Journal: Journal of Investigative Dermatology

Article Title: The p73 Gene Is an Anti-Tumoral Target of the RARβ/γ-Selective Retinoid Tazarotene

doi: 10.1111/j.0022-202x.2004.23498.x

Figure Lengend Snippet: Figure 1 Tazarotene (Taz) treatment induces TAp73 protein and downregulates DNp73. (A) C5N cells were treated with 2 mM Taz or with 2 mM all- trans-retinoic acid and lysed after 24 h. The lev- els of endogenous p53, DNp63, p73, and DNp73 were analyzed by immunoblotting using specific antibodies. An anti-actin immunoblot was per- formed to ensure that equal amounts of proteins were loaded. (B) HaCaT cells were left untreated or were treated with increasing concentrations of tazarotene for 24 h and lysed. Eighty micrograms of total cell extract was analyzed by immuno- blotting using anti-p73 or anti-p63 specific anti- bodies.

Article Snippet: The mouse monoclonal anti-p73 antibody (clone 1288) (Imgenex Inc., San Diego, California) recognizes all the known alternative splicing variants of the human and mouse p73 (Costanzo et al, 2002).

Techniques: Western Blot

Journal: Journal of Investigative Dermatology

Article Title: The p73 Gene Is an Anti-Tumoral Target of the RARβ/γ-Selective Retinoid Tazarotene

doi: 10.1111/j.0022-202x.2004.23498.x

Figure Lengend Snippet: Figure 2 Tazarotene (Taz) activates TAp73 and re- presses DNp73 transcription. (A) Schematic diagram representing the 50 portion of p73 gene and its two gene products. The locations of the P1p73 promoter (TA isoforms) and the P2p73 (DNp73 isoforms) are indicated by P1 and P2, respectively. (B) Five micrograms of total mRNA from HaCaT cells treated with the indicated con- centrations of Taz for 24 h were subjected to re- verse transcription using random hexamers. One-tenth of the RT product was amplified in a semiquantitative PCR using primers specific for TAp73, DNp73, or actin (left panel). The relative amount of TAp73 and DNp73 transcripts was assessed by densitometric quantitation of PCR bands (ImageQuant 2.1 software) after normal- ization with actin. Results are expressed as rel- ative expression after setting the untreated samples as 100% (right panel).

Article Snippet: The mouse monoclonal anti-p73 antibody (clone 1288) (Imgenex Inc., San Diego, California) recognizes all the known alternative splicing variants of the human and mouse p73 (Costanzo et al, 2002).

Techniques: Quantitation Assay, Software, Expressing

Journal: Journal of Investigative Dermatology

Article Title: The p73 Gene Is an Anti-Tumoral Target of the RARβ/γ-Selective Retinoid Tazarotene

doi: 10.1111/j.0022-202x.2004.23498.x

Figure Lengend Snippet: Figure 4 P73 gene products regulate the sensitivity of HaCat cells to tazarotene-induced cell death. (A) HaCat cells were transfected with 1 mg of an expression vector encoding for green fluorescent protein (GFP)–spectrin fusion protein together with 2 mg of the indicated expression vector and 5 mg of p73- or DNp73-specific small interfering RNA (siRNA). thirty-six hours after transfection, the expression of HAp73 or HA-DNp73 was evaluated by immunoblotting. Actin expression was used as loading and specificity control. (B) HaCaT cells were transfected with 0.5 mg of the bax-luc reporter plasmid, 50 mg of the pRL-null plasmid, and 1 mg of the indicated siRNA. The day after transfection, cells were treated with 2 mM tazarotene for 24 h and luciferase activity was determined as described in Materials and Methods. Results represent the mean of three experiments in duplicate. Error bars represent 1 SD. (C) HaCat cells were transfected with 1 mg of an expression vector encoding for GFP–spectrin fusion protein and 5 mg of the indicated siRNA. Eighteen hours after transfection, cells were mock treated or were treated with 2 mM tazarotene for 24 h. Cells were then harvested, stained with propidium iodide, and GFP expressing cells were evaluated for their DNA content by fluorescence-activated cell sorting analysis as described in Materials and Methods. Results express the percentage of cells with sub-G1 content of DNA and are representative of three independent experiments.

