Plod2

Whole-cell lysates were prepared in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Cell Signaling Technology, USA). The concentration of protein was quantified with the Bradford Method using Coomassie Blue Staining Solution (Beyotime, China). Total protein was then separated by 8% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes and probed with primary antibodies against HIF-1α (Abclonal, USA), PLOD2, β-catenin (Proteintech, USA), E-cadherin (Genetex, USA), FAK, p-FAK (Abcam, USA), AKT, phosphorylated-AKT, MMP2, MMP9 (Abcam, USA) and Vimentin (Cell Signaling Technology, USA). The bands were detected using Enhanced Chemiluminescence (ECL) (Bio-rad, USA) or the Odyssey IR Imaging System (LI-COR Bioscience, USA).

WB assay was done in accordance with prior literature descriptions (19 (link)). We used RIPA buffer to extract the proteins needed for western blotting. The subsequent antibodies were utilized: PLOD2 (Proteintech, China), GLUT3 (Abcam, UK) and GAPDH (ImmunoWay, USA).

Sections (4 μm) were cut from paraffin-embedded samples and then processed for immunohistochemistry. Briefly, the sections were deparaffinized, rehydrated, boiled in 1 mM EDTA (pH 8.0) for antigen retrieval and blocked with goat serum. Sections were incubated with primary antibodies overnight at 4°C (PLOD2 at 1:100; Proteintech; Wuhan, China), rinsed and incubated with poly-HRP secondary antibodies for 30 min. The substrate used for visualization was 3′-diaminobenzidine, and nuclei were counterstained with hematoxylin. Images were captured using an Olympus IX81 microscope. Blinded review of the specimens was performed independently by two pathologists. The immunoreactivity of PLOD2 was semiquantitatively scored with a well-established system incorporating both the percentage of positive tumor cells and the intensity of staining (5 fields of view per section). In short, a mean percentage of positive cells was determined among 5 fields at 200×, and the case was assigned to one of five categories: < 5% (0), 5–25% (1), 25–50% (2), 50–75% (3), and ≥ 75% (4). The mean intensity of staining was also determined in 5 fields and scored as: weak (1), moderate (2), and strong (3).

Paraffin sections (4 mm) were deparaffinized in 100% xylene and re-hydrated in descending ethanol series and water according to standard protocols. Heat-induced antigen retrieval was performed in 10 mM citrate buffer for 2 min at 100°C. Endogenous peroxidase activity and non-specific antigens were blocked with peroxidase blocking reagent containing 3% hydrogen peroxide and serum. The sections were then incubated with primary antibodies, including HIF-1 α (1:50; Novus Biologicals, USA), PLOD2 (1:100;Proteintech, USA), E-cadherin (1:100;Proteintech, USA), β-catenin (1:100; Proteintech, USA), and snail (1:100; Abcam, USA), at 4°C overnight. After washing, the sections were followed by incubation with biotin-labeled rabbit anti-goat antibody for 15 min at room temperature, and subsequently were incubated with streptavidin-conjugated horseradish peroxidase (HRP) (Maixin, Fuzhou, China). The peroxidase reaction was developed using 3,3-diaminobenzidine (DAB) chromogen solution in DAB buffer substrate. Sections were visualized with DAB and counterstained with hematoxylin, mounted in neutral gum, and analyzed using a bright field microscope equipped with a digital camera (Nikon, Japan).

Cells were fixed with 4% paraformaldehyde followed by permeabilisation and staining with primary antibodies for LOXL2 (1:100, R&D Systems), PLOD2 (1:100, Proteintech) and tetramethylrhodamine (TRITC)-conjugated Phalloidin (1:1000, Millipore UK Limited, Watford, UK). The secondary antibodies used were Alexafluor 488 and 647 (1:1000, BioLegend UK Ltd, London, UK). Cell nuclei were counterstained with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (1:1000, Millipore UK Limited, Watford, UK). Cells were imaged using an inverted confocal microscope (Leica TCS-SP5 Confocal Microscope, Leica Microsystems).

Cultured cells were washed twice in PBS and lysed in RIPA buffer (Beyotime, China) with complete protease inhibitors (Roche, Germany). Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% low-fat dry milk in TBST for 2 hours at room temperature and probed with the indicated primary antibodies overnight at 4°C; Antibodies for the following targets were used: PLOD2 (Proteintech, USA), Bax (Signalway Antibody, USA), P-gp(MDR1), MRP1, BCRP(ABCG2), Bcl2 (Santa Cruz, USA), and GAPDH antibody (Abcam, USA). After washed with TBST, the membranes were incubated with HRP-conjugated anti-IgG secondary antibody at room temperature for 1 hour. Signals were analyzed using an Enhanced Chemiluminescence (ECL) Detection System (Tanon, China).

The proteins were separated by 10% SDS-PAGE and transferred to PVDF Membranes (Millipore). The membranes were blocked in 5% BSA for 1 h at room temperature. The membranes were incubated with primary antibodies overnight at 4 °C, GAPDH(1:5000, #10494; Proteintech), β-tubulin(1:2000, #10068; Proteintech), LTBP1(1:500, #26855; Proteintech) and FN1(1:500, #66042; Proteintech), PLOD2 (1:500, #21214; Proteintech), THBS1(1:500, #bs-2715R; Bioss), RCN3(1:500, #ab204178; abcam), α-SMA (1:1000, #14395; Proteintech), Vimentin (1:1000, #5741; Cell Signaling), N-Cadherin (1:1000,#13116; Cell Signaling), E-Cadherin (1:1000, #3195; Cell Signaling), BCL2 (1:1000, #4223; Cell Signaling), BAX (1:1000, #5023; Cell Signaling). The membranes were washed thrice with TBST and incubated with HRP-conjugated secondary antibody (1:10000) 1 h at room temperature. The membranes were washed thrice with TBST and visualized using ECL (Thermo Scientific). The relative density was quantified by ImageJ.