P egfr

Cells were lysed in PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Cat# 17081) supplemented with phosphatase inhibitor cocktail (Gen DEPOT, Cat. No. p3200-001). Protein concentrations were then measured using a Bio-rad Bradford assay (Cat# 500-0006, Hercules, CA, USA). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Cat# 10600023). The membranes were blocked with Tris-buffered saline containing 0.05% Tween 20 and 5% non-fat dry milk for 1 h, and membranes were incubated overnight at 4 °C with primary antibody. HER2 (Cat# 2165, RRID:AB_10692490), p-HER2 (Cat# 2247, RRID:AB_331725), p-EGFR (Cat# 2234, RRID:AB_331701), Akt (Cat# 9272, RRID:AB_329827), p-Akt (Cat# 9271, RRID:AB_329825), ERK (Cat# 9102, RRID:AB_330744), and p-ERK (Cat# 9101, RRID:AB_331646) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA), EGFR (Cat# sc-03, RRID:AB_631420) was obtained from Santa Cruz, and ß-actin (Cat# A5316, RRID:AB_476743) was obtained from Sigma (Saint Louis, MO, USA). Then, horseradish peroxidase-conjugated secondary antibodies were incubated for 1 h at room temperature. Immunoreactive bands were detected with chemiluminescence reagent (Cat# RPN2106).

For immunostaining or western blotting, primary antibodies against the following proteins were used: MYOF (Santacruz); EGFR, pEGFR, EPHA2, and EPHA2-pS897 (Cell Signaling); VIM and E-cadherin (Abcam); and actin (Pierce). For western blot analysis, cells were washed once with ice-cold PBS and then lysed in cold RIPA buffer supplemented with protease inhibitor cocktail (Roche Applied Biosciences). Cell lysate was centrifuged at 12,000 × g for 30 min, boiled in 2 × loading sample buffer, separated on a 10% gradient SDS–PAGE gel and blotted onto a PVDF membrane. The membrane was blocked with 0.1% Tween-20 in TBS containing 5% non-fat milk and probed with the indicated primary antibody, followed by incubation with the appropriate secondary antibody conjugated to horseradish peroxidase (Sigma), and the signals were visualized via ECL (Millipore).

Gefitinib (CAS No. HY-50895) and Osimertinib (AZD-9291, CAS No. HY-15772) were purchased from MedChemExpress (NJ, USA). Cycloheximide (CAS No. 66-81-9) was purchased from A.G. Scientific (CA, USA). Yuanhuadine (YD; purity >98.5%) was isolated from a CHCl₃-soluble fraction of the flowers of Daphne genkwa, as described previously^{22 (link)}. All chemicals were dissolved in DMSO for in vitro experiments.
Antibodies against C-terminal AXL (sc-1096), EGFR (sc-03), p-ERK (sc-7383), ERK (sc-94), MET (sc-10), β-actin (sc-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-AXL (#5724), p-EGFR (#2234), p-MET (#3077), p-Akt (#9271), Akt (#9272), p-p70S6 Kinase (#9205), p70S6 Kinase (#9202), p-SAPK/JNK (#9091), SAPK/JNK (#9252), and snail (#3879) were obtained from Cell Signaling Technology (Danvers, MA, USA).

HM, NM, pCMV‐Vector and pCMV‐FAM83D cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (20 mmol/L Tris‐HCl [pH 7.5], 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, 1 mmol/L EGTA, 1% NP‐40, 1% sodium, deoxycholate, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β‐glycerophosphate, 1 mmol/L Na₃VO₄, 1 µg/mL leupeptin and 1 mmol/L PMSF). Total proteins (20‐30 µg) were separated through polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane using a semi‐dry transfer system (Bio‐Rad). The membrane was incubated at 4°C overnight with specific primary antibodies for FAM83D (Biorbyt, orb183501), AKT (Cell Signal Technology, CST, MA, cat#: 4691), p‐AKT (CST, MA, cat#:4060), ERK1/2 (CST, MA, cat#:9102), p‐ERK1/2 (CST, MA, cat#: 9101), c‐Raf (CST, MA, cat#: 9422), p‐c‐Raf (CST, MA, cat#: 9421), EGFR (CST, MA, cat#: 4267), p‐EGFR (CST, MA, cat#:2234), P38 (CST, MA, cat#:9212), p‐P38(CST, MA, cat#:9211), N‐cadherin (CST, MA, cat#:13116), ZO1 (CST, MA, cat#:8193) and beta‐actin (CST, MA, cat#: 4970) used at a dilution of 1:1000. Subsequently, the membrane was incubated with secondary antibody (Bio‐Rad, CA, cat#: 1706515 in 1:5000 dilution) for 1 hour at room temperature. Clarity™ Western ECL Substrate (Bio‐Rad, cat#: 1705060) was used for the detection of protein signals. The signals were captured using the ChemiDoc™ XRS + Imaging Systems (Bio‐Rad, CA).

Total proteins from MM1.S cells lysates, MM1.S exosome, conditioned medium of cells deprived of exosomes, patient’s exosomes, and RAW 264.7 lysates were extracted and analyzed by SDS-PAGE followed by western blotting. Antibodies used in the experiments were as follows: anti-EGFR, pEGFR (Cell Signalling Technology, Lane Danvers, MA, USA), anti-AREG (Novus Biologicals), and anti-GAPDH (Santa Cruz Biotechnology, CA, USA).