tissue: Epithelial, breast adenorcinoma cell line: Breast cancer cell line MDA-MB231 age: derived from breast adenocarcinoma of female 51 years adult treatment: control PTB shRNA, PTBP1 knockdown, Dox treated
Treatment protocol
In order to analyze the effect of PTBP1 depletion, two consecutive viral transductions were performed in both MDA-MB231 and A2780 cell lines. Cells were plated on 24-well plate (10-20×104 cells/well), maintained in culture for 16 hours, and then medium containing LV-tTR/KRAB-Red lentiviral particles was added. Following 16 h of incubation, cells were transduced a second time by LV-THM/PTBshRNA or LV-THM/LUCshRNA lentiviral particles. Clones expressing both red and green fluorescent protein (dsRED and GFP respectively) were selected and expanded. Following 16 h of incubation, cells were washed and split in two subcultures, one without doxycycline (PTBP1/-DOX; Control , express PTBP1 WT) and the other with Doxycycline (DOX) at a final concentration of 1 μg/ml (PTBP1/+DOX; PTBP1-KD). Doxycycline was prepared according to the manufacturer’s recommendations (Sigma-Aldrich, St. Louis, MO). Five days later, cells were analyzed by fluorescence microscopy, and PTBP1 gene expression was assessed using PCR and Western Blotting (data not shown). The cells that were transduced by LV-LUCshRNA express PTBP1 regardless of the presence of DOX (LUCshRNA/+DOX). Constructs and lentivirus preparation were performed as previously described
Growth protocol
Cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine in a humid environment at 37°C, with 5% CO2.
Extracted molecule
total RNA
Extraction protocol
For each of the cell lines, MDA-MB231 and A2780, total RNAs were extracted from four biological replicates of PTBP1-depleted cells, PTBP1-KD (4× PTBP1/+DOX) and eight biological replicates control cells (4× PTBP1/-DOX and 4× LUCshRNA/+DOX) by Direct-zol RNA kit (Zymo Research, Irvine, CA) . All paired samples consist of PTBP1-depleted cells (PTBP1-KD) and matched control cells. Qualities of RNA were assessed based on the RNA quality indicator (RQI ≥ 8) using Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA).
Label
biotin
Label protocol
Labeling of cRNA were prepared according to the standard Affymetrix protocol.
Hybridization protocol
Hybridization were performed following Affymetrix protocols
Scan protocol
standard Affymetrix protocol
Description
Gene expression data
Data processing
The raw data were normalized according to the Robust Multiple-array Average (RMA) technique using Affymetrix Power Tools (APT).
