Supplementary figure 1. The levels of F-actin andthe phosphorylation of cofilin and LIMKin Scr/H1299 and SiNUAK1/H1299 cells
(A)Time courses of relative F-actin content in Scr/H1299 and SiNUAK1/H1299 cells with EGF stimulation. EGF, 10ng/mL. The data were collected from a representative of at least three repeated experiments. Each data point was an average of triplicate assays.*P<0.05 (two-way ANOVA).(B)Western blot analysis of phosphorylation of cofilin and LIMK in total cell lysates from Scr/H1299 and SiNUAK1/H1299 cells with 10ng/mL EGF stimulation for 0, 15, 30, and 90 min. LIMK was used as a loading control. The results of Western blot were from a representative of at least three repeated experiments and quantification of relative protein levels is shown below the blots.
Supplementary figure 2. NUAK1 enhanced NSCLC cellsinvasion
(A)The invasiveness of Scr/H1299 and SiNUAK1/H1299 cells analyzed bytranswell invasion assay. Upper, representative pictures of penetrated cells. Bottom, quantification of indicated cells. Scale bars=50μm. *P<0.05 (two-way ANOVA).(B)Western blotanalysis of MMP-2, MMP-9and MT-MMP from indicated cells with EGF (10 ng/ml) stimulation. (C) Gelatin zymography analysisof MMP-2, MMP-9and MT-MMP from culture media of indicated cellswith EGF (10 ng/ml) stimulation.(D)Expression of pmTOR (Ser2448), p-4E-BP-1 (Ser65) and p-P70S6K (Thr389)in Scr/H1299 and SiNUAK1/H1299 cells with or without EGF (10 ng/ml)stimulation was analysed by Western blot.β-actin was used as a loading control.Each result is a representative from of at least three independent experiments.
Supplementary figure 3. Expression levels of NUAK1 in H1299 cells transfected with different miRNAs
According to the predictionof online miRNA target databases (miRNA.org and Targetscan), miRNAs(miR-204, miR-182, miR-96 and miR-708) which were predicted as potential miRNAs targeting NUAK1 and negative control (NC) mimics were transfected into H1299 cells. Western blot analysis showed that the NUAK1 protein levels were reduced drastically in miR-204-transduced cells but the enhanced miR-182, miR-96 and miR-708 in H1299 cells, however,could not repressNUAK1 protein expression. The result is a representative from of at least three independent experiments.
Supplementary figure 4. Lung metastasis in SCID mice implanted with Scr/H1299, SiNUAK1/H1299, miR-NC/H1299 and miR-204/H1299 cells
(A)Visualized metastasis foci on mouse lungs of indicatedSCID mice groups implanted with Scr/H1299, SiNUAK1/H1299, miR-NC/H1299 and miR-204/H1299 cells.(B) H&E staining of lung metastasis in SCID mice implanted with Scr/H1299, SiNUAK1/H1299, miR-NC/H1299 and miR-204/H1299 cells.