Cell culture

All cell lines used in this study were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea), where they were characterized by DNA-fingerprinting and isozyme detection, and cultured according to American Type Culture Collection instructions. All cell lines were used within 3 to 20 passages of thawing the original stocks and were tested every 3 months for mycoplasma contamination. The cell lines were maintained for no more than 3 passages between experiments. Human HEK293T and human breast cancer cell lines (MCF-7, MDA-MB 231) were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and 1% penicillin and streptomycin. Human breast cancer cell lines (T47D, BT549) were cultured in RPMI (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and 1% penicillin and streptomycin. Serum starvation experiments were performed by switching from normal medium to medium containing 0.2% FBS after 24 h of incubation and maintaining the culture for an additional 48 h.

Plasmid construction and transfection

The wild type Snail, HSC70, and LAMP2A plasmids were purchased from Sino Biological (Beijing, China). Site-directed mutagenesis was performed with a QuikChange mutagenesis kit (Stratagene, San Diego, CA, USA), according to the manufacturer’s instructions. For transient transfection, HEK293T cells were seeded in 6-well or 100-mm-diameter dish for 24 h and transfected with the indicated plasmid by using X-tremeGENE™ HP DNA transfection reagent (Roche, Basel, Switzerland) following manufacturer’s instruction. After 48 h, the cells were harvested and used for western blot analysis. Two different siRNA oligo duplexes for targeting human HSC70 (siHSC70-1; 5’-GCUGGUCUCAAUGUACUUATT-3’ and siHSC70-2; 5’-GUGCCAUGACAAAGGAUAATT-3’), LAMP2A (siLAMP2A-1; 5’-GCAGUGCAGAUGACGACAATT-3’ and siLAMP2A-2; 5’-GCCUUGGCAGGAGUACUUATT-3’), LAMP2B (siLAMP2B; 5’-GUGUUCGCUGGAUGAUGACACCATT-3’) or ATG7 (siATG7-1; 5’-CAGCUAUUGGAACACUGUATT-3’ and siATG7-2; 5’-CUCUUGAAAACCCUGUACUTT-3’), respectively, were purchased from Bioneer (Daejeon, Korea). Transient transfection of siRNA oligo duplex was accomplished using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer’s instruction. For stable transfection, MCF-7 cells were transfected with Flag-tag (Control), Flag-WT-Snail, or Flag-58AAAA-Snail expressing plasmid by using the X-tremeGENE™ HP DNA transfection reagent (Roche, Basel, Switzerland). After 48 h incubation, 500 µg/ml of G418 (Sigma, St. Louis, MO, USA) was added to the cultures to select for G418-resistant clones. Three to four weeks later, independent colonies were picked using cloning cylinder (Sigma, St. Louis, MO, USA), sub-cultured, and tested for Snail expression by western blot analysis.

Immunoprecipitation

Cells were lysed in lysis buffer: 20 mM Tris pH 7.4, 2 mM EDTA, 150 mM sodium chloride, 1 mM sodium deoxycholate, 1% Triton X-100, 10% glycerol, 2 pills protease inhibitor cocktail (Roche, Basel, Switzerland), mixed by vortexing and incubated 30 min on ice. Lysates were pre-cleared using protein A/G beads (Santa Cruz Biotechnology, Dallas, Texas, USA), incubated with the specific antibodies for overnight at 4 °C with gentle mixing and then incubated with beads for 2 h at 4 °C with gentle mixing. Beads were then washed 5 times with lysis buffer and eluted with 40 µl of 2 × SDS sample buffer. Western blot analysis was then performed. Primary antibodies used for IP were as follows: anti-Flag (Abm #G191; 2 µg/ml), anti-HA (Abm #G036; 2 µg/ml), mouse mAb IgG1 isotype control (Cell Signaling #5415; 2 µg/ml), anti-HSC70 (Santa Cruz Biotechnology, #sc-7298; 2 µg/ml) and anti-Snail (Cell Signaling #3895; 2 µg/ml).

