RNA stability assay Following sorting into CD44posCD24pos and CD44posCD24neg populations, cells have been seeded into six nicely dishes. One particular day later, cells have been treated with 10g ml Actin omycin D and collected at 0, 4, 8, or 16 hr. RNA was isolated applying Trizol. Changes in CD24 mRNA had been monitored by realtime RT PCR. Statistics Evaluation of variance was performed using StatView 5. 0. 1. For evaluation of realtime RT PCR data, technical replicates for every gene from each of 3 independent experiments have been averaged. Analysis of variance was performed around the resulting three independent values. Benefits CD24 expression is dynamically regulated in breast cancer cell lines In an effort to know the dynamics of CD24 expression in breast cancer cell lines, cells had been sorted based on their CD44 CD24 expression and also the CD44CD24 expression of their progeny was evaluated.
Nineteen breast cancer cells lines had been initially screened for their expression of CD44 and CD24. Four cell lines had been chosen to evalu ate the great post to read fluidity of CD24 expression in vitro. Cells were sorted into CD44posCD24neg and CD44posCD24pos populations and allowed to expand for two passages right after which their CD44CD24 expression was assessed by flow cytometry. For all 4 cell lines queried, CD44posCD24neg cells gave rise to CD44posCD24pos cells and vice versa. Data presented above suggests that CD24 expression is dynamically regulated in immortalized breast cancer cell lines. To evaluate in the event the CD24 gene was susceptible to dynamic transcriptional regulation, CpG methylation status in the CD24 promoter was queried in CD44posCD24neg and CD44posCD24pos populations sorted in the Ca1a cell line.
A region spanning 366 bases and 28 CpG dinucleotides was que ried by way of bisulfite sequencing. No variations in CpG methylation have been observed among CD44posCD24neg and CD44posCD24pos cells. This suggests that rapid changes in CD24 transcription can occur selleckchem without necessitating epige netic modification of its promoter. To further have an understanding of the regulation of CD24 expression, sta bility of the transcript was compared between CD44posCD24neg and CD44posCD24pos FACS sorted Ca1a cells. Following sorting, transcription was inhibited with Actin omycin D as well as the price of CD24 mRNA disappearance was evaluated. As indicated in Figure 1c, variations in CD24 abundance in between CD44posCD24neg and CD44posCD24pos cells is not achieved by altered mRNA stability.
CD24 expres sion as evaluated by flow cytometry could also be regulated in the translational level or by cell surface localization in the pro tein. However, offered that cells devoid on the protein at the cell surface have markedly depressed levels of CD24 transcript indicates that transcriptional regulation plays a considerable role in regulat ing CD24 protein expression.