Fig 2: BOS/BOR mutations and their transcriptional effects. (A) Amino acid alignment of the N-terminal region of Xenopus laevis Six1, human SIX1 and Drosophila Sine oculis (SO) shows a high level of identity across species; human and frog are 100% identical in this region; differences from fly are in white. The sequence shown begins with the six domain (SD), which contains six α-helices (blue bars), and ends with the homeodomain (HD, black bar). Amino acid substitutions/deletions that have been reported in human BOS/BOR patients are indicated with arrows; red arrows indicate the four mutations that were examined in this study. (B) Expression of Six1+Eya1 caused a significant ∼7-fold increase in luciferase activity when compared to activity of control vector (P<0.0001), Six1WT alone (P<0.0001) or Eya1 alone (P<0.0001). Each mutant plus Eya1 failed to significantly induce luciferase activity relative to control (V17E, P=0.27122; R110W, P=0.99999; W122R, P=0.99764; Y129C, P=0.99947) or in the absence of Eya1 (V17E, P=0.99988; R110W, P=0.99999; W122R, P=0.99999; Y129C, P=0.99999). Experiments were repeated five independent times and subjected to a one-way ANOVA with Tukey post hoc multiple comparisons test. Bars=mean±s.d. (C,I,O). HEK293T cells transfected with only Myc-Eya1 show both cytoplasmic (arrowheads) and nuclear localization. (D,J,P) Cells co-transfected with both Six1WT-FLAG and Myc-Eya1 show nuclear colocalization of both proteins. (E,K,Q) Those transfected with both V17E-FLAG and Myc-Eya1 showed nuclear colocalization and some cytoplasmic Eya1 (arrowheads). Those transfected with (F,L,R) R110W-FLAG and Myc-Eya1, (G,M,S) W122R-FLAG and Myc-Eya1, or (H,N,T) Y129C-FLAG and Myc-Eya1 showed exclusive nuclear colocalization. Scale bars: 10 µm.

Fig 3: SIX1 facilitates NSCLC cells growth in vitro. (A) The protein and mRNA level of SIX1 on baseline in four NSCLC cells and one normal bronchial epithelial cell. (B) SIX1 expression in A549 and H1299 cells transfected with SIX1-overexpressing lentivirus. (C) SIX1 expression in PC-9 cells transfected with SIX1 siRNA. (D) Immunofluorescence staining of SIX1 in indicated cells. (E) DNA synthesis in indicated cells by EdU assays. (F) (G) (H) The growth curves of indicated cells by CCK-8 assays. *p< 0.05, **p < 0.01, ***p < 0.001. Graphs show mean ± SD.

Fig 4: Correlation of SIX1 expression and clinicopathological features in NSCLC patients. (A) SIX1 was significantly upregulated in LUAD and LUSC in the UALCAN database. (B) SIX1 expression in LUAD and LUSC with different lymph node metastatic states. (C) SIX1 expression in LUAD and LUSC with stage Ⅰ to stage Ⅳ diseases. (D) SIX1 expression in patients with different stages in the GSE31210 database. (E) The correlation between SIX1 expression and RFS in NSCLC patients. (F) The correlation between SIX1 expression and OS in NSCLC patients. *p< 0.05, **p < 0.01, ***p < 0.001.

Fig 5: SIX1 mRNA expression in pan-cancer. (A) SIX1 expression in multiple cancers were analyzed using the TIMER database. (B) SIX1 expression in multiple cancers were analyzed using the Oncomine database. (C) SIX1 expression was increased in LUAD, LUSC, lung carcinoid tumor and LCLC tumor tissues. *p< 0.05, **p < 0.01, ***p < 0.001.