Article Snippet: The mouse monoclonal anti-p73 antibody (clone 1288) (Imgenex Inc., San Diego, California) recognizes all the known alternative splicing variants of the human and mouse p73 (Costanzo et al, 2002).

Techniques: Transfection, Expressing, Plasmid Preparation, Small Interfering RNA, Western Blot, Control, Luciferase, Activity Assay, Staining, FACS

Journal: Scientific reports

Article Title: Assessment of the toxic effect of benzalkonium chloride on human limbal stem cells.

doi: 10.1038/s41598-025-96919-2

Figure Lengend Snippet: Fig. 3. (A) Agarose gel electropherogram of amplification products (passage 1). Lane 1: DNA ladder (M), lanes 2–3: KRT3 (101 bp), lanes 4–5: KRT12 (190 bp), lane 6: DNA ladder (M), lanes 7–8: TP63 (96 bp), lanes 9–10: ABCG2 (144 bp), lanes 11–12: ACTβ (131 bp), lane 13: DNA ladder (M). NC, negative control. (B) Visualization of lack of expression of CK3, and CK12 and expression of positive markers p63 and ABCG2, in LSCs cultured in a medium supplemented with H/I/E. FITC-conjugated specific antibodies (green); DAPI- stained cell nuclei (blue). IC, isotype control. Scale bars = 15 μm.

Article Snippet: After rinsing with PBS, the cells were incubated with blocking buffer for 1 h, rinsed again, and labelled with rabbit anti-ABCG2 polyclonal (1:50; 27286-AP; Proteintech, USA), rabbit anti-p63 polyclonal (1:50; 12143-1-AP; Proteintech, USA), mouse anti-CK3 monoclonal (1:50; ab68260; Abcam, UK), and rabbit anti-CK12 monoclonal (1:50; ab185627; Abcam, UK) primary antibodies at 4 °C overnight.

Techniques: Agarose Gel Electrophoresis, Amplification, Negative Control, Expressing, Cell Culture, Staining, Control

Journal: Oncogene

Article Title: S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation.

doi: 10.1038/sj.onc.1209401

Figure Lengend Snippet: Figure 1 S100A2 expression is induced during keratinocyte differentiation. (a) RNA was extracted from HaCaT cells at the indicated time points after the induction of differentiation and subjected to RT–PCR analysis. Amplification of aldolase-A was used for normalization. (b) Detection of protein levels of p73 during HaCaT differentiation. (c) Quantitative analysis of TAp63 and DNp63 transcripts during differentiation of HaCaT cells. Protein levels of TAp63 and DNp63 during differentiation are also shown. TAp63 protein is detectable only after long exposure (L.E.). S.E.: short exposure. (d) Proteins were extracted from HaCaT cells at the indicated time points after the induction of differentiation and subjected to Western blot analysis. Equal loading of protein amount for each lane was determined by probing with anti-Hsp70 antibody.

Article Snippet: For supershift analysis, anti-p73 (sc-7237 and sc7238 from Santa Cruz) and anti-p63 (sc-8343 from Santa Cruz) polyclonal antibodies were used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Oncogene

Article Title: S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation.

doi: 10.1038/sj.onc.1209401

Figure Lengend Snippet: Figure 3 In vivo binding of p73b and p63 to the regulatory regions of S100A2 gene. (a) Crosslinked chromatin derived from prolifer- ating (P) or terminally differentiated (TD) HaCaT cells was immunoprecipated with antibodies to p73 or p63 or in the absence of antibody and analysed by PCR with specific primers for the indicated regulatory regions. Input, non-immunoprecipitated crosslinked chromatin. (b) Crosslinked chromatin derived from proliferating HaCaT cells (0 h) or from HaCaT cells after 12 h of differentiation (12 h) was immunoprecipated with antiacetylated histone H4 antibody or in the absence of antibody and analysed by PCR with specific primers for the indicated regulatory regions. Input, non-immunoprecipitated crosslinked chromatin.

