Journal: BMC Dermatology

Article Title: SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes

doi: 10.1186/s12895-017-0060-y

Figure Lengend Snippet: Exposure to repeated UVB plus heat significantly decreased acetylated p53 levels in keratinocytes. Representative immunohistochemical staining of ex vivo skin either untreated, or exposed to heat, UVB or UVB plus heat, for nuclear DNA (DAPI, blue ), CPD ( red ) and ( a ) phosphorylated SIRT1 (SIRT1-p), ( b ) caspase (casp-3), ( c ) total p53 or ( d ) acetylated p53 (p53-a382). Inset images are an enlarged view of cells positive for CPD and SIRT1 ( red arrows ), CPD and Casp-3 ( orange arrows ), CPD and p53 ( green arrows ) or CPD and p53-a382 ( blue arrows ). Broken lines denote the epidermal/dermal border. All images are at 400X magnification

Article Snippet: The membrane was probed with rabbit monoclonal p53 antibody or p53 acetyl K382 and detected using Clarity Western ECL Blotting Substrate detection kit (Bio-Rad) as per the manufacturer’s instructions.

Techniques: Immunohistochemical staining, Staining, Ex Vivo

Journal: BMC Dermatology

Article Title: SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes

doi: 10.1186/s12895-017-0060-y

Figure Lengend Snippet: Effect of UVB and/or heat exposure in keratinocytes of the ex vivo skin models or NHEK in vitro

Article Snippet: The membrane was probed with rabbit monoclonal p53 antibody or p53 acetyl K382 and detected using Clarity Western ECL Blotting Substrate detection kit (Bio-Rad) as per the manufacturer’s instructions.

Techniques: Ex Vivo

Journal: BMC Dermatology

Article Title: SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes

doi: 10.1186/s12895-017-0060-y

Figure Lengend Snippet: Effect of UV and/or heat exposure on p53 downstream gene targets and cell proliferation. a-b Fold change on mRNA expression of BAX, Survivin, ERCC1 or XPC in keratinocytes of the ( a ) skin and ( b ) in vitro, relative to untreated controls. c-d Bar graphs of the percentage mean (+/− SD) of keratinocytes positive for ki67 per field of view either ( c ) in ex vivo skin or ( d ) in vitro. Statistically significant differences are indicated with ** p < 0.001 and/or *** for p -values p < 0.0001

Article Snippet: The membrane was probed with rabbit monoclonal p53 antibody or p53 acetyl K382 and detected using Clarity Western ECL Blotting Substrate detection kit (Bio-Rad) as per the manufacturer’s instructions.

Techniques: Expressing, In Vitro, Ex Vivo

Journal: BMC Dermatology

Article Title: SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes

doi: 10.1186/s12895-017-0060-y

Figure Lengend Snippet: Inhibition of SIRT1 in UVB plus heat significantly increased acetylated p53 protein expression and apoptosis of keratinocytes in vitro. a Immunoblot showing levels of total p53, and acetylated p53 protein in UVB, UVB plus heat and UVB plus heat with the SIRT1 inhibitor (Ex-527). b Quantification of protein levels by relative average density standardised by β-actin. c Representative immunohistochemical staining of cells positive for CPD and p53 ( white arrows ), CPD and p53-a382 ( green arrows ) in primary keratinocytes exposed to UVB or UVB plus heat with or without SIRT1 inhibitor Ex-527. All images are at 400X magnification. d Bar graphs of percentage mean (+/− SD) of DNA damaged (CPD), apoptotic, p53 or p53-a382 positive primary keratinocytes exposed to UVB or UVB plus heat with or without Ex-527. Statistically significant differences are indicated with ** for p < 0.001 and/or *** for p -values p < 0.0001 respectively

Article Snippet: The membrane was probed with rabbit monoclonal p53 antibody or p53 acetyl K382 and detected using Clarity Western ECL Blotting Substrate detection kit (Bio-Rad) as per the manufacturer’s instructions.

Techniques: Inhibition, Expressing, In Vitro, Western Blot, Immunohistochemical staining, Staining

Journal: iScience

Article Title: TSC22D3 as an immune-related prognostic biomarker for acute myeloid leukemia

doi: 10.1016/j.isci.2023.107451

Figure Lengend Snippet:

Article Snippet: Rabbit Monoclonal anti-p53 , PTM Biolabs , Cat#PTM-6319.

Techniques: Virus, Recombinant, Staining, Reverse Transcription, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Apoptosis Assay, Cell Cycle Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Sequencing, shRNA, Software

Journal: Nature Communications

Article Title: Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis

doi: 10.1038/s41467-018-02950-5

Figure Lengend Snippet: Cisplatin induces K472 acetylation and S461 phosphorylation of PFKFB3. a DNA damage signals induced PFKFB3 K472 acetylation. Flag-tagged PFKFB3 was expressed in HEK293T cells, which were then treated with etoposide (10 μM), adriamycin (1 μM), UV irradiation (10 J/m 2 ) and cisplatin (50 or 100 μM) for 24 h. Flag-PFKFB3 was immunoprecipitated with Flag beads and immunoblotting was performed with the antibodies indicated. Relative PFKFB3 K472 acetylation and phosphorylation were normalized by Flag protein. b The amino acid sequence near K472 of PFKFB3 displays high similarity with the sequence near K320 of TP53. c Cisplatin treatment induces PFKFB3 cytoplasmic accumulation. HEK293T cells were treated with or without cisplatin (50 μM) for 24 h before harvest. Cells were then suspended in PBS and treated with a gradient concentration of digitonin. Supernatant and precipitate were collected for immunoblotting with indicated antibodies. S, supernatant; P, precipitate. d Cisplatin induces K472 acetylation and S461 phosphorylation of endogenous PFKFB3. Endogenous PFKFB3 protein was purified from HEK293T cells after cisplatin treatment as indicated for 24 h. e Cisplatin or etoposide treatment enhances K472 acetylation in SIRT1 knockout cells. Endogenous PFKFB3 protein were purified from WT or SIRT1 knockout HEK293T cells treated with EX527 (10 μM), cisplatin (50 μM) or etoposide (10 μM) for 24 h. f Combined knockdown of PCAF and GCN5 abolishes cisplatin- or etoposide-induced PFKFB3 K472 acetylation. HEK293T cells were transfected with siRNAs targeting PCAF and GCN5. After 60 h, cells were treated with cisplatin (50 μM) or etoposide (10 μM) for 24 h. g , h Cisplatin treatment induces pan-acetylation of PCAF and GCN5. Flag-tagged PCAF or GCN5 was expressed in HEK293T cells, which were treated with cisplatin for the duration indicated at a concentration of 50 μM. Relative pan-acetylation level of PCAF or GCN5 was normalized by Flag protein. i Cisplatin increases acetyltransferase activity of PCAF and GCN5. Flag-tag PCAF or GCN5 was purified from HEK293T cells treated with or without cisplatin (50 μM) for 24 h, then incubated with recombinant His-PFKFB3 in acetylation assay buffer. Purified proteins visualized by Coomassie blue staining are shown (lower panel). Data are representative of at least two independent experiments

Article Snippet: Antibody against acetyl-p53 (Lys320, 06–1283, 1:1000) was purchased from Merck Millipore.

Techniques: Phospho-proteomics, Irradiation, Immunoprecipitation, Western Blot, Sequencing, Concentration Assay, Purification, Knock-Out, Knockdown, Transfection, Activity Assay, FLAG-tag, Incubation, Recombinant, Acetylation Assay, Staining