Western blot analysis

Protein samples were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with indicated primary antibodies for overnight at 4 °C. After washing with TBS-T (TBS containing 0.1% Tween-20), membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000) for 1 h at room temperature. Blots were developed with enhanced chemiluminescence (ECL, Bio-Rad, Hercules, CA, USA) reaction according to manufacturer’s instructions. Primary antibodies used were: anti-Snail (Cell Signaling, #3879; 1:1,000), anti-HSC70 (Santa Cruz Biotechnology, #sc-7298; 1:500), anti-LAMP2A (Abcam. #ab125068; 1:1000), anti-LAMP2B (Abcam. #ab18529; 1:1000), anti-LAMP1 (Cell Signaling, #9091; 1:1,000), anti-Flag (Abm, #G191; 1:2,000), Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) (Sigma-Aldrich, #A8592; 1:2,000), anti-HA (Abm, #G036; 1:2,000), anti-HA High Affinity (Roche, #11867423001; 1:1,000), anti-Histone H3 (Cell Signaling, #4499; 1:1,000), anti-α-tubulin (Sigma-Aldrich, #T6199; 1:10,000), anti-β-actin (Cell Signaling, #4967; 1:1,000), anti-LC3B (Cell Signaling, #3868; 1:1,000), anti-Ubiquitin (linkage-specific K48) (Abcam. #ab18529; 1:1,000), anti-ATG7 (Cell Signaling, #8558; 1:1,000), anti-p62/SQSTM1 (Sigma-Aldrich, #P0067; 1:1,000), anti-MEK1/2 (Cell Signaling, #8727; 1:1,000), anti-Lamin A/C (Santa Cruz Biotechnology, #sc-376248; 1:500), anti-E-cadherin (BD Biosciences, #610181; 1:10,000), anti-Occludin (Invitrogen, #71-1500; 1:1,000), anti-ZO-1 (Invitrogen, #40-2200; 1:1,000), anti-Fibronectin (BD Biosciences, #610078; 1:5,000), anti-N-cadherin (BD Biosciences, #610921; 1:5,000), anti-Vimentin (Santa Cruz Biotechnology, #sc-6260; 1:500), anti-Claudin-1 (Cell Signaling, #13995; 1:1,000), anti-p-PKD1 (Ser744/748) (Cell Signaling, #2054; 1:1,000), anti-PKD1 (Cell Signaling, #90039; 1:1,000), anti-p-PAK1 (Thr423)/PAK2 (Thr402) (Cell Signaling, #2601; 1:1,000), anti-PAK1 (Cell Signaling, #2602; 1:1,000), anti-GSK3β (Ser9) (Cell Signaling, #5558; 1:1,000), anti- GSK3β (Cell Signaling, #9315; 1:1,000), anti-p-Akt (Ser473) (Cell Signaling, #9271; 1:1,000), anti-Akt (Cell Signaling, #9272; 1:1,000).

Total RNA extraction and qRT-PCR

Total RNA was extracted from the cultured cells using RNeasy Mini Kit (GeneAll, Seoul, Korea) following the manufacturer’s instructions. Reverse transcription was performed using iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. qRT-PCR was performed using iQ™ SYBR®q RT/PCR PreMix (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. Amplification was performed using CFX96 Touch Real-Time Detection System. Primers used were: Snail (F: 5’-ATCGGAAGCCTAACTACAGC-3’; R: 5’-CAGAGTCCCAGATGAGCATT-3’), GAPDH (F: 5’-GTGGTCTCCTCTGACTTCAAC-3’; R: 5’-TCTCTTCCTCTTGTGCTCTTG-3’).

Immunofluorescence and confocal microscopy

The cells were washed once in PBS and then fixed in fresh 4% paraformaldehyde at room temperature for 15 min. To ensure permeability and prevent non-specific antibody binding, the cells were treated with PBS containing 0.1% Triton X-100 and 5% bovine serum albumin (BSA) for 1 h. Following that, the cells were incubated overnight at 4 °C with the appropriate antibody. The cells were washed four times with PBS and then incubated for 1 h with Alexa Fluor-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Samples were washed and mounted on microscope slides with a drop of VECTASHIELD (Vector Labs, Newark, CA, USA) and sealed with medical adhesive. Samples were examined with laser-scanning confocal microscope (Olympus, Japan).

Cycloheximide (CHX) pulse-chase assay

HEK293T and MCF-7 stable cells were seeded on 12-well plate at a density of 2.5 × 105 cells per well. After culturing overnight, the cells were transfected with plasmids or siRNAs or treated with inhibitors as desired. Two days after transfection, the cells were treated with 100 µg/ml of cycloheximide before harvest. Total protein lysates were collected at different time points and subjected to immunoblotting for Snail.