Article Snippet: For supershift analysis, anti-p73 (sc-7237 and sc7238 from Santa Cruz) and anti-p63 (sc-8343 from Santa Cruz) polyclonal antibodies were used.

Techniques: In Vivo, Binding Assay, Derivative Assay, Immunoprecipitation

Journal: Oncogene

Article Title: S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation.

doi: 10.1038/sj.onc.1209401

Figure Lengend Snippet: Figure 5 Silencing of S100A2 impairs keratinocyte differentiation. (a) The efficiency of inhibition of S100A2 expression by S100A2- pRetroSuper or LacZ-pRetroSuper (negative control) was assessed by immunoblotting in H1299 cells. (b–d) RNA was extracted from HaCaT cells, stably transfected with p73-pRetroSuper or control lacZ-pRetroSuper, at the indicated time points after the induction of differentiation, and subjected to RT–PCR analysis. Specific primers for the detection of S100A2, involucrin and keratin 10 (K10) transcripts were used. Quantification by densitometry and normalization on aldolase-A expression were performed. Results represent the folds of induction over the 0 h time point. Histograms show the mean of three experiments; bars indicate s.d.

Article Snippet: For supershift analysis, anti-p73 (sc-7237 and sc7238 from Santa Cruz) and anti-p63 (sc-8343 from Santa Cruz) polyclonal antibodies were used.

Techniques: Inhibition, Expressing, Negative Control, Western Blot, Stable Transfection, Transfection, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Oncogene

Article Title: S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation.

doi: 10.1038/sj.onc.1209401

Figure Lengend Snippet: Figure 6 S100A2 gene is not induced in response to DNA damage. (a) Proteins were extracted from HaCaT cells at the indicated time points after the addition of cisplatin (CDDP) (2.5, 5 and 7.5 mg/ml) and subjected to Western blot analysis. Cell death was determined with PARP cleavage. Equal loading of protein amount for each lane was determined by probing with anti-Hsp70 antibody. (b) Percentages of subG1 fractions were quantified by cytofluorimetry in HaCaT cells treated as in (a). Histograms represent the mean of three experiments. (c) RNA was extracted from HaCaT cells at the indicated time points after treatment with CDDP 2.5 mg/ml and subjected to RT–PCR analysis. Amplification of aldolase-A was used to normalize each cDNA sample. (d) Crosslinked chromatin derived from HaCaT cells at 0 or 24 h after CDDP treatment was immunoprecipated with anti-p73 antibody or in the absence of antibody and analysed by PCR with specific primers for the indicated regulatory regions. Input, non-immunoprecipitated, crosslinked chromatin.

Article Snippet: For supershift analysis, anti-p73 (sc-7237 and sc7238 from Santa Cruz) and anti-p63 (sc-8343 from Santa Cruz) polyclonal antibodies were used.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Immunoprecipitation

Journal: Frontiers in Oncology

Article Title: p53-Dependent Anti-Proliferative and Pro-Apoptotic Effects of a Gold(I) N -Heterocyclic Carbene (NHC) Complex in Colorectal Cancer Cells

