Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Impact of PTCH1b 5'UTR size and CGG-repeat number in gene reporter assays. pGL3-Promoter-derived constructs containing PTCH1b 5'UTRs (188 or 300 nucleotides) fused to the Firefly luciferase (Fluc) were transiently transfected in HCT116 p53+/+ (A), MCF7 (B) and HEK 293T cells (C). Presented are the average fold inductions relative to the empty vector and standard deviations of at least 3 biological replicates. The luciferase values are expressed relative to the value obtained with an empty control vector. The same constructs were used to measure relative Fluc mRNA transcript levels in transiently transfected HCT116 p53+/+ (D), MCF7 (E) and HEK 293T cells (F). Presented is the average Fluc mRNA expression normalized for the average plasmid copy number and for relative cDNA synthesis efficiency as revealed by the amplification of GAPDH and B2M mRNAs. For each UTR size type, results were compared to the corresponding reference allele with (CGG)7; *P < 0.05

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Derivative Assay, Construct, Luciferase, Transfection, Plasmid Preparation, Control, Expressing, cDNA Synthesis, Amplification

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Impact of PTCH1b 5'UTR size and CGG-repeat number on cap-independent translation. Bicistronic pRuF-derived constructs were used to study the potential of the PTCH1b 5'UTR to drive cap-independent translation of the Firefly luciferase gene in transiently transfected MCF7 (A) or HEK 293T cells (B). Presented are the average ratios and standard deviations between Firefly (Fluc) and Renilla luciferase (Rluc) relative light units, normalized with the results obtained for empty pRuF vector, and standard deviation of at least 3 replicates. The PTCH1b 5'UTR size and CGG-repeat number are indicated. (C, D) The bicistronic nature of the transcript expressed by pRuF constructs was estimated measuring the Fluc/Rluc mRNA ratio by a qPCR approach. A ratio equal to 1.0 would strongly argue against the presence of a cryptic promoter within cloned PTCH1b 5'UTR resulting in a monocistronic Fluc mRNA transcript. A pRuF-type plasmid with cloned c-MYC 5'UTR was used as a positive control. For each UTR size type, results were compared to the corresponding reference allele with (CGG)7; *P < 0.05

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Derivative Assay, Construct, Luciferase, Transfection, Plasmid Preparation, Standard Deviation, Clone Assay, Positive Control

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: An internal ribosome entry site (IRES) motif maps in the 3' end of the PTCH1b 5'UTR. (A) Putative PTCH1b IRES motif cloned into pRuF-type plasmid is not sufficient to obtain Firefly luciferase (Fluc) activity observed with the complete PTCH1b 5'UTR in MCF7 cells, while the remaining part of PTCH1b 5'UTRs (188ΔIRES and 300ΔIRES) retains certain levels of IRES activity. (B) Similar results were obtained in HEK 293T cells. (C) The ratios between Fluc and Renilla luciferase (Rluc) mRNA for remodeled pRuF PTCH1b 5'UTR plasmids in transfected MCF7 cells didn't deviate from 1.0. (D) The same results were obtained in HEK 293T cells. Results are presented as described in Figure 4.

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Transfection

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Hypoxia enhances PTCH1b 5'UTR mediated translation. MCF7 and HEK 293T cells transiently transfected with the different pRuF reporter constructs were grown in normoxic (dark gray) or severe hypoxic (light gray) conditions for 16 hours. Luciferase assays were performed as described in Materials and Methods and shown as those presented in Figure 5.

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Transfection, Construct, Luciferase

Journal: RNA Biology

Article Title: Regulation of human PTCH1b expression by different 5' untranslated region cis -regulatory elements

doi: 10.1080/15476286.2015.1008929

Figure Lengend Snippet: Higher PTCH1b mRNA relative translation efficiency during hypoxia in HEK 293T cells overexpressing GLI1. HEK 293T cells were transfected with an empty vector or a construct overexpressing GLI1 and then cultured in normoxia or hypoxia for 16 hours (48 hours post transfection). (A). Total RNA levels of the indicated mRNAs, quantified by qPCR as described in the method section and presented as relative expression compared to the reference genes (ΔCq). c-MYC mRNA was included as an example of mRNA whose 5'UTR is considered to possess IRES activity, while PCNA was included as negative control for IRES function. Error bars plot the average and standard deviations of 3 replicates (B). Cytoplasmic lysates of HEK 293T cells transfected and cultured as for panel A, were separated by sucrose-gradient equilibrium density; subpolysomal (SUB) and polysome-associated (POL) mRNAs were identified and collected by UVC scanning and the indicated transcripts were quantified by qPCR, as for panel A. (C). The results from panel B, are also plotted as ratio between polysome-associated and subpolysomal mRNAs as a function of the different treatment. This ratio is considered an estimate of relative mRNA translation efficiency. (D). Global relative mRNA translation rates in HEK 293T cells cultures in normoxia or hypoxia for 16 hours were assessed by incorporation of an immune-detectable methionine analog, as described in Materials and Methods. (E). Western blot analysis of PTC1-L protein and PCNA. Transfection of the GLI1 overexpression plasmid and treatment are indicated. Beta-tubulin was used as reference protein.

Article Snippet: Human breast adenocarcinoma-derived MCF7 and Human Embryonic Kidney HEK 293T cells were obtained from the InterLab Cell Line Collection (ICLC, Azienda Ospedaliera Universitaria San Martino-IST, Genoa, Italy).

Techniques: Transfection, Plasmid Preparation, Construct, Cell Culture, Expressing, Activity Assay, Negative Control, Western Blot, Over Expression