Ab1416

Tumor tissues in the PDX assays and MiniPDX assays were fixed in buffered 10% formalin and routinely stained with hematoxylin and eosin (H&E) and examined by a certified pathologist.
For immunofluorescence studies, cellularized tumor cells (2 × 10⁴ cells, 200 L) were cytospun onto a slide, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 in PBS for 30 min, and then blocked with 5% normal goat serum for 1 h at room temperature. The cells were then divided into three fractions and incubated with primary mouse monoclonal antibodies at 4 °C overnight against the following proteins: pan-cytokeratin, indicating carcinoma components [27 (link), 28 (link)] (1:200, AE1/AE3, sc-81714, Santa Cruz Biotechnology, Santa Cruz, CA, US), E-cadherin, generally found in gastric adenocarcinomas [29 (link)] (1:50, HECD-1, ab1416, Abcam, Cambridge, UK), and MG7, a marker of gastric cancer [30 (link)] (1:300, NOTA-MG7) [30 (link)]. Subsequently, the cells were probed with secondary antibody donkey anti-mouse IgG H&L (Alexa Fluor^® 488) (1:200, ab150105, Abcam). Finally, the cells were mounted with DAPI-containing mounting medium (S36973, Thermo Fisher, MA, US). Images were captured with a fluorescence microscope (Leica, Germany) with Leica Application Suite V4 software and edited with Photoshop (Adobe, US).

The cell lysates were collected, and 30 mg protein of each sample was separated using 10% SDS-PAGE. The protein was then electrotransferred onto Polyvinylidene Fluoride (PVDF) membrane (Millipore, Boston, MA, USA). The PVDF membrane was sealed in 10% skimmed milk. Then the membrane was incubated with primary antibodies, including anti-TNC (Abcam, ab108930, 1:1000 dilution), anti-GAPDH (Abcam, ab181602, 1:10,000 dilution), and anti-ERK (Abcam, ab184699, 1:1000 dilution)), anti-p-ERK (Abcam, ab201015, 1:1000 dilution), anti-MMP9 (Abcam, ab76003, 1:1000 dilution), anti-MMP2 (Abcam, ab92536, 1:1000 dilution), anti-E-cadherin (Abcam, ab1416, 1:1000 dilution), anti-N-cadherin (Abcam, ab18203, 1:1000 dilution), anti-vimentin (Abcam, ab92547, 1:1000 dilution), anti-snail (Abcam, ab216347, 1:1000 dilution), anti-clumping (Abcam, ab27568, 1:1000 dilution) and anti-TWIST1 (Abcam, ab50887, 1:1000 dilution) overnight. Then the membrane and horseradish peroxidase-conjugated secondary antibody (Abcam AB6721, 1:10,000 dilution) were incubated for 2 h at room temperature. Chemiluminescence detection reagent (Millipore, Boston, MA, USA) was used to visualize protein bands.

OC cells were lysed in cell lysis buffer (Beyotime) containing protease and phosphatase inhibitors to extract the total protein. The protein concentration was evaluated by a bicinchoninic acid (BCA) kit (Thermo Fisher). Then, 30 μg protein sample was run on 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After being blocked in 5% bovine serum albumin, the membranes were cultured with the primary antibodies against E-cadherin (1:50, ab1416, Abcam, Inc., Cambridge, MA, USA), N-cadherin (1:5000, ab76011, Abcam), Vimentin (1:1000, ab92547, Abcam); PLK1 (1:500, #4535, Cell Signaling Technology (CST), Beverly, MA, USA), KDM4B (1:5000, ab191434, Abcam), H3K9me3 (1:1000, ab176916, Abcam), and GAPDH (1:5000, ab8245, Abcam) at 4 °C overnight. Then, the membranes were stained with the secondary antibodies anti-mouse IgG H&L (HRP) (1:10,000, ab205719, Abcam) and goat anti-rabbit IgG H&L (HRP) (1:10,000, ab205718, Abcam) at 37 °C for 45 min. The protein blots were developed by enhanced chemiluminescence (Millipore) and examined on a gel imaging system (Bio-Rad, Hercules, CA, USA). GAPDH was set as the control, and the signal intensity was examined by Image J.