cells were seeded at 1 106 cells per 10 cm plate and allowed to attach for 5 h

cells were seeded at 1 106 cells per 10 cm plate and allowed to attach for 5 h. EGFR-amplified cell line (MDA-MB-468) were less sensitive to caPeptide treatment with the exception of the UACC-893 cell line. Triple-negative breast malignancy cell lines trended toward being more sensitive to caPeptide with the exception of the EGFR-amplified MDA-MB-468 cell line. The resistant BT-474 cell line was the only line positive for all those 4 cell surface receptors considered, being amplified for Her2 as well as positive for EGFR, ER and PR. caPeptide can be endogenously expressed in MDA-MB-231 cells MDA-MB-231 cells constitutively expressing the reverse tetracycline-controlled transactivator protein (rtTA) were stably transfected with pCaPeptide (Fig. 2). The resulting cell pool was named 231-caPep. Features of pCaPeptide include sequence coding for a caPeptideCnuclear localization signal repeat fusion peptide (caPeptide-3xNLS), followed by sequence for 2a peptide and a mCherry reporter gene; all under transcriptional control of the tetracycline regulated promoter, pTight. The transcript is usually translated in a single run with the 2a peptide serving to separate equimolar product of caPeptide-3xNLS from mCherry. Fluorescent microscopy was used to monitor caPeptide-3 NLS expression via the mCherry reporter after induction with doxycycline (Dox; Fig. 2c). Reverse transcription PCR was used to confirm transcription of caPeptide (Fig. 2d). Two different PCRs were used to amplify the cDNA product of the reverse transcription reaction. One reaction used primers that annealed to the 5 end and 3 end of the mCherry sequence in the cDNA template. The product from this reaction was 770 bp reflecting the full length of the mCherry sequence. WM-1119 The other reaction used an upstream primer that annealed WM-1119 to the 5 end of the caPeptide sequence and the same downstream primer as in the first reaction. The resulting 930 bp sequence reflects the full length of the mCherry plus the caPeptide-3 NLS and 2a peptide sequence. A small amount of transcriptional leakage can be detected in the uninduced sample (Fig. 2d, reaction #2, -Dox lane). Open in a separate windows Fig. 2 a Nucleotide sequence of caPeptide placed into WM-1119 expression vector along with the corresponding amino acid translation. The amino acids corresponding to a.a. 126C133 of caPCNA are fold change, value of Students test) caPeptide expression coupled with CDDP treatment leads to cell death Combining caPeptide expression with low levels of CDDP causes cell death as measured by cellular growth assay (Fig. 5a). 231-caPep cells were plated at a starting concentration of 1 1 105 cells per 60-mm plate. Each time point and condition was plated in triplicate. Dox-treated samples were treated with 1 g/ml Dox every other day starting on Day 1. Cisplatin at 2, CACNB2 3.5 or 5 M was added every fourth day starting on Day 1. The media was replenished every fourth day on schedule with the CDDP and every other Dox treatment. Triplicate plates were collected and counted on Day 1, 5, 9 WM-1119 and 13. Consistent with the CDDP resistance of the parental MDA-MB-231 cell line, 231-caPep cells treated with CDDP and without induction of caPeptide grew rapidly and joined exponential growth albeit at an increasingly delayed time as the CDDP burden increased from.

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