Fig. 2. ASNS enhances cellular invasiveness independent of endogenous asparagine products.

In different lung-cancer cell lines, ASNS were tested in protein (A) and mRNA (B) levels, ASNS mRNA level was analyzed by Student’s t test. 95C and 95D cell lines were derived from the same lung-cancer patient and domesticated into high metastasis (95D) and low metastasis (95C) cell lines (C); H1299 and A549 cell lines were also paired cell lines, their invasive and migrative ability were tested (D), Student’s t test was used for statistic test. ASNS protein was knocked down in 95D and H1299 (E, F), cell growth number was recorded (G, H), and the different growth speeds were tested by Student’s t test. Knockdown-ASNS cells were seeded into Matrigel-coated Transwell chambers and cultured in FBS gradient medium for 36 h. The count of invasive cells was recorded in 95D group (I) and H1299 group (J), the different invasive abilities were tested by Student’s t test. At the same time, knockdown-ASNS cells were seeded into colony assay (K, L), then cultured for 2–3 weeks. M aligning ASNS protein sequence in different species. And building ASNS^WT and ASNS^C2A cell lines (N). ASNS^C2A cells defected glutamine metabolism and asparagine synthesis according to RNA sequencing data (O). The ASNS overexpressed cells were seeded into invasive and migrative assay in normal asparagine medium (P) and low asparagine medium (Q). At the same time, these cells were seeded into crystal violet staining assay (R) and colony formation assay (S), then cultured for 2–3 weeks. The clone formations were analyzed by Student’s t test. Error bars were expressed as mean ± S.D. P < 0.05 is significant statistic difference, *p < 0.05, **p < 0.01, ***p < 0.001. Representative images from three independent experiments are shown.