Manufacturer’s protocol. Basically, the essential steps applied for BT-20 cells

Manufacturer’s protocol. Basically, the essential steps applied for BT-20 cells were the same as the ones used for MCF-7/AZ cells, differing only in the use of protein G magnetic beads instead of non-magnetic beads for simplicity of use. However, for the tumour sample, some alterations in the basic protocol were employed. Briefly, the tumour sample, that 25033180 was frozen at 280uC since surgical extraction, was thawed and immediately fixed in 1 formaldehyde for 25 minutes, followed by the addition of 16 glycine solution for 5 minutes, washed in 16 PBS twice, frozen in liquid nitrogen, and finally pulverized mechanically. The following steps were the same used for Licochalcone-A breast cancer cell lines.Statistical AnalysisData are expressed as mean values of at least three independent experiments 6 s.d. Student’s t-tests were used to determine statistically significant differences (*P,0.05).Western BlotCells were lysed and the concentration of total protein was determined by Bradford quantification. Western Blot was performed as earlier described [17,18]. For MCF-7/AZ cell line, due to its lower expression of P-cadherin, 50 mg of total protein lysate has been loaded; for BT-20, due to its P-cadherin overexpression, the gel loading was done only with 20 mg of protein lysate. Membranes were incubated with primary antibodies according to the conditions described in Table S1.Results P-cadherin is co-expressed with C/EBPb and is regulated by this transcription factor in breast cancer cellsUsing a large cohort of invasive breast carcinomas, the expression of C/EBPb was previously demonstrated to be significantly associated with P-cadherin expression in about 60 of the cases [18]; however, the cellular co-expression of these two proteins was not verified. Thus, based on the hypothesis that C/ EBPb directly activates the CDH3 gene promoter, a double immunostaining was performed in all invasive breast carcinomas that previously showed strong positivity for both proteins. As represented in Figure 1A, C/EBPb expression was found in the nuclei of the same cells that were expressing P-cadherin at the cell membrane, pointing 1081537 for a putative functional relationship between both proteins. Based on these results, two different breast cancer cell models were used to demonstrate if P-cadherin expression could be affected by C/EBPb: 1) MCF-7/AZ, which is an ER+/luminal type breast cancer cell line expressing moderate levels of Pcadherin, and 2) BT-20, an ER-negative/basal-like breast cancer cell line, highly positive for P-cadherin [17]. The siRNA mediatedknock-down of C/EBPb induced a significant downregulation of all C/EBPb isoforms (LAP1, LAP2 and LIP) in both cell lines. Interestingly, P-cadherin expression was also affected by the reduction of C/EBPb isoforms, being this effect more pronounced in MCF-7/AZ cells (Figure 1B). According with these results, andSite-Directed MutagenesisAll the C/EBPb binding sites mutations in CDH3 promoter were performed in order to impair the binding of any predicted transcription factor: bioinformatic prediction tools were used to blast all point mutated sequences. To introduce point mutations in the CDH3 promoter region, the QuickChange Site-directed Mutagenesis Protocol (SIS 3 site Stratagene, Cedar Creek, USA) was followed, and the oligos used are listed in Table S2. The PCR cycles were set as follows: 95uC for 30 seconds; 16 cycles of 95uC for 30 seconds, 55uC for 1 minute, and 68uC for 5 minutes. Following PCR reaction, products were incubated w.Manufacturer’s protocol. Basically, the essential steps applied for BT-20 cells were the same as the ones used for MCF-7/AZ cells, differing only in the use of protein G magnetic beads instead of non-magnetic beads for simplicity of use. However, for the tumour sample, some alterations in the basic protocol were employed. Briefly, the tumour sample, that 25033180 was frozen at 280uC since surgical extraction, was thawed and immediately fixed in 1 formaldehyde for 25 minutes, followed by the addition of 16 glycine solution for 5 minutes, washed in 16 PBS twice, frozen in liquid nitrogen, and finally pulverized mechanically. The following steps were the same used for breast cancer cell lines.Statistical AnalysisData are expressed as mean values of at least three independent experiments 6 s.d. Student’s t-tests were used to determine statistically significant differences (*P,0.05).Western BlotCells were lysed and the concentration of total protein was determined by Bradford quantification. Western Blot was performed as earlier described [17,18]. For MCF-7/AZ cell line, due to its lower expression of P-cadherin, 50 mg of total protein lysate has been loaded; for BT-20, due to its P-cadherin overexpression, the gel loading was done only with 20 mg of protein lysate. Membranes were incubated with primary antibodies according to the conditions described in Table S1.Results P-cadherin is co-expressed with C/EBPb and is regulated by this transcription factor in breast cancer cellsUsing a large cohort of invasive breast carcinomas, the expression of C/EBPb was previously demonstrated to be significantly associated with P-cadherin expression in about 60 of the cases [18]; however, the cellular co-expression of these two proteins was not verified. Thus, based on the hypothesis that C/ EBPb directly activates the CDH3 gene promoter, a double immunostaining was performed in all invasive breast carcinomas that previously showed strong positivity for both proteins. As represented in Figure 1A, C/EBPb expression was found in the nuclei of the same cells that were expressing P-cadherin at the cell membrane, pointing 1081537 for a putative functional relationship between both proteins. Based on these results, two different breast cancer cell models were used to demonstrate if P-cadherin expression could be affected by C/EBPb: 1) MCF-7/AZ, which is an ER+/luminal type breast cancer cell line expressing moderate levels of Pcadherin, and 2) BT-20, an ER-negative/basal-like breast cancer cell line, highly positive for P-cadherin [17]. The siRNA mediatedknock-down of C/EBPb induced a significant downregulation of all C/EBPb isoforms (LAP1, LAP2 and LIP) in both cell lines. Interestingly, P-cadherin expression was also affected by the reduction of C/EBPb isoforms, being this effect more pronounced in MCF-7/AZ cells (Figure 1B). According with these results, andSite-Directed MutagenesisAll the C/EBPb binding sites mutations in CDH3 promoter were performed in order to impair the binding of any predicted transcription factor: bioinformatic prediction tools were used to blast all point mutated sequences. To introduce point mutations in the CDH3 promoter region, the QuickChange Site-directed Mutagenesis Protocol (Stratagene, Cedar Creek, USA) was followed, and the oligos used are listed in Table S2. The PCR cycles were set as follows: 95uC for 30 seconds; 16 cycles of 95uC for 30 seconds, 55uC for 1 minute, and 68uC for 5 minutes. Following PCR reaction, products were incubated w.