Journal: International Journal of Molecular Sciences

Article Title: Hypermethylation-Mediated Silencing of CIDEA , MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies

doi: 10.3390/ijms23074021

Figure Lengend Snippet: Effect of AZA + VAL or DEC + VAL combinations on CiDEA, MAL and PCDH17 protein expression in CLBL-1 cells. ( A ) Whole protein lysates from treated and untreated cells were subjected to immunoblotting, using GAPDH as the loading control. The image is representative of six independent experiments (independent cell cultures). MCF7, HepG2 and MDCK cell lines as well as CF2Th cells transfected with canine CiDEA and MAL full sequences have been used as positive controls (human and canine). Thirty and ~15 µg of total proteins were loaded in each well for the positive controls and CLBL-1 cells, respectively. ( B ) For the protein quantification of each sample, the integrated optical density (IOD) of the specific bands was normalized first to the corresponding GAPDH IOD and subsequently to the canine positive control, selected as the calibrator (CF2Th_CiDEA for CiDEA, CF2Th_MAL for MAL and MDCK for PCDH17). The results of the densitometric analysis are expressed in arbitrary units (AU) as the mean ± SEM of six independent experiments (independent cell cultures). Statistical analysis: Mann Whitney U-test (*: p < 0.05). L: ladder.

Article Snippet: Cf2Th canine normal thymus cell line obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, UK, Ref No. 90110521) was used for the heterologous transfection.

Techniques: Expressing, Western Blot, Control, Transfection, Positive Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Hypermethylation-Mediated Silencing of CIDEA , MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies

doi: 10.3390/ijms23074021

Figure Lengend Snippet: Luciferase assays of pCpGL-basic constructs containing CiDEA ( A ), MAL ( B ) and PCDH17 ( C ) CpG islands (CpGI) in CF2Th transfected cells. Luciferase activity values are expressed in arbitrary units (AU) as the fold activation relative to pCpGL-basic-mock-transfected cells (mean ± SEM of three independent experiments). On the right, a schematic diagram of CiDEA , MAL and PCDH17 gene structure and CpG-rich region position respect to the transcription starting site (TSS) is reported.

Article Snippet: Cf2Th canine normal thymus cell line obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, UK, Ref No. 90110521) was used for the heterologous transfection.

Techniques: Luciferase, Construct, Transfection, Activity Assay, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Hypermethylation-Mediated Silencing of CIDEA , MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies

doi: 10.3390/ijms23074021

Figure Lengend Snippet: Luciferase activity of in vitro methylated CiDEA_CpGI1 ( A ), MAL_CpGI1 ( B ), PCDH17_CpGI1 ( C ) and PCDH17_CpGI3 ( D ) plasmids in CF2Th cells. Plasmids containing the CpG sites mostly involved in the regulation of CiDEA , MAL and PCDH17 transcription were subjected to in vitro methylation. SssI, HhaI and HpaII methylation enzymes were used separately or in combination (HhaI + HpaII). Luciferase activity values (mean ± SEM of three independent experiments) are expressed as the percentage of the negative control (CTRL-, the respective unmethylated plasmid) activity, to which an arbitrary value of 100% was assigned. Statistical analysis: ANOVA + Bonferroni post hoc test (*: p < 0.05; ***: p < 0.001).

Article Snippet: Cf2Th canine normal thymus cell line obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, UK, Ref No. 90110521) was used for the heterologous transfection.

Techniques: Luciferase, Activity Assay, In Vitro, Methylation, Negative Control, Plasmid Preparation