N cadherin 22018 1 ap

Paraffin-embedded tissue was cut into 5 μm thick slices, dewaxed with xylene, and hydrated with ethanol. Then, the slides were heated at 95°C in a 0.01 M citrate buffer (pH = 6.0) and quenched for peroxidase activity with 3% hydrogen peroxide for 20 min to retrieve antigens. After being treated with 10% goat serum, the slides were incubated overnight with antibodies at 4°C. PBS washing was followed by incubation with goat anti-rabbit IgG for 1 h and then stained with 3,3-diaminobenzidine. When all sections were dehydrated and sealed, we selected images with an inverted microscope (ZEISS). Immunohistochemistry was performed with the following antibodies: Ki67 (27309-1-AP; Proteintech), MALT1 (66225-1-Ig; Proteintech), p-p65 (AB11014, phosphor-Ser536), fibronectin (66042-1-Ig; Proteintech), N-cadherin (22018-1-AP; Proteintech), E-cadherin (20874-1-AP; Proteintech), vimentin (10366-1-AP; Proteintech) Snail1 (26183-1-AP; Proteintech), and Snail2 (12129-1-AP; Proteintech).

Radioimmunoprecipitation assay lysis buffer (China Institute of Biotechnology, Beyotime) was added to tissues and SMMC-7721 cells to prepare protein samples. Bicinchoninic acid protein quantification kits (Bio-Rad) were utilized for detection of protein concentration. The lysate was mixed with the loading buffer and denatured in boiling water. Next, the same amount of sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to the polyvinylidene fluoride membrane. After blocking the membrane with 5% skim milk at room temperature for 30 min, it was placed with the primary antibody at 4 °C overnight. Next, the membrane and horseradish peroxidase-coupled secondary antibody (ab205718; Abcam) were incubated at room temperature for 1 h. Finally, protein bands were developed using enhanced chemiluminescence Blot substrates (Promega). The antibody information was as follows: PTMA (YN2871, immunoway), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP) (Proteintech), Snail (ab53519), GAPDH (ab8245) (Abcam)^{20 (link)}.

Sorafenib was purchased from LC labs, Rapamycin was purchased from MedChemExpress. Antibodies against HA (MMS101P, Covance), FLAG (F1804, Sigma), GST (IT003 M, M&C Gene Technology), His (66005-1-Ig, Proteintech), TGN46 (13573-1-AP Proteintech), AFP (4550-1-AP, Proteintech), GP73(sc-365817, Santa Cruz), E-cadherin (sc-8426, Santa Cruz), N-cadherin (22018-1-AP, Proteintech), MMP9 (10375-2-AP, Proteintech), PTEN (9188, CST), p-AKT (4060, CST), AKT (9272, CST), β-actin (AC026, ABclonal).

A172 and SHG44 cells were lysed with RIPA buffer for half an hour at 4°C. The supernatant was collected and boiled at 95°C for 5–8 min in SDS loading buffer. Then, they were subjected to electrophoresis in 10% SDS-polyacrylamide gels and transferred to the polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in phosphate-buffered saline (PBS) for 1 h before being incubated with the primary antibody at 4°C overnight. The primary antibodies for western blotting used in this study were GAPDH, ETV2 (ab181847, Abcam), N-cadherin (22018-1-AP, proteintech), and vimentin (10366-1-AP, Proteintech). Then the cells were washed three to four times with 0.1% PBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) for 1 h at room temperature. The membranes were washed in 0.1% PBST four times before exposure. Chemiluminescent HRP substrate was purchased from Millipore (Catalog: WBKLS0500). Images were acquired in a Bio-Rad Universal Hood II machine with Image Lab software.

After lysis with lysis buffer, 20 μg of protein per sample was loaded onto a lane of a sodium dodecyl sulphate-polyacrylamide gel for electrophoresis. The proteins were electrotransferred to polyvinylidene fluoride membranes. The transferred membrane was treated with blocking buffer and incubated with primary antibodies (E-cadherin [20874-1-AP; Proteintech, Chicago, IL, USA)], N-cadherin [22018-1-AP; Proteintech, Chicago, IL, USA], Cyclin D1 [60186-1-lg; Proteintech, Chicago, IL, USA], β actin [MAB1501R; Millipore, Burlington, VT, USA]) for 2 h at room temperature. Secondary antibodies (goat anti-rabbit [AP132P; Millipore, Burlington, VT, USA] and goat anti-mouse [AP124P; Millipore, Burlington, VT, USA]) were then added, and the cells were incubated for 90 min at room temperature. An enhanced chemiluminescence solution (205–14,621; Revvity, Burlington, VT, USA) was used to detect specific bands using a MINICHEMI (Thermo) system.

IHC assays were performed as described previously [5 (link)]. In brief, tumor sections (5 μm thick) were, respectively, treated with antibody of LC3-II (cat. no. #3868), (1 : 500, Cell Signaling Technology, Beverly, MA, USA), beclin-1 (cat. no. 11306-1-AP), p62 (cat. no. 18420-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), vimentin (60330-1-Ig), and TWIST1 (25465-1-AP) (1 : 200; Proteintech, USA) overnight at 4°C. Then, sections were incubated with a corresponding secondary antibody for 30 min and treated with the ABC reagent for 30 min and then treated with 3,3′-diaminobenzidine for 10 min. Finally, the detected indexes were observed and documented (400×) by a microscope (Leica, Solms, Germany). The quantification of IHC assays was calculated through the rate of positive cell number to total cell number at 5 fields which were selected in each slide randomly.

Fetal bovine serum was purchased from Serana (Germany), and RPMI-1640 medium was purchased from Hyclone (Beijing, China). Puromycin and polybrene were purchased from Beyotime (Shanghai, China). Total RNA extraction kits were purchased from Tiangen (Beijing, China). Reverse transcription kits and quantitative real-time PCR kits were purchased from Vazyme (Nanjing, China). LINC00922 siRNA lentivirus (si-LINC00922) and negative control (NC) lentivirus (viral titer: 8 × 10⁸TU/ml) were purchased from GenePharma (Shanghai, China). The sequences of siRNA were shown in Supplementary Table S2. The 24 well plates and the matrigel (356231) for transwell assay were purchased from Corning Incorporated (USA). The antibodies include GAPDH (60004-1-Ig), E-Cadherin (20874-1-AP), N-Cadherin (22018-1-AP), and vimentin (10366-1-AP) antibodies were purchased from Proteintech (Hubei, China).