doi: 10.3389/fonc.2019.00438

Figure Lengend Snippet: MC3-indued cytotoxicity appears to be independent of the tumor suppressor, TAp73. (A) Western blot analysis reveals a strong reduction in the two major isoforms of p73, TAp73 and ΔNp73, in WT and p53 −/− HCT116 cells treated with either 5 μM of MC3 or the vehicle (0.1% DMF) for 24 h. β-actin and vinculin were used as loading controls. Numbers show values obtained from densitometric analyses of the presented blots normalized to the respective loading controls (β-actin). (B) 24 h of treatment with MC3 at the indicated concentrations down-regulates the TP73 gene in the three CRC cell lines. mRNA levels were analyzed by qRT-PCR and the relative gene expression was determined using the ΔΔ Ct method where the Ct values of TP73 were normalized to those of β-actin. 0.1% DMF was used as mock. Error bars indicate the SEM of three biological replicates. (C) Knock-down of p73 in HCT116 p53 −/− does not hamper MC3-induced PARP degradation as well as p21 induction. p53-deficient HCT116 cells were transfected with anti-p73 siRNA (HCT116 p53 −/− p73KD) or a negative control (HCT116 p53 −/− siRNA NC), thereafter were treated with 0.1% DMF or MC3 (5 μM) for 24 h. Transfection efficiency was confirmed by a clear reduction in p73 protein expression. β-actin and vinculin served as loading controls. Numbers represent densitometric analyses of the presented blots normalized to the respective loading controls (vinculin). (D) Viability of HCT116 p53 −/− p73KD cells after 24 h treatment with 5 μM of MC3 is reduced in a similar manner to HCT116 p53 −/− siRNA NC as well as the parental cell line, measured by MTT assay. Percentage cell viability was determined by normalizing the results of MC3-treated cells to those of vehicle (0.1% DMF)-treated ones. Data represent mean ± SD from two independent experiments, each was done in triplicates.

Article Snippet: Primary antibodies against p21 Waf1/Cip1 (#2947), PARP (#9542), Bcl-xL (#2764), Bax (#5023), Bcl-2 (#2872), Puma (#12450), cytochrome c (#4272), COX IV (#11967), pp38 MAPK (T180/Y182, #9211), p73 (#14620) were obtained from Cell Signaling Technologies (NEB, Germany), whereas those against p53 (SC-126), β-actin (SC-47778) and vinculin (SC-73614) were from Santa Cruz Biotechnology.

Techniques: Western Blot, Quantitative RT-PCR, Gene Expression, Knockdown, Transfection, Negative Control, Expressing, MTT Assay

Journal: Cell Death & Disease

Article Title: c-Abl phosphorylation of ΔNp63 α is critical for cell viability

doi: 10.1038/cddis.2009.15

Figure Lengend Snippet: ΔNp63 α is a direct target of the c-Abl tyrosine kinase. ( a ) In vitro kinase assay employing recombinant ΔNp63 α and active, recombinant c-Abl was carried out as described in Experimental Procedures. Kinase reactions were fractionated by SDS-PAGE and analyzed by western blot with indicated antibodies (p63 α =p63 α isoform specific antibody). ( b ) 293 cells were co-transfected with indicated plasmids or relevant empty vector and Flag-ΔNp63 α immunoprecipitated with Flag antibody. Immunoprecipitates and representative whole cell lysate (Input) were fractionated by SDS-PAGE and analyzed by western blot with indicated antibodies. ( c ) Sequences from peptides containing phosphorylated tyrosines which overlapped from mass spectrometric analysis of ΔNp63 α of both in vivo and in vitro phosphorylation by c-Abl (see Supplementary Figure S1) were compared across species: human= Homo sapien ; mouse= Mus musculus ; chicken= Gallus gallus ; frog=Xenopus laevis; zebrafish= Danio rerio . Phosphorylated tyrosine indicated in red. ( d ) 293 cells were transfected with c-Abl and either empty vector, ΔNp63 α or indicated tyrosine to phenalanine mutant construct. Whole-cell lysates were fractionated by SDS-PAGE and analyzed by western blot with indicated antibodies (p-p73Y99=antibody to Tyrosine 99 phosphorylated p73, see text)

Article Snippet: Anti- actin, c-Myc (A14), p63 α (H129), pan-p63 (4A4), tubulin, YAP (H-125) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho p73 (Tyr99) antibody, phospho-c-Abl (Tyr245) (Cell Signalling Technology, Beverly, MA, USA), c-Abl (Ab-3) antibody (Calbiochem, San Diego, CA, USA) and phosphotyrosine (4G10) antibody (Upstate, Millipore) were used as manufacture's recommendation; mouse anti-human proliferating cell nuclear antigen (PCNA) antibody (Research Monoclonal Antibody Service, Cancer Research UK, 1 : 1000).

