Fig 2: SIX1 activates the Notch signaling pathway in NSCLC cells. (A) The SIX1 expression was positively correlated with the Notch signaling pathway in NSCLC by GSEA. (B) The SIX1 and HES1 mRNA expression was positively correlated in TCGA LUAD samples. (C) The SIX1 and HES1 mRNA expression was positively correlated in TCGA LUSC samples. (D) (E) The protein level of Notch1, Notch2 and Hes1 in indicated cells. (F) The protein level of Notch1, Notch2, Hes1 E-cadherin, Vimentin and SIX1 upon treated with Notch pathway inhibitor (DAPT, 10uM). (G) Cell migration and invasion assays of indicated cells with or without the DAPT treatment by transwell chamber. *p< 0.05, **p < 0.01, ***p < 0.001. Graphs show mean ± SD.

Fig 3: SIX1 facilitates the growth of A549 cells in vivo. (A) Tumor xenografts of each group. n=5. (B) Tumor growth curve in each group. (C) Tumor volumes on day 24 were shown for groups containing 5 mice each. (D) (E) IHC staining was performed to determine the expression of SIX1, Hes1, Vimentin and Ki-67 in the tumor. *p< 0.05, **p < 0.01, ***p < 0.001. Graphs show mean ± SD.

Fig 4: Schematic diagram of the mechanism of ITCH/SIX1 regulating CDC27/cyclinB1 signaling pathway in NPC.

Fig 5: ITCH was involved in ubiquitination degradation of SIX1 in NPC cell lines. (A) Molecular docking analysis of the interaction between ITCH and SIX1. (B) Immunofluorescence showed colocalization of ITCH (green) and SIX1 (Red) in 6-10B cells. (C,D) The western blot demonstrated the effect of gradient overexpression of ITCH on SIX1 protein expression in 6-10B cells. The statistical chart was displayed below it. (E,F) COIP assays showed the interaction between SIX1 and ITCH in 293T and 6-10B cells. G The western blot demonstrated the efficiency of ITCH overexpression in 6-10B cells. (H) Immunofluorescence showed the effect of ITCH overexpression on SIX1 colocalization in 6-10B cells. (I,J) COIP experiments confirmed that the E3 ubiquitination ligase ITCH promoted the ubiquitination level of SIX1. *P < 0.05.