Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 1 Cloning strategy for the production of the pCCL.shRNA-UPF1. WPRE lentiviral vector. The pRS-shRNA-UPF1 construct was subjected to enzymatic digestion by the restriction enzymes NaeI and SalI with the purpose to isolate the 410 bp insert containing U6 promoter and UPF1 shRNA.pCCL.PGW vector, as the result of cutting by the SalI and EcoRV endonucleases, was deleted of a region containing the GFP gene and the relative PGK promoter. That region was replaced by the 410 bp insert deriving from the pRS-shRNA-UPF1, obtaining the final vector pCCL.shRNA-UPF1.WPRE

Article Snippet: Membranes were washed three times for 5 min each with 30 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with primary rabbit monoclonal antibody against UPF1 (1:15.000) (Origene, Rockville, MD,) in 15 ml primary antibody dilution buffer (TBS, 0.1% Tween20, 5% BSA) with gentle agitation over-night at 4 °C.

Techniques: Cloning, shRNA, Plasmid Preparation, Construct

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 2 Quantification of the insertion of pCCL.shRNA-UPF1.WPRE and pCCL.shRNA-scramble.WPRE vectors in the obtained clones. Analysis of the integration of pCCL.shRNA-UPF1.WPRE vector in β039.m5 (a) and βwt.wt3 (c) derived clones and of pCCL.shRNA-scramble.WPRE construct in β039.m5 (b) and βwt.wt3 (d) clones. Histograms show the integration of the lentiviral vectors understood as fold compared to insertions already present in the respective original cells, used as reference samples. The data represent the averages ± SD of three independent determinations

Article Snippet: Membranes were washed three times for 5 min each with 30 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with primary rabbit monoclonal antibody against UPF1 (1:15.000) (Origene, Rockville, MD,) in 15 ml primary antibody dilution buffer (TBS, 0.1% Tween20, 5% BSA) with gentle agitation over-night at 4 °C.

Techniques: shRNA, Clone Assay, Plasmid Preparation, Derivative Assay, Construct

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 3 Quantification of the UPF1 mRNA content in UPF1- and scramble clones. Histograms show the UPF1 mRNA content in UPF1- (a) and scramble (b) β039.m5 clones and UPF1- (c) and scramble (d) βwt.wt3 clones and in their original cell lines, β039.m5 and βwt.wt3, used as reference samples. The data were obtained by Real Time qRT-PCR using expression assays for UPF1 and GAPDH cDNAs, the last of which was used for the normalization. The data represent the averages ± SD of three independent determinations

Article Snippet: Membranes were washed three times for 5 min each with 30 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with primary rabbit monoclonal antibody against UPF1 (1:15.000) (Origene, Rockville, MD,) in 15 ml primary antibody dilution buffer (TBS, 0.1% Tween20, 5% BSA) with gentle agitation over-night at 4 °C.

Techniques: Clone Assay, Quantitative RT-PCR, Expressing

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 4 Quantification of the β globin mRNA content in UPF1- and scramble β039.m5 clones. Histograms show the UPF1 mRNA content in UPF1- clone 3, scramble clone a and in the original cell line β039.m5, used as reference samples. The data were obtained by Real Time qRT-PCR using expression assays for β globin and GAPDH cDNAs, the last of which was used for the normalization. The data represent the averages ± SD of three independent determinations

Article Snippet: Membranes were washed three times for 5 min each with 30 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with primary rabbit monoclonal antibody against UPF1 (1:15.000) (Origene, Rockville, MD,) in 15 ml primary antibody dilution buffer (TBS, 0.1% Tween20, 5% BSA) with gentle agitation over-night at 4 °C.

Techniques: Clone Assay, Quantitative RT-PCR, Expressing

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 5 Evaluation of the content of UPF1 protein in UPF1- clones. The UPF1 protein accumulation in UPF1- clones 3 (a) and 1 (b) compared to the respective control cells, β039.m5 and βwt.wt3, was determined by Western Blotting using scalar quantities of protein extracts and monoclonal antibodies against UPF1 and p70 proteins. The image obtained was analysed by densitometry, which evaluated the relative amount of protein staining and quantified the results in terms of optical density: the histogram shows the reduction in UPF1 protein content in clone 3 and 1, meant as a percentage compared to the corresponding control cells (c). Values were normalized using p70 protein. The data represent the averages ± SD of three independent determinations

Article Snippet: Membranes were washed three times for 5 min each with 30 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with primary rabbit monoclonal antibody against UPF1 (1:15.000) (Origene, Rockville, MD,) in 15 ml primary antibody dilution buffer (TBS, 0.1% Tween20, 5% BSA) with gentle agitation over-night at 4 °C.

Techniques: Clone Assay, Control, Western Blot, Bioprocessing, Staining

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 6 Effects of geneticin (G418) on the β globin production in UPF1- clone 3, scramble clone a and relative original cell line β039.m5. After a 3 day incubation with 400 ng/μl G418, cells were labeled with the monoclonal antibody Hemoglobin β-PE and analysed by FACS. Histogram a shows the proportion of β globin producing cells and the fold of that proportion in β039.m5, clone 3 and clone a cells, untreated and treated with 400 ng/μl G418. Histogram b shows the fluorescence median of all the samples and its shift after treatment. The data represent the averages ± SD of three independent experiments

Article Snippet: Membranes were washed three times for 5 min each with 30 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with primary rabbit monoclonal antibody against UPF1 (1:15.000) (Origene, Rockville, MD,) in 15 ml primary antibody dilution buffer (TBS, 0.1% Tween20, 5% BSA) with gentle agitation over-night at 4 °C.

Techniques: Incubation, Labeling, Fluorescence

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 7 Effects of geneticin (G418) on the β globin production in UPF1- clone 1, scramble clone c and relative original cell line βwt.wt3. After a 3 day incubation with 400 ng/μl G418, cells were labelled with the monoclonal antibody Hemoglobin β-PE and analysed by FACS. Histogram A shows the proportion of β globin producing cells and the fold of that proportion in K562.βwt.wt3, clone 1 and clone c cells, untreated and treated with 400 ng/μl G418. Histogram B shows the fluorescence median of all the samples and its shift after treatment. The data represent the averages ± SD of three independent experiments

Article Snippet: Membranes were washed three times for 5 min each with 30 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with primary rabbit monoclonal antibody against UPF1 (1:15.000) (Origene, Rockville, MD,) in 15 ml primary antibody dilution buffer (TBS, 0.1% Tween20, 5% BSA) with gentle agitation over-night at 4 °C.

Techniques: Incubation, Fluorescence

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 8 Effects of geneticin (G418) on the content of β globin mRNA in β039 and βwt clones. Histograms show the β globin mRNA content in β039 (a) and βwt (b) clones, UPF1 or scramble silenced, and in the original cell lines, β039.m5 and βwt.wt3 respectively, untreated and treated with 400 ng/μl G418. The data were obtained by Real Time qRT-PCR using expression assays for β globin and GAPDH cDNAs, the last of which was used for the normalization. The data represent the averages ± SD of three independent experiments

Article Snippet: Membranes were washed three times for 5 min each with 30 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with primary rabbit monoclonal antibody against UPF1 (1:15.000) (Origene, Rockville, MD,) in 15 ml primary antibody dilution buffer (TBS, 0.1% Tween20, 5% BSA) with gentle agitation over-night at 4 °C.

Techniques: Clone Assay, Quantitative RT-PCR, Expressing