Journal: American Journal of Cancer Research

Article Title: Indisulam exerts anticancer effects via modulation of transcription, translation and alternative splicing on human cervical cancer cells

doi:

Figure Lengend Snippet: Indisulam treatment induce p73-dependent mitochondrial pathway of apoptosis in HeLa cells. A. HeLa cells treated with different concentration of indisulam for 24 h were subsequently detected using co-staining with Annexin V-FITC and PI flow cytometry. B. Expression of p73 splicing isoforms ΔNp73 and TAp73 were analysed by western blot assay. β-Actin protein was used as an internal control. C. Quantitative analysis of the ΔNp73 and TAp73 is represented by column graphs. D. Expression of apoptosis-associated proteins, including Bcl-2, Bax, procaspase-3, cleaved caspase 3 and cytochrome c were analysed by western blot assay. β-Actin protein was used as an internal control. E. Quantitative analysis of the Bcl-2, Bax, procaspase-3, cleaved caspase 3 and cytochrome c proteins were represented by column graphs. Data represent the mean ± SD of three independent experiments each conducted in triplicate. ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (One-way ANOVA).

Article Snippet: The membranes were blocked with 5% skim milk for 1 h and then incubated with antibodies against RBM39 (Abcam, ab244254, 1:1000), caspase-3 (Abcam, ab184787, 1:2000), ΔNp73 (Novus, 38C674.2, 1:1000), p73 (Novus, 5B429, 1:1000), Cytochrome C (Immunoway, YM3402, 1:1000), Bax (Immunoway, YT0455, 1:1000), Bcl-2 (Immunoway, YT0470, 1:1000), Cleaved Caspase-3 (Cell Signaling Technology, 9661S, 1:1000), p21 (Abcam, ab109199, 1:1000), Cyclin D1 (Abcam, ab16663, 1:200), and β-Actin (Bioss, bs-10966R, 1:5000).

Techniques: Concentration Assay, Staining, Flow Cytometry, Expressing, Western Blot, Control

Journal: American Journal of Cancer Research

Article Title: Indisulam exerts anticancer effects via modulation of transcription, translation and alternative splicing on human cervical cancer cells

doi:

Figure Lengend Snippet: Model of potential targets of RBM39 in cancer. Indisulam treatment induced RBM39 degradation results in perturbations of transcription, translation and especially alternative splicing events in multiple cellular processes in HeLa cells. Surprisingly, indisulam treatment was able to alter the ΔNp73 to TAp73 ratio by redirecting the alternative splicing of p73 to induce mitochondrial apoptosis in HeLa cells independent of the P53 gene.

Article Snippet: The membranes were blocked with 5% skim milk for 1 h and then incubated with antibodies against RBM39 (Abcam, ab244254, 1:1000), caspase-3 (Abcam, ab184787, 1:2000), ΔNp73 (Novus, 38C674.2, 1:1000), p73 (Novus, 5B429, 1:1000), Cytochrome C (Immunoway, YM3402, 1:1000), Bax (Immunoway, YT0455, 1:1000), Bcl-2 (Immunoway, YT0470, 1:1000), Cleaved Caspase-3 (Cell Signaling Technology, 9661S, 1:1000), p21 (Abcam, ab109199, 1:1000), Cyclin D1 (Abcam, ab16663, 1:200), and β-Actin (Bioss, bs-10966R, 1:5000).

Techniques: Alternative Splicing

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: 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