Anti hmga2

Protein was extracted from indicated cells with ice-cold radioimmunoprecipitation lysis solution for 30 min followed by refrigerated centrifugation for 15 min to remove cell debris. The Bicinchoninic Acid Kit (Sigma, St. Louis, MO, USA) was used for concentration measurement. The samples were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyvinylidene fluoride (PVDF) membrane was used for transfer. The background signal was briefly masked with 5% nonfat milk. Primary antibody (anti-HMGA2, #5269, 1:1000, anti-Wnt3a, #2391, 1:1000, anti-p-β-Catenin, #9562, 1:1000; anti-p-β-Catenin, #9561, 1:1000, anti-c-Myc, #9402, 1:1000, anti-β-Actin, #4967, 1:1000, anti-GAPDH, #2118, 1:1000 were obtained from Cell Signaling Technology, Danvers, MA, USA) incubation was performed at 4°C for 12 h. The PVDF membrane was rigorously washed and subjected to hybridization with HRP-conjugated secondary antibody (anti-rabbit, #7074, 1:5000 obtained from Cell Signaling Technology) for 1 h at room temperature. Blots were visualized using an enhanced chemiluminescence method (ECL, Millipore, Billerica, MA, USA). GAPDH and β-actin were employed as loading controls.

Protein samples from the Huh-7 or HepG2 cells transfected with miRNA mimics were extracted using radioimmunoprecipitation assay reagent supplemented with protease inhibitors (both Beyotime Institute of Biotechnology, Haimen, China). The protein was quantified by using BCA Protein Assay kit (Beyotime Institute of Biotechnology). Denatured protein (40 µg) was separated on 8% SDS-PAGE gels and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with a buffer containing 5% skimmed milk in PBS with 0.05% Tween-20 for 1 h at room temperature. The nitrocellulose membranes were incubated with primary antibody (anti-HMGA2, 1:1,000, 7777; and anti-β-actin, 1:6,000, 4970; both Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. Then the membranes were incubated with peroxidase-conjugated secondary antibodies (1:3,000; A0208; Beyotime Institute of Biotechnology). Subsequently, the membranes were incubated with an enhanced chemiluminescence detection system (EMD Millipore).

Formalin fixed tumor samples were sectioned, stained with hematoxylin and eosin (H&E), or the following antibodies: anti-Ki67 (mouse: D3B5, Cell Signaling Technology; human: MIB-1, Dako), anti-PAX8 (Proteintech Cat# 10336–1-AP, RRID:AB_2236705), anti-p53 (mouse: CM5, Novacastra; human: DO-7, Dako), anti-PanCK (mouse: polyclonal, Abcam; human: AE1/3, Dako), anti-vimentin (Cell Signaling Technology Cat# 5741, RRID:AB_10695459), anti-HMGA2 (Cell Signaling Technology Cat# 8179, RRID:AB_11178942), anti–N-cadherin (Abcam Cat# ab18203, RRID:AB_444317), anti-ZEB1 (Novus Cat# NBP1–05987, RRID:AB_2273178), anti-human CD8 (C8/144B, Dako). H&E and IHC slides were scanned digitally at ×20 magnifications using the Pannoramic 1000 scanner (3DHISTECH Ltd.). Ki67 and CD8 IHC were quantified using CellProfiler (Broad Institute).

Whole protein extracts were lysed and run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membrane (BioRad, Hercules, CA, USA). After blocking, the membranes were incubated with the following primary antibodies: anti-HMGA2 (1:1,000), anti-LCN2 (1:1,000), and anti-GAPDH (1:1,000) (Cell Signaling Technology, Danvers, MA, USA), and anti-MSI2 (1:1,000), anti-LOXL2 (1:1,000), and anti-PBX1 (1:1,000) (GeneTex, Irvine, CA, USA). After washing with Tris-buffered saline containing Tween-20 (TBST) and incubation with secondary antibodies, signals were developed using an enhanced chemiluminescence kit (Pierce, Waltham, MA, USA).