Fig 1: NRF-1 and E2F2 bind to the CALB2 promoter(A) Nucleotide sequence comparison reveals high homology in the CALB2 promoter regions of different species in the stretch containing E2F2/NRF-1 predicted binding sites. The analysis was performed using the ClustalW2 software. Asterisks indicate conserved nucleotides. (B) EMSA assay showing that a 25bp-oligo containing E2F2/NRF-1-like sites forms three DNA-protein complexes (C1, C2, C3) when incubated in with the ZL55 cells-derived nuclear extracts. Addition of a NRF-1 antibody but not of one against E2F2 resulted in a supershift (SS). (C) qPCR analysis on CDC25A and CALB2 promoter regions after ChIP-experiments. Data of two independent experiments were normalized to input. (D) NRF-1 protein expression in different cell lines. Actin is used as loading control. (E) NRF-1 silencing resulted in downregulation of calretinin expression.
Fig 2: Promoter methylation does not regulate calretinin expression in cell lines and tumor samples(A) ZL55 and SPC111 cells were treated for 7 days with the hypomethylating agent 5-Aza-CdR (100 nM and 250 nM). Analysis of mRNA showed no change in CALB2 expression but strong upregulation of MAGE-C1 and CTAG1B expression. Mean ± SD, n = 3. (B) Calretinin protein levels decreased upon 5-Aza-CdR treatment in both ZL55 and SPC111 cells. Representative of three independent experiments. (C) CpG sites in the promoter region of CALB2 from the Illumina HumanMethylation450 array are shown in 5' to 3' order versus methylation beta-value which represents the percent methylation of the sample. Mesotheliomas are stratified by tumor histology, epithelioid (n = 57), and biphasic (n = 23).
Fig 3: Effect of site-directed mutagenesis of transcription factor binding sites on the -161/+80bp CALB2 promoter activity(A) Predicted potential TF binding sites (highly conserved nucleotides selected for site-directed mutagenesis are colored in red). (B) A series of mutant constructs harboring mutated nucleotides (in red) of predicted E2F2, NRF-1 and CREB binding sites. Numbers indicate the position of the first (from the left to the right) mutated nucleotide. (C) The wild-type reporter plasmid expression was arbitrarily set to 100% and the reporter activity of the mutants was expressed as a percentage of the wild-type construct. Mean ± SD n = 3; *p < 0.05; **p < 0.01; ***p < 0.005.
Fig 4: Differential expression of calretinin in a panel of 13 cell lines(A) Quantitative RT-PCR analysis of CALB2 expression in 11 mesothelioma cell lines, one immortalized mesothelial cell line (MET5A) and HEK293 cells using histones as an internal control. Levels are shown relative to the HEK293 cells according to the –??Ct method. (B) Western blot analysis of calretinin protein levels in the same panel of cell lines. Actin was used as loading control. (C) Relative mRNA levels are plotted against the relative protein levels; each dot represents a cell lines as in A and B.
Fig 5: Transcriptional activity of the minimal CALB2 promoter is proportional to calretinin expressionTranscriptional activity of different promoter constructs was assessed by transient transfection and dual luciferase assay using pTK-RL as an internal control. (A) 5'-deletion constructs of the CALB2 promoter engineered in the pGL3-basic (pGL3-B) luciferase reporter plasmid. Lines represent CALB2 promoter fragment; the name and the size are indicated by nucleotide position upstream (-) or downstream (+) relative to the transcription start site (+1). (B) CALB2 promoter activity in ONE58, SPC111, ZL55 and NCI-H226 cells, which express different levels of calretinin. To allow for comparison of the absolute promoter activity between the cell lines, Firefly light units were multiplied by a correction factor that compensates for differences in the transfection efficacy. The correction factor was based upon the Renilla Light Units (ReLU) of the internal control, pTK-RL, and was calculated by dividing the mean ONE58 ReLU by the mean of ZL55, SPC111 and H226 ReLU for each construct. Mean ± SD, n = 3. (C) Relative mRNA abundance is plotted versus the relative Firefly light units reflecting absolute -161/+80bp CALB2 activity for the indicated cell lines.
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