Snail

After 0.3 × 10⁶ cells were seeded in 6-well plates, the cells were incubated with WF for 24 h; the cell morphology was analyzed by microscopic examination. In addition to the morphological analysis, the EMT was examined using rt-PCR. The following primers were selected (all primers were purchased from Thermo Fisher Scientific): Snail (Cat. No. Hs00195591_m1), Snail 2 (Cat. No. Hs00950344_m1), vimentin (Cat. No. Hs00958111_m1), Twist (Cat. No. Hs01675818_s1), E-cadherin (Cat. No. Hs01023894_m1), N-cadherin (Cat. No. Hs00983056_m1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cat. No. Hs02758991_g1) was used as a housekeeping gene. The first step was total RNA extraction with RNeasy Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s protocol. This was followed by reverse transcription of the RNA into cDNA (Super Script Reverse Transcriptase, Invitrogen). For the PCR we used SYBR Green Real-Time-PCR-Master-Mix (Thermo Fisher Scientific, Waltham, MA, USA). The PCR protocol was as follows: for 40 cycles, 50 °C for 2 min, 95 °C for 15 s, and 60 °C for 1 min; the first denaturation step was 10 min. The 2^−ΔΔCq method was used to quantify the proteins [39 (link)].

Five micrometer-thick longitudinal heart tissues were cut from apical to basilar and mounted on a glass slide. To define the fibrotic area of heart, Masson’s trichrome stained area was measured according to standard protocol by using ImageJ software. To determine the number of apoptosis and necrosis in the infarcted myocardium, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Assay Kit—BrdU-Red (Abcam, #ab66110) was used according to the manufacturer’s instructions. TUNEL images were blindly captured and counted at least 4 parts of the left ventricle using a virtual microscope (BX51 Dot Slide; Olympus, Tokyo, Japan). Immunohistochemistry was performed to assess the relationship between EGCG and ischemic myocardium. Sections were deparaffinized and incubated with 1% H₂O₂ to lose endogenous peroxidase. Heart tissues were blocked for 1 h with a mixture of 1% (w/v) BSA and 5% (v/v) horse serum and incubated with Snail (Invitrogen, Carlsbad, CA, USA, #MA5-14801) or CD31 (Sigma-Aldrich, St. Louis, MO, USA) at a ratio of 1:200. After washing 3 times with PBS, the samples were incubated with secondary antibodies with fluorescent dyes (Alexa Fluor 488, #ab150077 at a ratio of 1:250, Abcam) for 1 h at room temperature, mounted with DAPI, and imaged with a confocal microscope (LSM 700, Carl Zeiss, Oberkochen, Germany).

FGFR1 protein expression was evaluated with IHC using a rabbit polyclonal anti-FGFR1 antibody (Clone ab10646, 1:1500 dilution, Abcam, Cambridge, UK) on 4-μm TMA tissue sections on a Ventana Bench Mark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA). FGFR1 staining pattern (cytoplasmic, membranous, or nuclear) and intensity (0, negative; 1, weak; 2, moderate; and 3, strong), and the percentage of positively stained tumor cells (0–100%) were evaluated. Staining pattern of normal squamous epithelial cells and stromal cells adjacent to or separated from tumors was compared to that of tumor cells. In addition, IHC was performed for Snail (dilution 1:200; Invitrogen, Thermo Fisher Scientific, CA, USA) and Twist (dilution 1:200; Abcam, Cambridge, UK), which are transcription factors related to epithelial mesenchymal transition (EMT). IHC expression of FGFR1, Snail, and Twist was analyzed using the semi-quantitative H-score method, which yields a possible score range of 0–300 obtained by multiplying the dominant intensity score with the percentage of positive tumor cells.
In total, 171 samples subjected to p16 IHC (a mouse monoclonal antibody, clone E0037, Ventana, AZ, USA) at the time of diagnosis were reviewed. p16 expression was scored as positive upon detection of at least 70% nuclear and cytoplasmic expression, with at least moderate to strong intensity [29 (link)].