Fig 2: STT3A knockout inhibits tumor cell growth. (A) Establishment of STT3A knockout cell lines in PC-9 and H1299 verified by Western blot. (B) STT3A knockout significantly inhibited PC-9 and H1299 cell proliferation. (C, D) STT3A knockout also significantly affected the colony formation ability of these cell lines but did not lead to apoptosis. (E) Effect of STT3A knockout on the G0/G1 cell-cycle transition was detected in the PC-9 and H1299 cells by PI staining and flow cytometry. (F) The quantification data was analyzed by ModFit 5. (G) STT3A regulated the expression of the cell-cycle transition mediators, including CDK4, CDK6, p21, and p27. Staining method for (C): 0.1% crystal violet. The data are presented as mean ± standard deviation according to the Student’s t-test. **, P<0.01; ***, P<0.001. sgNC, small guide RNA negative control; sgSTT3A, small guide RNA of STT3A; STT3A, STT3 oligosaccharyltransferase complex catalytic subunit A; PI, propidium iodide.

Fig 3: STT3A knockout inhibits tumor invasion and migration via EMT. (A) Knockout of STT3A inhibited PC-9 and H1299 cell migration detected by transwell assays: cell counts for the transwell assays are indicated below. Scale bar: 100 µm. (B) Transwell assays with Matrigel were used to confirm the effects of STT3A on tumor cell invasiveness. Scale bar: 100 µm. (C,D) The migration ability of the transfected PC-9 and H1299 cells was verified by a wound-healing experiment. The quantification data are also indicated, Scale bar: 500 µm. (E) Western blot analysis of EMT-related proteins E-cadherin, N-cadherin, vimentin. Staining method for (A,B): 0.1% crystal violet; magnification, ×100. The data are presented as mean ± standard deviation according to the Student’s t-test. **, P<0.01; ***, P<0.001. sgNC, small guide RNA negative control; sgSTT3A, small guide RNA of STT3A; STT3A, STT3 oligosaccharyltransferase complex catalytic subunit A; EMT, epithelial-mesenchymal transition.

Fig 4: STT3A is highly expressed in lung cancer tissues and correlated with the poor prognosis of LUAD patients. (A) Heat map of DE glycosyltransferase in LUAD and normal adjacent tissues; (B) STT3A expression in TCGA database (normal: 59; tumor: 535); (C) Western blot analysis of the expression levels of STT3A in the tumor tissues and adjacent normal tissues of 10 clinical patients with LUAD; (D) the relative expression of STT3A in clinical specimens was detected by qRT-PCR (n=10); (E) IHC staining was performed in paired clinical specimens, and the results showed that STT3A was highly expressed in lung cancer tissues (scale bar: 20 µm); (F) the expression levels of STT3A in NSCLC cancerous cell lines, including H3122, H1975, H1299, HCC827, H292, H1650, A549, and PC-9 were examined by Western blot, and compared to normal human bronchial epithelial cell lines (BEAS-2B and MRC-5); (G) Kaplan-Meier analysis of OS according to TCGA database; the high expression of STT3A is correlated with a worse survival rate (P=0.009); (H) Kaplan-Meier analysis of OS according to the GEO cohort (P=0.00033); (I) representative images of different immunohistochemical staining intensities of STT3A in TMA. Scale bar: 20 µm. (J) High STT3A expression was associated with poor OS in LUAD patients in the TMA samples. **, P<0.01; ***, P<0.001; ns, not significant. STT3A, STT3 oligosaccharyltransferase complex catalytic subunit A; LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas; qRT-PCR, quantitative real-time polymerase chain reaction; IHC, immunochemistry; NSCLC, non-small cell lung cancer; OS, overall survival; GEO, Gene Expression Omnibus; TMA, tissue microarray.

Fig 5: The effect of STT3A inhibition on the progression of LUAD was verified in vivo. (A-C) STT3A knockout inhibited xenograft tumor formation in vivo. Representative xenograft tumor images (A), tumor weight (B), tumor volume (C) and body weight (D). (E) Representative IHC staining of STT3A, EGFR, and Ki67 for indicated xenograft tumors. Quantification data were also indicated. Scale bar: 50 µm. Mice were treated with the STT3A inhibitor NGI-1 and compared to a placebo group (7 mice for each group). (F-I) NGI-1 inhibited xenograft tumor formation in vivo. Representative xenograft tumor images (F), tumor weight (G), tumor volume (H) and body weight (I). The data are presented as the mean ± standard deviation according to the Student’s t-test. **, P<0.01; ***, P<0.001. LUAD, lung adenocarcinoma; sgNC, small guide RNA negative control; sgSTT3A, small guide RNA of STT3A; IHC, immunohistochemistry.