Table 1：primer sequence for full length of MTDH coding sequence

Gene / MTDHCDS (1749bp)
Forward / GCCATGGCTGCACGGAGCTGGCAGGAC
Reverse / TCACGTTTCTCGTCTGGCTTTTTTCTT

Table 2：sequences ofsiRNAs for MTDH and negative control

Gene / Forward / Reverse
siMTDH / GCAAUUCCUUGGAUCUUAUTT / AUAAGAUCCAAGGAAUUGCTT
siNC / UUCUCCGAACGUGUCACGUTT / ACGUGACACGUUCGGAGAATT

Antibodies. Antibodies against E-cadherin (Bioworld), Vimentin (Epitomics), Claudin-1, Slug, Snail, ZO-1, ZEB1 and β-actin (Cell Signaling Technology), Phalloidin(Sigma-Aldrich), and Metadherin (Abcam) were purchased. TGF-β(PEPRO-TECH) was purchased.

Quantitative real-timePCR (Q-PCR). Total RNA was isolated from the cells using TRIzol(Takara) according to the manufacturer’s protocol. To analyze mRNA levels, Q-PCR assays were performed after reverse transcription with RevertAidTM First Strand cDNA Synthesis Kit (Fermentas) using a SYBR-Green–based system (Roche) and Mx3000PTM Real-time PCR System (Stratagene). The relativeamount of tissue MTDH mRNA wasstandardized against glyceraldehyde 3-phosphatedehydrogenase (GAPDH) mRNA.See Supplemental Table 3 for primers sequences.

Immunohistochemistry(IHC) staining.After deparaffinization with xylene and gradual rehydration, antigen retrieval was achievedby pressure cooking inpH9.0Tris-EDTAbuffer for 10 min. Slides wereincubated with the primary antibody (rabbit polyclonal anti-MTDH; dilution 1:100) at 4ºC overnight. Slide omitting the primaryantibodywas used as negative control. Detection took place by theEnVision detection system(Dako) with diaminobenzidine peroxidase serving as chromogen. Theslides were briefly counterstained with hematoxylin and aqueouslymounted.

Immunofluorescencestaining.Cells were cultured on Poly-L-Lysine (Sigma-Aldrich)-coated coverslips and then stained with DAPI (Sigma-Aldrich), fluorescein isothiocyanate labeled Phalloidin, and anti-MTDH antibody. A Zeiss LSM 510 confocal microscope was used tocapture fluorescent images of Matrigel cultures. Fluorescence-stained slides were imaged on anOlympusmicroscope equipped with a CoolSnap HQ2 camera (Zeiss).

Westernblotting.Protein was obtained from cells usingcell lysis solution (Fermentas)according to the manufacturer’s protocol. Cell lysates were separated by SDS-PAGE, transferred onto PVDF membrane, and then incubated with primary antibodies and HRP-conjugated secondary antibodies. Protein bands were visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and photographed by Gel Logic 2200 PRO imaging system.

Cell culture. Human lung cancer cells (A549, H1299 and H1650) were cultured inDulbecco's Modified Eagle Medium (Gibco) with 10% FBS (Gibco) and 1%Penicillin-Streptomycin(Gibco). Cells were transfected using LipofectamineTM 2000 Reagent (Invitrogen). After transfected with MTDH overexpression vector and control vector,H1299 and H1650 cells were cultured inDMEM with 10% FBS in presence of 1000ug/ml G418 (Gibco) until the overexpression colony formed.

MTT and Edu proliferation assay. For MTT assay, 2 × 103 cells were planted into 96-well plates with 6 duplications. Cell viability was tested by Thiazolyl BlueTetrazolium Bromide (Sigma-Aldrich) at 24h, 48h, 72h and 96h. For Edu assay, 1 × 105 cells were planted into 6-well plates. Cells were starved without serum for 48h to synchronize cell cycle. Six hours after addition of serum, newly synthesized DNA was tested by EdU Alexa Fluor® 488 Imaging Kit(Life Technologies).

