Anti pan cytokeratin antibody

Invasion of tumor cells through collagen I matrices including TIF or M-CAF cells was performed as described [6 (link),58 (link)]. Briefly, fibroblasts were embedded in a neutralized collagen I solution, extracted from tendon of adolescent rat tails, and allowed to contract matrices in 35 mm plates in DMEM-10% FBS for 3 days (TIFs) or for a week (M-CAFs), in the presence or absence of Pep 1 or Pep 2, as indicated. Then, HT1080-GFP or MDA-MB-231 were grown on matrix top for 2 days in 24 well plates and matrices transferred to an air-liquid interface on a steel greed in 60 mm plates for the invasion phase. After 7 days, matrices were fixed in 4% PFA, paraffin embedded, sectioned and processed for H&E or DAPI (4′,6-diamidino-2-phenylindole) staining. MDA-MB-231 cells were stained with anti-pan-cytokeratin antibody (Sigma-Aldrich C2562) and with a secondary donkey anti-mouse antibody (Alexafluor 594). For quantitation of matrix invasion by HT1080-GFP or MDA-MB-231 cells, only images of fluorescence-emitting samples from a wavelength between 375 and 495 nm or 561 and 594 nm, respectively, were employed. Invading cells were counted in rectangular shapes selected on each matrix section image. The number of infiltrating cells into matrices was counted by ImageJ software and compared to untreated matrices and to the initial inoculus in the “seeding phase” (T0).

The samples described in this work were prepared from a triple-negative breast cancer primary culture and its normal counterpart as described in Pece et al. (2004 (link)). The epithelial origin of the cultures was confirmed by immunofluorescence with an anti-Pan cytokeratin antibody (Sigma-Aldrich). All tissues were collected at the European Institute of Oncology via standardized operative procedures approved by the Institutional Ethical Board, and informed consent was obtained for all tissue specimens. Total RNA, including small species, was isolated through the miRNeasy mini kit (Qiagen). One microgram of total RNA was used to prepare Small RNA libraries following the Illumina TruSeq™ Small RNA Sample Preparation Guide, as by manufacturers’ instructions. The libraries were sequenced at 50 bp single-read mode and 80 million read depth on an Illumina HiSeq 2000 platform. All the relevant steps of the IsomiRage analysis workflow are fully described in the text. Sequencing results are listed in Table S1 in Supplementary Material. Raw data together with detailed description of the procedures are available in GEO database (GSE21090).