Ubiquitination assay

Ubiquitination assay was done following an immunoprecipitation protocol. After culturing overnight, HEK293T cells were transfected with plasmids or siRNAs as desired. Two days after transfection, cells were treated with 10 µM MG132 (Sigma-Aldrich, #C2211) for 8 h to block proteasomal degradation of the Snail protein before lysed with Triton X-100 lysis buffer. Cell lysates were then collected and immunoprecipitated with anti-Flag antibody (Abm, #G191; 2 µg/ml) to specifically pull-down Flag-Snail protein. Pulled down samples were subject to immunoblotting with anti-HA (Ubiquitin) to visualize polyubiquitinated Snail protein bands.

Cellular fractionation and lysosome isolation

HEK293T cells were cultured at a density of 1 × 106cells per dish in 100 mm diameter dishes and transfected with plasmids 24 h later. After 48 h of cultivation, cells were harvested. 12 h before cell harvest, the cells were treated with NH4Cl (Sigma-Aldrich, #A9434; 20 mM) and leupeptin (Santa Cruz Biotechnology, #sc-295358; 100 µM). Cellular fractionation was carried out using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, #78835) following the manufacturer’s instructions. Lysosome isolation was performed using the Minute™ Lysosome Isolation Kit for Mammalian Cells/Tissues (Invent, #LY-034) following the manufacturer’s instructions.

Proliferation assay

Cells were seeded in 6-well plates at 1 × 105 cells/well. After incubation for 1 to 4 days, cells were trypsinized and resuspended in 1 ml of appropriate medium. The viable cells were stained with trypan blue and counted with a hemocytometer.

Migration assay

Cells were seeded into Culture-Insert (Ibidi, Fitchburg, WI, USA) at 5 × 105 cells/insert. After the cells were confluent, the Culture-Insert was removed and washed with PBS for three times to rinse off the detached cells. Cells were then cultured with appropriate fresh media for further 24 h. The wound closure was observed and photographed at indicated times, using a phase-contrast microscope with digital camera.

Invasion assay

Invasion assays were assessed using QCM™ 24-Well Cell Invasion Assay (Fluorometric) kit (Millipore, Bedford, MA, USA) following the manufacturer’s instruction. Cells were serum-starved for 24 h and 2.5 × 105 cells in 250 µl of serum-free medium were seeded into upper chambers. The lower chambers were filled with 500 µl of appropriate media containing 20% fetal bovine serum. 30 h after incubation, non-invaded cells/medium remaining on the upper chambers were removed by pipetting. The upper chambers were transferred into a clean well containing 225 µl of prewarmed Cell Detachment Solution, and incubated for 30 min at 37 °C. The upper chambers were removed from the well. 75 µl of Lysis Buffer/Dye solution (CyQuant GR Dye 1:75 with 4X Lysis Buffer) was added into each well and incubated for 15 min at room temperature. Two hundred microliters of the mixture were transferred into a 96-well plate and assessed with a fluorescence plate reader using a 480/520 nm filter set.

Mice and animal housing

Female BALB/c nude mice at 6 weeks of age were purchased from DooYeol Biotech and housed in a pathogen-free barrier room in Animal Care Facility at Korea Research Institute of Bioscience and Biotechnology (KRIBB). All experiments using animals were conducted under the Institutional Animal Care and Use Committee (IACUC)-approved protocols at KRIBB in accordance with institutional guidelines.

Xenografts studies

For metastasis analysis of MCF-7 human breast cancer models, 2 × 106 of MCF-7 (Control), wild-type Snail or mutant Snail-expressing MCF-7 (Snail-WT) and MCF-7 (Snail-58AAAA) cells were injected into the lateral tail-vein of BALB/c female nude mice (n = 7 for each group). Six weeks after the injection, the mice were euthanized, and stained H&E at lung.

Statistical analysis

Quantitative data in this study are presented as means ± S.D. and were analyzed by Student’s t-test, one-way ANOVA or two-way ANOVA. P < 0.05 was considered statistically significant. For quantification of protein stability following treatment of cycloheximide, Snail and α-tubulin proteins detected by immunoblotting were quantified using ImageJ software. For normalization, α-tubulin expression was used as a control. GraphPad Prism version 7 and SPSS version 26 software were used in this study. All experiments were repeated at least three independent times. Animal studies were performed with adequate n numbers to ensure statistical evaluation. No statistical method was used to predetermine sample size. Sample size was chosen on the basis of literature in the field.