Vimentin

Cinnamaldehyde (Aladdin, Shanghai, China) has the chemical formula C₉H₈O, a relative molecular mass of 132.16, and a purity of >99.5%. EGF was purchased from MedChemExpress (MCE, Shanghai, China).
Antibodies against mTOR, p-mTOR (Ser2448), pan-AKT, p-AKT (Ser473), PI3K, p-PI3K(Tyr458), E-cadherin, N-cadherin, vimentin, and Snail were purchased from Affinity Biosciences (Affinity Biosciences, OH, United States). Antibodies against Ki67, MMP9, EGFR (Tyr1068), cleaved-PARP, PARP, cleaved-caspase-3, and caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, United States). The antibody against β-actin was purchased from Proteintech (Proteintech, Chicago, United States).

PTL (> 99%) was provided by Shangdeyaoyuan Co. (Tianjin, China). DEX sodium phosphate (> 98.5%) was purchased from Meilun Biological Technology Co. (Dalian, China), and BLM sulfate (> 91%) was obtained from Meilun Biological Technology Co. (Dalian, China). The NF-κB, Snail, β-actin, GAPDH, E-cadherin, vimentin, MMP1, α-SMA and Col-1 antibodies were purchased from Affinity Biosciences Co. (Beijing, China). The mouse TNF-α, mouse IL-4, mouse TGF-β1, and mouse interferon gamma ELISA Kits were purchased from Meilian Biological Technology Co. (Shanghai, China). Chlorine ammonia T (> 97.08%) and p-dimethylaminobenzaldehyde (> 97.08%) were obtained from (> 99.71%). Reverse-4-hydroxy-l-proline (> 99.4%) was purchased from Bailingwei Technology Co. (Beijing, China). Perchloric acid (> 70%) was obtained from Jingchun Biological Technology Co. (Shanghai, China).

The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology Co., Ltd. P0027) was used to extract nuclear and plasma proteins from the tissues and cells. All lysates contained protease inhibitors. The quantified proteins were separated by 10% SDS-PAGE. After transferring the protein to the PVDF membrane and blocking with 5% BSA, the PVDF membrane was blocked at room temperature with the following primary antibodies: HIF-1α (Affinity, Changzhou, China), TGFΒ1 (Affinity, Changzhou, China), SAMD3 (Affinity, Changzhou, China), p-SMAD3 (Affinity, Changzhou, China) E-cadherin (Affinity, Changzhou, China), Vimentin (Affinity, Changzhou, China), Lamin B (Affinity, Changzhou, China) and GAPDH (Affinity, Changzhou, China). GAPDH and Lamin B were used as loading controls. After 4 h, the excess primary antibody was removed, and the PVDF membrane was incubated with HRP-labelled secondary antibody at room temperature for 2 h. Protein intensity was detected with an Image Lab instrument (Bio-Rad, USA). Each experiment was performed in triplicate.

Neoprzewaquinone A (purity ≥98%) was obtained from Herbpurify (Chengdu, China). SGI-1776 and Netarsudil (purity ≥98%) were purchased from Selleck (Chengdu, China). RPMI 1640, DMEM, DMEM/F12 media and Fetal Bovine Serum (FBS) were acquired from Gibco (NewYork, NY, USA). Paraformaldehyde (PFA; 4%) was purchased from Biosharp (Hefei, China). The BCA protein assay kit, cell cycle analysis kit, annexin V-FITC/PI apoptosis detection kit and Hoechst 33258 were obtained from Beyotime (Shanghai, China). The trypsin, crystal violet (1%), and Mondansylcadaverine (MDC) assay kit were purchased from Solarbio (Beijing, China). The primary antibodies against ROCK1, ROCK2, mTOR/p-mTOR, MYPT1/p-MYPT1, PIM1, BAD/p-BAD, STAT3/p-STAT3, E-cadherin, Vimentin, cyclin B1, cyclin D1, Beclin1, ATG5, LC3B, GAPDH, β-actin, and HRP-conjugated secondary antibodies were purchased from Affinity (Jiangsu, China). The electrochemiluminescence (ECL) western blot detection kit was purchased from Abbkine (Redlands, CA, USA).

Cellular proteins were lysed using standard methods. Cellular proteins were separated by 10% SDS‐PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with TBS containing 0.1% Triton X‐100 and 5% nonfat milk for 2 hours at RT and then were incubated with Trop2 (R＆D SYSTEMS), E‐cadherin (Invitrogen), fibronectin (Affinity), vimentin (Affinity), β‐catenin (Santa Cruz Biotechnology), and β‐actin antibody (Abcam) at 4°C overnight. After being washed, the membranes were incubated with HRP‐conjugated anti‐IgG at RT for 1 hour. Signal detection was carried out with an ECL system (Tanon, shanghai, China). BGC823 and MGC803 were transfected with different plasmids and then lysed in cellular lysis buffer (Sangon Biotech, Shanghai, China) for 15 minutes on ice. Extracts were clarified by centrifugation at 13 000 g for 25 minutes at 4°C for co‐immunoprecipitation. After centrifugation, the supernatant was collected, and then incubated with protein G PLUS‐agarose immunoprecipitation beads (Santa Cruz Biotechnology) at 4°C for 1 hour. Then, they were incubated with special antibodies against Trop2, β‐catenin at 4°C on a rocker platform. The antibody‐coated beads were then incubated with the lysates at 4°C overnight. Immunoprecipitates were collected, washed, lysed, and boiled. The boiled samples were analyzed by Western blot analysis as describe above.