunoblotting. (C) Expression levels of p53 (n = 5), p21 (n = 6), and p16 (n = 6) were determined via quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Values are represented as the mean ± SEM. The between-the-group differences were assessed using the Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Results are representative of at least two independent experiments.

Article Snippet: For immunohistochemical staining, the sections were deparaffinized in xylene, dehydrated in alcohol, epitope unmasked by heating, immersed in H 2 O 2 , and stained with antibodies against p53 (#sc-6243, Santa Cruz Biotechnology), p21 (#sc-6246, Santa Cruz Biotechnology), p16 (#sc-6243, Santa Cruz Biotechnology), SA-β-gal (#sc-377257, Santa Cruz Biotechnology), tnmd (#ab203676, Abcam), and BrdU (#GTX128091, GeneTex) in combination with chromogen 3-amino-9-ethylcarbazole (Zymed Laboratories).

Techniques: Expressing, Activity Assay, Isolation, Injection, Western Blot, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Amelioration of experimental tendinopathy by lentiviral CD44 gene therapy targeting senescence-associated secretory phenotypes

doi: 10.1016/j.omtm.2022.06.006

Figure Lengend Snippet: Expression levels of CD44, matrix degrading enzymes, inflammatory mediators, extracellular matrix (ECM)-related molecules, and senescent markers in LVCD44-transduced rat primary tenocytes (A) Immunoblotting for CD44, matrix metalloproteinase-1 (MMP)-1 and -3, cyclooxygenase-2 (COX-2), phospho-nuclear factor-κB (p-NF-κB), NF-κB, collagen type I alpha 1 (COL1A1), tenomodulin (tnmd), p53, p21, and p16 expression levels in LVSin- (the control vector), and LVCD44-transduced tenocytes. LVCD44- and LVSin-transduced tenocytes were stimulated with IL-1β (10 ng/mL) or left unstimulated for 5 days (except for phospho-NF-κB, which was stimulated for 30 min). (B) Supernatant was isolated and subjected to enzyme-linked immunosorbent assay (ELISA) for detecting IL-6 levels (n = 3). (C) The underlying tenocytes were subjected to SA-β-gal activity assay. SA-β-gal was identified and counted in five high-power fields (200×) to determine the average percentages of SA-β-gal-positive cells corresponding to total cells (n = 6). (D) qRT-PCR to determine CD44, IL-6, MMP-1, COX-2, COL1A1, and tnmd expression levels. LVCD44- and LVSin-transduced tenocytes were stimulated with IL-1β (10 ng/mL) or left unstimulated for 24 h (n = 3). Results are representative of at least three independent experiments. Scale bars represent 50 μm in ×100 magnifications. Values are represented as the mean ± SEM. The difference within groups were assessed using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison tests. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See Figure 2 for other definitions.

Article Snippet: For immunohistochemical staining, the sections were deparaffinized in xylene, dehydrated in alcohol, epitope unmasked by heating, immersed in H 2 O 2 , and stained with antibodies against p53 (#sc-6243, Santa Cruz Biotechnology), p21 (#sc-6246, Santa Cruz Biotechnology), p16 (#sc-6243, Santa Cruz Biotechnology), SA-β-gal (#sc-377257, Santa Cruz Biotechnology), tnmd (#ab203676, Abcam), and BrdU (#GTX128091, GeneTex) in combination with chromogen 3-amino-9-ethylcarbazole (Zymed Laboratories).

Techniques: Expressing, Western Blot, Plasmid Preparation, Isolation, Enzyme-linked Immunosorbent Assay, Activity Assay, Quantitative RT-PCR

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Amelioration of experimental tendinopathy by lentiviral CD44 gene therapy targeting senescence-associated secretory phenotypes

