Plasmids and antibodies
The Plvx-GFP plasmid was purchased from Sunny Biotechnology (Shanghai, China). The Plvx-GFP-ALPL expression plasmid was constructed using the following primers: forward: 5’- CCG CTC GAG GCC ACC ATG ATT TCA CCA TTC TTA GTA CTG GCC − 3’, reverse: 5’- CCG GAA TTC GGT GAA CAG GAC GCT CAG GGG − 3’. The Plvx-GFP-RhoA expression plasmid was constructed using the following primers: forward: 5’- CCG CTC GAG GCC ACC ATG GCT GCC ATC CGG AAG A -3’, reverse: 5’- CCG GAA TTC ACC CAA GAC AAG GCA CCC AGA T -3’. The RhoA promoter-driven luciferase reporter was constructed into PGL3 Basic vector using primers, forward: 5’- CGG GGT ACC ACT TCC TGT ATC CTG TTG TTT GTG T -3’, reverse: 5’- CCC AAG CTT CAA ATG ACA ATG ACA CAG GAC ATA C -3’. The c-Myc plasmid was from our research group . The antibody against ALPL (GTTX100817) was purchased from Genetex (Irvine, CA, USA). The antibodies against RhoA (2117S), ERK1/2 (4695S), and p-ERK1/2 (4370S) were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibodies against c-Myc (sc-764) and p-c-Myc (sc-377552) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against GAPDH (GTX100118) was purchased from Genetex.10X Cell Lysis Buffer (9803) was purchased from Cell Signaling Technology.
Cell culture and transfection
Normal bronchus epithelial cell line Beas-2B and lung cancer cell lines A549, H1975, HCC827, H1299, and SK-MES-1 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cells were subjected to DNA analysis and authenticated before use in these studies . A549 and SK-MES-1 cells were cultured in F12K (21127-022; Gibco/ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (10437-028; Gibco). H1975 cells were cultured in a 1:1 mixture of DMEM/Ham’s F12 medium (10565-018; Gibco) supplemented with 5% FBS. HCC827 and H1299 cells were cultured in RPMI 1640 (11875-093; Gibco) supplemented with 10% FBS, and Beas-2B cells were cultured in DMEM (11995-065; Gibco) supplemented with 10% FBS. Cell transfections were performed using PolyJet DNA In Vitro Transfection Reagent (SignaGen Laboratories, Rockville, MD, USA) according to the manufacturer's instructions. For stable cell line selection, cells were treated with G418 (500–1000 µg/mL) or puromycin (0.2–0.3 µg/mL) depending on the antibiotic resistance plasmid used. Cells surviving the antibiotic selection were pooled as stable mass transfectants .
Total RNA was extracted using TRIzol reagent (Invitrogen/ThermoFisher Scientific) according to the manufacturer’s instructions. Next, 5 µg total RNA was used for first-strand cDNA synthesis with the SuperScript First-Strand Synthesis system and oligo (dT) primers (Invitrogen), as described previously .
Fast SYBR Green Master Mix (4385614; Applied Biosystems/ThermoFisher Scientific) was used for real-time PCR with the 7900HT Fast Real-Time PCR system (Applied Biosystems) . The primers used in this study were as follows: human ALPL (forward: 5’- AAC CGA GAT ACA AGC ACT CCC ACT − 3’, reverse: 5’- TCC GTC ACG TTG TTC CTG TTC AG -3’), human RhoA (forward: 5’- ACT ATG TGG CAG ATA TCG AGG TGG A -3’, reverse: 5’- CTA TCA GGG CTG TCG ATG GAA AAA C -3’), human c-Myc (forward: 5’- GGA GGA ACA AGA AGA TGA GGA AGA AA -3’, reverse: 5’- TGA GGA CCA GTG GGC TGT GAG G -3’), and human GAPDH (forward: 5’- GAC TCA TGA CCA CAG TCC ATG C -3’, reverse: 5’- CAG GTC AGG TCC ACC ACT GA -3’).
Western blot analysis
Whole cell extracts were prepared in cell lysis buffer [10 mM Tris-HCl (pH 7.4), 1% SDS, and 1 mM Na3VO4]. Western blotting was performed as described previously [26, 29]. Briefly, blots were incubated with primary antibodies diluted 1:500–1:1000 in 5% BSA for 12–16 h at 4 °C, and then incubated with secondary antibody diluted 1:1000–1:2000 in 5% skim milk for 2–3 h at 4 °C. The images were acquired by scanning with the Phosphoimager Typhoon FLA 7000 (GE, Pittsburgh, PA, USA)  .
The anchorage-independent growth ability of the cells was evaluated in soft agar, as described . Briefly, 3 mL of 0.5% agar (214010; Becton Dickinson) in basal modified Eagle’s medium supplemented with 10% FBS was layered onto each well of 6-well tissue culture plates. Cells (3 × 104) suspended in 1 mL normal medium (B9638; Sigma-Aldrich) were mixed with 2 mL of 0.5% agar in basal modified Eagle’s medium supplemented with 10% FBS, and 1 mL of the mixture was added into each well on top of the 0.5% agar layer. Plates were incubated at 37 °C in 5% CO2 for 3 weeks, and the colonies were counted. The data is presented as the number of colonies/104 cells .
Cell migration and invasion assay
Cell migration and invasion were evaluated using kits from BD Falcon. The assay was performed according to the manufacturer’s instructions, as described previously [26, 30]. Briefly, cells were seeded in the inserts in 400 µL serum-free medium. The inserts were placed in wells containing 700 µL medium with 10% FBS and incubated for 24 h. Then, cells on the upper surface of the filters were removed with a cotton swab. The inserts were fixed in methanol and stained with Giemsa, imaged with a microscope, and then the cells were counted. The data shown are representative of three independent experiments .
Lung metastasis assay
Female euthymic (nu+/nu+) mice were purchased from Shanghai Silaike Experimental Animal Company (license no. SCXK, Shanghai 2010 0002; Shanghai, China). At age 4–5 weeks, the mice were randomized and injected with cells [A549 (vector), A549 (ALPL), HCC827 (vector), and HCC827 (ALPL)] via the lateral tail vein (3 × 106 cells in 100 µL PBS/mouse). Detailed experimental methods have been described previously .
Luciferase promoter reporter assay
The RhoA luciferase reporter and pRL-TK were each transiently co-transfected into A549 and HCC827 cells. Luciferase activity was determined 24 h after transfection using the Luciferase Assay System (Promega, Madison, WI, USA), as described [26, 32, 33]. The values were normalized to the internal thymidine kinase (TK) signal. All experiments were conducted in triplicate, and the results were expressed as the mean ± standard error .
Fixed lung tissues were dehydrated using an alcohol gradient, paraffin-embedded, and then stained with H&E, as described in detail previously . Images were captured using the Nikon Eclipse Ni microsystem (Nikon DS-Ri2; Tokyo, Japan).
In total, 36 pairs of human LUAD tissues and corresponding adjacent normal tissues were obtained. The authorized case information is shown in Supplementary Table S1. Preoperative biopsy and postoperative pathological examination of the above patients confirmed LUAD. The tissue samples were snap-frozen in liquid nitrogen at the time of surgery, RNA was extracted, and cDNA was synthesized and stored at − 80 °C.
Student’s t-tests were used to determine significant differences. The differences were considered to be significant at P < 0.05.