Twist1

Full-length FOXQ1, SNAIL1, TWIST1, ZEB2, and DDR2 plasmids were purchased from Open Biosystems. These genes were subcloned into the pENTR vector (RRID:Addgene_149548) and transferred into a pLenti6 vector (BRID: Addgene_21691) via homologous recombination. The lentivirus for the full-length gene was then generated using the lentivirus-expression system (Invitrogen). In addition, a set of short hairpin RNA (shRNA) clones for DDR2, FOXQ1, or SNAIL1 was purchased from Open Biosystems. The information for the effective shRNA is available in Supplementary Primers and shRNA information. The lentivirus for the shRNA was then generated using the Trans-Lentiviral packaging system (Addgene). The generated lentivirus was then used to infect the targeted model cells. Stable cells were generated after being selected with Blasticidin (10 μg /mL) for the overexpression model or puromycin (12 μg/mL; Invivogen) for the knockdown model.

The ISH, IHC and TUNEL staining of FFPE tissues were performed as described previously.^{14 (link)} The primary antibodies used in IHC were as follows: RHOF (1 : 200, ab101349, Abcam), TWIST1 (1 : 200, PA5-49688, Invitrogen), E-cadherin (1 : 400, 3195, CST), Vimentin (1 : 100, 5741, CST). Semiquantitative scoring of ISH and IHC was based upon the staining intensity (I: negative, 0; weak, 1; moderate, 2; intense, 3) and the percentage of positive-staining cells (P: 0–5%, scored 0; 6–35%, scored 1; 36–70%, scored 2; and >70%, scored 3) to obtain a final score (Q) defined as the product of I × P. Low expression was defined as Q<4 and high expression with Q ≥ 4. Two senior pathologists performed the scorings independently in a blinded manner.

Excised tumor and adjacent tissues were fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and cut into sections. Consecutive 4-μm-thick sections were analyzed using primary antibodies against ZEB1 (human; ab181451; 1:200; Abcam), phospho-STAT3 (human; #9145; 1:100; Cell Signaling Technology), STAT3 (human; #4695; 1:100; Cell Signaling Technology), TWIST-1(human; PA5-49688; 1:100; Invitrogen)and a biotin-conjugated goat anti-rabbit polyclonal antibody (1:50; ZSGB-BIO, Beijing, China) as the secondary antibody. Images were obtained by light microscopy (Olympus, Japan) at 100× and 400× magnification and quantified using Image-Pro Plus. Five random fields were examined per animal.

Total RNA and cDNA were prepared as previously stated^{56 (link)}. Transcript levels were measured using a MyiQ Cycler (Bio-Rad). Taqman Probes for SNAI1, SLUG, ZEB1, ZEB2, TWIST1, CJUN, JUNB, CFOS, FRA1(FOSL1), FOSL2, and B2M were purchased from Life Technologies. Primers for DNMT1, DNMT2, DNMT3, EZH2, EHMT1, EHMT2, SETDB1, SUV39H, HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, HDAC9, and GAPDH were obtained from PrimerBank and utilized with Cyber-Green-based qPCR system. Amplification was carried out for 40 cycles of 15 seconds at 95 °C, 30 seconds at 55 °C and 30 seconds at 72 °C. All samples were run in triplicate, and the relative gene expression levels were determined by normalizing their expression to B2M or GAPDH. Expression data are presented as fold-change values relative to the normalized expression levels in a reference sample using the following equation: RQ = 2^−ΔΔCt.

Total RNA was extracted from the cell lysates using the RNeasy Micro Kit (Qiagen) without DNase treatment. The total amount of RNA was converted into cDNA using the SuperScript VILO Mastermix (Invitrogen). Following a gene-specific pre-amplification, qPCR was done in duplicates in a 10 µL total reaction volume using TaqMan™ UniversalMastermix II and exon spanning TaqMan™ assays (EpCAM, BPIFA1, FAM83A, PTHLH, ERBB3, TWIST1, NANOG, PROM1, MET, UCHL1, TERT, CDH5, and GRP; Life Technologies). The qPCR was performed on the ViiA7 Real-Time PCR System with standard thermal cycling parameters (50 °C for 2 min; 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min). A qPCR specific for CK19 was performed at 65 °C annealing/extension with forward and reverse primers that corresponded to published primer sequences and with a FAM-labeled hydrolysis probe (5′-TgTCCTgCAgATCgACAACgCCC-3′) [21 (link)]. Raw data were analyzed using the ViiA7 Software v1.1 (Applied Biosystems, Waltham MA, US) with the automatic threshold setting and baseline correction. If the fluorescent signal did not reach the threshold in both duplicate reactions, the sample was regarded as negative for that respective transcript.

TaqMAN: ZEB1 (Thermo Fisher Scientific, Hs00232783_m1), ZEB2 (Thermo Fisher Scientific, Hs00207691_m1), SNAI1 (Thermo Fisher Scientific, Hs00195591_m1), SNAI2 (Thermo Fisher Scientific, Hs00950344_m1), TWIST1 (Thermo Fisher Scientific, Hs01675818_s1), GAPDH (Thermo Fisher Scientific, 4326317E).
SYBR Green: KRT14 (IDT, Hs.PT.58.4592110), KRT19 (IDT,Hs.PT.58.4188708), MEG3 ex 10-11 (IDT, Hs.PT.58.25190740), GAPDH (IDT, Hs.PT.39a.22214836), KRT5 (IDT, Hs.PT.58.14446018), TP63 (IDT, Hs.PT.58.2966111), CDH3 (IDT, Hs.PT.58.39234242).

For generation of the gene expression lentiviral plasmids, synthesized DNA fragments of Slug (807 bp; NM_003068.4), Snail (795 bp; NM_005985.3) and Twist1 (609 bp; NM_000474.3) (Thermo Fisher Scientific, Waltham, MA, USA) were cloned into the pLex-MCS lentiviral vector. The shRNA-containing lentiviral vectors were provided by the National RNAi Core Facility, Academia Sinica, Taiwan. The identifier (ID) numbers of the two shRNA clones used for E-cadherin knockdown are as follows: TRCN0000130433 (shE-cad #1), TRCN0000131097 (shE-cad #2). The lentiviral particles for all expression plasmids and shRNAs were prepared by co-transfection with SPAX2 and pMD2G plasmids into HEK293T cells. Stable clones of individual infected cell lines were established by using puromycin (1–3 μg/mL).