Twist2

For all cell lines, total RNA was extracted from 10⁶ viable cells. For tumour xenografts, it was extracted from 20 frozen sections of 5 µm. Total RNA was extracted using RNeasy‐Mini‐Kit (#74104; Qiagen, France) and quantified on NanoDrop (LabTech, France). Human PROM2 gene expression was assessed using the human PROM2 primer Hs00376331_m1 (ThermoFisher). EMT gene expression was assessed using human ZEB1 primers (Hs00232783_m1), ZEB2 (Hs00207691_m1), SNAI1 (Hs00195591_m1), SNAI2 (Hs00950344_m1), TWIST1 (Hs01675818_s1), TWIST2 (Hs02379973_s1), CDH1 (Hs01023894_m1) and VIM (Hs00185584_m1) (ThermoFisher). Human CAV1 gene expression was assessed using the human CAV1 primer Hs00971716_m1 (ThermoFisher). Total RNA was reverse‐transcribed before qPCR amplification using random primers with GoScript™ Reverse Transcriptase (#A5001; Promega, France). The qPCR reactions were performed using fluorescent probes on a CFX96 Real Time System (Bio‐Rad, France). A blank sample (no cDNA) was included and the experiments were performed in triplicate for each gene. The housekeeping genes TBP (Hs00427620_m1) and GAPDH (Hs02758991_g1) were used to normalise gene expression results. Each RT‐qPCR assay was performed according to the MIQE guidelines
²⁴ (link) and conducted in triplicate.

All cells were lysed in buffer composed of 50 mm Tris (pH 7.4), 5 mm EDTA, 150 mm NaCl and 0.5% Triton X-100 containing 1 μg/mL leupeptin and aprotinin and 1 mm phenylmethyl sulfonyl fluoride. The protein content of cell lysates was quantified by using the Pierce BCA protein assay kit, and equal amounts of the total protein were dissolved in Laemmli SDS-PAGE sample buffer prior to separation by 15% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad), and Western blot analysis was performed by using standard techniques with enhanced chemiluminescence detection (Roche/Boehringer Mannheim, Indianapolis, IN, USA). The following antibodies were used in this experiment: E-cadherin, vimentin, B-catenin, TWIST1, B-actin (Cell Signaling, Cat #-3195T, 5741T, 8480T, 46702, 4967S) and TWIST2 (ThermoFisher 66544-1-IG).