doi: 10.1016/j.omtm.2022.06.006

Figure Lengend Snippet: Effects of CD44 overexpression in the collagenase-induced rat Achilles tendinopathy model (A) Three days after the intratendinous collagenase injection, the rats randomly received three consecutive intratendinous injections of LVCD44 or LVSin within a 2-week interval. The ultrasound examination was done 2 months after collagenase injection. The semiquantitative scores of echogenicity, neovascularization, and calcification in B-mode and color doppler were measured and recorded. Yellow dashed square: region of interest for neovascularization. White asterisk: calcification. Values are represented as the mean ± SEM (n = 8). (B) H&E, histological score, tnmd, and BrdU immunohistochemical stainings and the quantitative analyses of LVSin- and LVCD44-treated tendinopathic tendon. Scale bars shown at ×100 magnification correspond to 50 and 200 μm. The blue-boxed areas are higher-magnification views of the red-boxed areas. CR, calcified region; arrowhead, chondrocyte-like cell; arrow, neovascularization. Values are represented as the mean ± SEM (n = 8). (C) Immunoblotting for MMP-1 and -3, COX-2, p-NF-kB, IL-6, COL1A1, SA-β-gal (64 kDa), p53, p21, and p16 expression levels in LVSin- and LVCD44-injected tendinopathic tendons (n = 2). Results are representative of at least three independent experiments. The between-the-group differences were assessed using the Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: For immunohistochemical staining, the sections were deparaffinized in xylene, dehydrated in alcohol, epitope unmasked by heating, immersed in H 2 O 2 , and stained with antibodies against p53 (#sc-6243, Santa Cruz Biotechnology), p21 (#sc-6246, Santa Cruz Biotechnology), p16 (#sc-6243, Santa Cruz Biotechnology), SA-β-gal (#sc-377257, Santa Cruz Biotechnology), tnmd (#ab203676, Abcam), and BrdU (#GTX128091, GeneTex) in combination with chromogen 3-amino-9-ethylcarbazole (Zymed Laboratories).

Techniques: Over Expression, Injection, Immunohistochemical staining, Western Blot, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Amelioration of experimental tendinopathy by lentiviral CD44 gene therapy targeting senescence-associated secretory phenotypes

doi: 10.1016/j.omtm.2022.06.006

Figure Lengend Snippet: Working model of CD44 gene therapy in tendinopathy In vitro CD44 gene transfer by lentiviral vectors in IL-1β-stimulated tendinopathic tenocytes resulted in CD44 overexpression that mediated the downstream effects, including the blocking of NF-κB activation and gene expression levels of senescence markers (p53, p21, P16, COX-2, and SA-β-gal) and SASPs (IL-6, and MMP-1 and -3), whereas the expression levels of tendon healing molecules (tnmd and COL1A1) were increased under the IL-1β-stimulated condition. In vivo gene therapy via intratendinous injection of LVCD44 in rat collagenase-treated tendinopathic tendons reduced the ultrasound and histological scores as well as senescence and SASPs and then facilitated tendon healing.

Article Snippet: For immunohistochemical staining, the sections were deparaffinized in xylene, dehydrated in alcohol, epitope unmasked by heating, immersed in H 2 O 2 , and stained with antibodies against p53 (#sc-6243, Santa Cruz Biotechnology), p21 (#sc-6246, Santa Cruz Biotechnology), p16 (#sc-6243, Santa Cruz Biotechnology), SA-β-gal (#sc-377257, Santa Cruz Biotechnology), tnmd (#ab203676, Abcam), and BrdU (#GTX128091, GeneTex) in combination with chromogen 3-amino-9-ethylcarbazole (Zymed Laboratories).

Techniques: In Vitro, Over Expression, Blocking Assay, Activation Assay, Expressing, In Vivo, Injection

Journal: Stem Cell Research & Therapy

Article Title: Thioredoxin mitigates radiation-induced hematopoietic stem cell injury in mice

doi: 10.1186/s13287-017-0711-2

Figure Lengend Snippet: TXN mitigates TBI-induced cell senescence in vivo and in vitro. Mice were irradiated with 7.25 Gy and then treated with saline or thioredoxin ( TXN ) as described in the text. a At 3 weeks after radiation, the Lin – cells were isolated and stained with β-galactosidase, analyzed by FACS and b quantified. c Primary fibroblast cell lines were irradiated with 3 and 5 Gy and cultured with and without TXN for 3 days and then stained with β-galactosidase kit. Cells were photographed under a light microscope (magnification, 200×) and d β-gal-positive cells were quantified. e The levels of p16 protein expression were analyzed by Western blot and f quantitative densitometry of the protein expressions. Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01. IR ionizing radiation, n.s. not significant, TBI total body irradiation

Article Snippet: Senescence β-galactosidase (SA-gal) staining kit, γ-H2AX, p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK (Thr180/Tyr182), p16, β-actin, and the corresponding secondary antibodies were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: In Vivo, In Vitro, Irradiation, Saline, Isolation, Staining, Cell Culture, Light Microscopy, Expressing, Western Blot