1. The p73 Gene Is an Anti-Tumoral Target of the RARβ/γ-Selective Retinoid Tazarotene 
Marina Papoutsaki, Mauro Lanza, Barbara Marinari, Steven Nisticò, Francesca Moretti, Massimo Levrero, Sergio Chimenti, Antonio Costanzo 
Journal of Investigative Dermatology 
Volume 123, Issue 6, Pages (December 2004)
DOI: /j X x
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Tazarotene (Taz) treatment induces TAp73 protein and downregulates ΔNp73. (A) C5N cells were treated with 2 μM Taz or with 2 μM all-trans-retinoic acid and lysed after 24 h. The levels of endogenous p53, ΔNp63, p73, and ΔNp73 were analyzed by immunoblotting using specific antibodies. An anti-actin immunoblot was performed 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 immunoblotting using anti-p73 or anti-p63 specific antibodies.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Tazarotene (Taz) activates TAp73 and represses ΔNp73 transcription. (A) Schematic diagram representing the 5′ portion of p73 gene and its two gene products. The locations of the P1p73 promoter (TA isoforms) and the P2p73 (ΔNp73 isoforms) are indicated by P1 and P2, respectively. (B) Five micrograms of total mRNA from HaCaT cells treated with the indicated concentrations of Taz for 24 h were subjected to reverse transcription using random hexamers. One-tenth of the RT product was amplified in a semiquantitative PCR using primers specific for TAp73, ΔNp73, or actin (left panel). The relative amount of TAp73 and ΔNp73 transcripts was assessed by densitometric quantitation of PCR bands (ImageQuant 2.1 software) after normalization with actin. Results are expressed as relative expression after setting the untreated samples as 100% (right panel).
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
The P1p73 promoter is activated by tazarotene (Taz). (A) Schematic representation of the P1p73 promoter reporter plasmids. The presence of putative retinoic acid receptor binding sites as well as E2F sites and TATA box is indicated. (B) HaCaT cells were transfected with 1 μg of the indicated luciferase encoding reporter plasmid and with 50 ng of the Renilla encoding pRL-null plasmid. Eighteen hours after transfection, cells were treated with tazarotene (2 μM) for 24 h. Cells were lysed and luciferase activity was quantified by the Dual Luciferase reporter kit (Promega Inc.). Renilla activity was used to normalize the transfection efficiency. Results are expressed as luciferase activity relative to Renilla activity. Results represent the mean of three independent experiments performed in duplicate. Error bars represent 1 standard deviation. (C) Schematic representation of the P2p73 promoter reporter plasmids. (D) HaCaT cells were transfected with 1 μg of the indicated luciferase encoding reporter plasmid and with 50 ng of the Renilla encoding pRL-null plasmid. Eighteen hours after transfection, cells were treated with tazarotene (2 μM) for 24 h. Luciferase activity was measured as in Figure 3. (e) HaCaT cells were cotransfected with the P2p73 promoter and expression vectors encoding for c-Jun or its mutant the c-Jun-Δ169. Eighteen hours after transfection, cells were left untreated or were treated with tazarotene 2 μM for 24 h, and the luciferase activity was measured as described in Materials and Methods.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
P73 gene products regulate the sensitivity of HaCat cells to tazarotene-induced cell death. (A) HaCat cells were transfected with 1 μg of an expression vector encoding for green fluorescent protein (GFP)–spectrin fusion protein together with 2 μg of the indicated expression vector and 5 μg of p73- or ΔNp73-specific small interfering RNA (siRNA). thirty-six hours after transfection, the expression of HAp73 or HA-ΔNp73 was evaluated by immunoblotting. Actin expression was used as loading and specificity control. (B) HaCaT cells were transfected with 0.5 μg of the bax-luc reporter plasmid, 50 μg of the pRL-null plasmid, and 1 μg of the indicated siRNA. The day after transfection, cells were treated with 2 μM 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 μg of an expression vector encoding for GFP–spectrin fusion protein and 5 μg of the indicated siRNA. Eighteen hours after transfection, cells were mock treated or were treated with 2 μM 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.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions