Genechip

The reagents used for microarrays were from “GeneChip” Affymetrix mouse 2.0 ST bioarrays—35 k genes for mRNAs and “Gene Chip” miRNA 4.0 Array for miRNAs (Affymetrix Inc., Santa Clara, CA, USA), following the manufacturer's protocols. Briefly, 1 μg of total RNA was transcribed to cDNA by in vitro transcription with the primers T7-(N)6 oligo(d) and complementary double-stranded cRNA and subsequently labeled with biotin. Fragmented cRNA samples were prepared for hybridization on GeneChip Probe Arrays (Affymetrix, USA) and incubated for 17 h. Then, the chips were washed and stained with streptavidin Cy5 and scanned in a Fluidics Station 450 (Affymetrix). Signals were detected and evaluated using GeneChip Operating Software (GCOS). Spots below the detection level of the negative controls were excluded, as well as spots with irregular shapes or intensities close to background levels.

Total RNA was extracted from FSCN1^KD or FSCN1^CON cells using QIAGEN RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s instructions. Integrity of the extracted RNA was determined using the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) before the cDNA synthesis using High Capacity RNA-to-cDNA Kit (Applied Biosystems, Paisley, UK). The cDNA was then subjected for global gene transcription using GeneChip (Affymetrix, Santa Clara, CA, USA), where generated fluorescent oligonucleotide probes were hybridized to the GeneChips^® Human Genome HG-U133 Array in a GeneChip^® hybridization oven as per the manufacturer’s instructions. This array has around 55,000 probe sets, which represent over 39,000 transcripts from 33,000 previously identified human genes. Gene Array scanner (Affymetrix) was used for visualization and GeneChip^® Operating Software was utilized for image quantitation.

Total RNA was purified from xenograft using TRIzol^® Reagent (Gibco, Life Technologies) according to the manufacturer. Briefly, 50–100 mg of fresh frozen tissue per ml of TRIzol^® was disrupted using a homogenizer followed by a single step of phenol/chloroform purification. Total RNA was quantified using the Nanodrop spectrophotometer (NanoDrop Technologies, Inc), and RNA Integrity Number (RIN) was calculated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA samples that reached a RIN between 8 and 10 were used for microarray hybridization (GeneChip; Affymetrix Inc., Santa Clara, CA). The GeneChip^® Human Gene 2.0 ST Arrays were washed and stained using the Affymetrix GeneChip fluidic station 450 (protocol EukGE‐WS2v5_450) and were scanned using a GeneChip scanner 3000G7 (Affymetrix Inc., Santa Clara, CA). GeneChip operating software version 1.4 (Affymetrix Inc., Santa Clara, CA) was used to obtain chip images and for quality control. Background subtraction and normalization of probe set intensities were performed using the method of Robust Multi‐array Analysis (RMA; Irizarry et al, 2003). Microarray analysis was performed by the CHU de Québec Research Center Gene Expression Platform (Quebec City, Quebec, Canada). Seventeen samples of the cohort were previously published (Duconseil et al, 2015) as GEO accession numbers GSE55513 and GSE89792.