Techniques: In Vitro, Kinase Assay, Recombinant, SDS Page, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, In Vivo, Phospho-proteomics, Mutagenesis, Construct

Journal: eLife

Article Title: Transcriptional activator TAp63 is upregulated in muscular atrophy during ALS and induces the pro-atrophic ubiquitin ligase Trim63

doi: 10.7554/eLife.10528

Figure Lengend Snippet: ( A ) Quantification: Proteins were extracted from C2C12 myoblasts treated with compounds: FCCP, Tunicamycin (Tun), Etoposide (Eto), menadione (Men). Western blot analysis revealed TAp63 expression. Bars correspond to means with SD (n = 3). *p<0.01. ( B ) C2C12 myoblasts were transfected with expression vectors encoding transcription factors involved in the ER or mitochondrial stress pathway (CHOP, ATF6, ATF4, XBP1s). RNA levels for TA isoforms of Trp63 , TA isoforms of P73 and P53 were followed by RT-qPCR. Bars represent means (relative induction versus Ct) with standard deviation (n = 3).*p<0.01. DOI: http://dx.doi.org/10.7554/eLife.10528.016

Article Snippet: *p<0.01. ( C, D ) Chromatin immunoprecipitation (ChIP) assay was performed on the Trim63 promoter using RT-qPCR on RE1/2 and RE4. p53 immunoprecipitation (C) was performed using p53 antibody IC12 (Cell Signalling), p73 immunoprecipitation was performed using p73 antibody IMG-259a (Imgenex).

Techniques: Western Blot, Expressing, Transfection, Quantitative RT-PCR, Standard Deviation

Journal: eLife

Article Title: Transcriptional activator TAp63 is upregulated in muscular atrophy during ALS and induces the pro-atrophic ubiquitin ligase Trim63

doi: 10.7554/eLife.10528

Figure Lengend Snippet: ( A ) mRNA levels of Trim63 in C2C12 cells following transfection with siRNA control and siRNA directed against p73, p53 and a mix of siRNA against P53, and the TA isoforms of Trp63 and P73 (siMIX). Bars represent means (relative induction versus Ct) with standard deviation (n = 3). *p<0.01. ( B ) mRNA level for TA isoforms of Trp63 , TA isoforms of P73 and P53 in C2C12 cells following transfection with siRNA control and siRNA directed against p63, p73, and p53. Bars represent means (relative induction versus Ct) with standard deviation (n = 3). *p<0.01. ( C, D ) Chromatin immunoprecipitation (ChIP) assay was performed on the Trim63 promoter using RT-qPCR on RE1/2 and RE4. p53 immunoprecipitation (C) was performed using p53 antibody IC12 (Cell Signalling), p73 immunoprecipitation was performed using p73 antibody IMG-259a (Imgenex). Bars correspond to means with SD (n = 3). *p<0.01 as calculated by a one-way ANOVA test followed by a Tukey post-test. DOI: http://dx.doi.org/10.7554/eLife.10528.019

Article Snippet: *p<0.01. ( C, D ) Chromatin immunoprecipitation (ChIP) assay was performed on the Trim63 promoter using RT-qPCR on RE1/2 and RE4. p53 immunoprecipitation (C) was performed using p53 antibody IC12 (Cell Signalling), p73 immunoprecipitation was performed using p73 antibody IMG-259a (Imgenex).

Techniques: Transfection, Control, Standard Deviation, Chromatin Immunoprecipitation, Quantitative RT-PCR, Immunoprecipitation