Soft agar assay. 1 × 104cells (in 0.3% agar with DMEM and 10% FBS) were seeded into 6-well plates layered with 0.7% agar (with DMEM and 10% FBS), and colonies were photographed, and counted21 dayslater. A cluster of cells with either more than 50 cells or diameter greater than 100um was consideredas a colony.

Cell cycle assay.5 × 105 cells were planted into 6cm dish. Cells were starved without serum for 48h to synchronize cell cycle. 12 hours after addition of serum, cell cycle was tested by PI staining (Sigma-Aldrich) and analyzed by flow cytometer (BD Biosciences).

Migration and invasion assay.For scratch assay, 2 × 105 cells were planted into 24-well plates. After 24 hours of incubation, cells were scratched and incubated without serum. Then the scratch was photographed and measured at 10h, 24h and 48h. For migration and invasion transwell assay, 2 × 104 cells were cultured in the upper wells of Transwell (Corning)without orwith Matrigel (BD Biosciences), respectively, and allowed to migrate toward 10% FBS in the bottom wells. After 12 hours and 24 hours of incubation, migratoryand invading cells were respectively stained with 0.1% crystal violet(Beyotime), photographed, and counted.

Animal work. For proliferation experiment, 2×106cells(in 100 µl phosphate-bufferedsaline (PBS)per mouse)that had been stably transfected with MTDH overexpression or empty vectorswere injected subcutaneously in the right flank of 6-week-old BALB/c Nude mice (n=12 per group). Mice were monitored for tumor growth, sacrificed at 8 weeks, and necropsied to isolate primary tumors.For metastasis experiment, 1×106cells (in 100 µl PBS per mouse) that had been stably transfected with MTDH overexpression or empty vectors were injected was injected into the lateral tail veins of 6-week-old BALB/c Nude mice(n=5 per group), sacrificed at 12 weeks, and necropsied to isolate primary tumors and sites of metastasis, which were confirmed histologically by analysis of formalin-fixed tissues.

Table 3：primer sequence for realtime PCR

Human / Forward / Reverse
GAPDH / CCATGTTCGTCATGGGTGTGAAC / GCCAGTAGAGGCAGGGATGATGTTC
MTDH / GTAAACGTGATAAGGTGCTGACT / CGGTGGTAACTGTGATGGTATTT
Claudin-1 / GACCCCAGTCAATGCCAGGTACG / CCCGCTGGAAGGTGCAGGTTT
E-cadherin / ACCCCACAGCCCCGCCTTAT / CCCGCCTCTCTCGAGTCCCC
Snail / TCGGAAGCCTAACTACAGCGA / AGATGAGCATTGGCAGCGAG
Vimentin / GAACGCCAGATGCGTGAAATG / CCAGAGGGAGTGAATCCAGATTA
Slug / AAGCATTTCAACGCCTCCAAA / GGATCTCTGGTTGTGGTATGACA
ZEB1 / GATGATGAATGCGAGTCAGATGC / ACAGCAGTGTCTTGTTGTTGT
ZO-1 / GAGGACCAGCTGAAGGACAG / ATATGGCTTGCCAATCGAAG

Table 4：Clinical characteristics of 38 NSCLC patients analyzed by quantitative real-timePCR

Characteristics / Data
No. of patients / 38
Gender
Female/Male / 11/27
Histology
Non-squamous/Squamous / 19/19
Differentiation
Well and moderate/Poor / 19/19
T Stage
T1/T2/T3/T4 / 9/21/3/5
N Stage
N0/N1/N2 / 26/2/10
TNM Stage
I/II/III / 16/9/13

Table 5：Clinical characteristics of 40 NSCLC patients analyzed by immunohistochemistry

Characteristics / Data
No. of patients / 40
Age(years,mean±SD) / 58.75 ±11.4
≤65/>65 / 26/14
Gender
Female/Male / 14/26
Smoke
Never/Ever / 22/18
Histology
Non-squamous/Squamous / 22/18
Lymph metastasis
None/Ever / 18/22
TNM Stage
I/II/III/IV / 14/16/9/1
Overall Survival
Days(median) / 529