Gsk3b

HCC827OR and PC9OR cells were treated with FYN at IC₅₀ for 48 h to determine protein expression levels. The cells were then lysed in ice-cold RIPA buffer (Beyotime Inc., China, P0013B), and a BCA Protein Assay Kit (Beyotime Inc., China, P0010) was used to quantify protein concentrations. Total proteins in different groups were subjected to SDS-PAGE gel separation, then transferred to polyvinylidene difluoride membranes (Merck Millipore). After blocking with 5% skim milk powder at room temperature for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight. Secondary antibodies were then added, and the preparations were incubated for 2 h. The probed membranes were developed with ECL solution (Sangon, Shanghai, China, C510043-0100). Primary antibodies against SRSF1 (Cat# 14908), GSK3B (Cat# 5558), β-catenin (Cat# 8480), and GAPDH (Cat# 5174) were acquired from Cell Signaling Technology. The primary antibody against PCNA (Cat# sc-56) was acquired from Santa Cruz Biotechnology.

MDA-MB-231 wt and miR-21 KO cells were seeded into 6-well plates and allowed to settle overnight; the cells were washed with 1× PBS and fixed with 4% formaldehyde for 20 min at room temperature. Cells were washed with 1× PBS twice, then blocked with 5% w/v bovine serum albumin (Invitrogen, Loughborough, UK) for 30 min at room temperature. The wells were washed with PBS twice and primary antibodies E-cadherin, Zeb1, Snail (20C8) (Invitrogen, Waltham, MA, USA), vimentin and GSK3B (Cell Signaling Technology; Danvers, MA, USA) ALDH1 (B-5) and GATA2 (H6) Santa Cruz Biotechnology; CA, USA), Wnt-11 (GeneTex; CA, USA) added and incubated for one hour. After washing the cells, either goat anti-rabbit IgG Alexa Fluor or anti-mouse IgG (Thermo Fisher, Oxford, UK) was added and incubated for an hour. Following another washing step with 1× PBS, ribonuclease A 100 mg/mL (Sigma-Poole, Dorset, UK) was added and incubated, gently rocking for 20 min. For counterstaining, 5 µL/mL of 1 nM To-Pro-3 (Thermo Fisher Scientific, Oxford, UK) was dispensed into each well and set gently rocking, then washed twice with PBS for 5 min gently rocking. The results generated were taken from the three biological and technical repetitions. 3–4 mL of 1× PBS were added to each well, Leica TCS SP2 (Leica Microsystems; Milton Keynes, UK) confocal microscope was used to analyze the cells.

Conducted using standard techniques. Immunofluorescence images were acquired on a Leica DMi8 inverted microscope equipped with a HCX PL Fluotar 100X /1.30 oil objective. Images were analyzed using ImageJ (NIH, Wayne Rasband, http://rsb.info.nih.gov/ij/). Cells were grown on glass coverslips coated with poly-d-lysine. Cells were fixed in 10% formalin for 10 minutes, permeabilized with 0.3% Triton X-100 in PBS for 1 hour, and incubated with primary antibody cocktail overnight. Primary antibodies used: MAP2 (ab5392; Abcam), AT-8 (MN1020; Thermo Scientific), MC-1 (Peter Davies lab), LC3b (ab48394; Abcam), p62 (23214; Cell Signaling Technologies), pGSK3b (Ser9) (9336; Cell Signaling Technologies), GSK3b (9832; Cell Signaling Technologies, pAMPK (Thr172) (2535; Cell Signaling Technologies), AMPK (2532; Cell Signaling Technolgies), Tomm20 (ab56783; Abcam), pJNK (Thr183/ Tyr185 (9255; Cell Signaling Technologies), and JNK (9252; Cell Signaling Technologies). Lipid droplets were detected using HCS LipidTox Red following manufacturer’s protocol (H34476; Thermo Scientific).
Sholl analysis was conducted using the SNT plugin (https://imagej.net/plugins/snt/) for ImageJ.