Fig 2: Six1 reduction can inhibit Bcl-2 and activate apoptosis executor caspase-7. (A) The relative mRNA levels of apoptosis related factors were detected by qRT-PCR with Six1 silence. (B) Western blot assay detected the protein levels of FL- caspase-7, c-caspase-7 and its downstream effector c-PARP with Six1 reduction. FL and c referred to full length and cleaved proteins respectively. ß-actin was the internal control. (C) Caspase-3 protein level was not affected with Six1 knock-down. (D) Bcl-2, Bax, P53 protein expression were detected with Six1 silence. Six1 reduction decreased Bcl-2 protein level but had slight effect on Bax and P53. (E) Schematic diagram displayed the mechanism of Six1-mediated mitochondrial pathway apoptosis.

Fig 3: Six1 expression is increased in gastric cancer samples and has clinical significance. (A) qRT-PCR results showed Six1 mRNA levels in 40 paired gastric cancer tissue specimens. GAPDH was the internal control. The relative fold change of the Six1 mRNA level was performed log2-transformed. (B) Relative mRNA levels of Six1 in gastritis (n=15), TNM I&II (n=8) and TNM III&IV (n=32) specimens respectively. (C) Western blot assay was conducted to detect Six1 protein expression in 10 paired gastric cancer tissues. T was the tumor samples and N was its adjacent normal counterpart. (D) IHC staining evaluated Six1 expresson and distribution in paired gastric cancer samples. (E) Online K-M plotter was used to conduct survival analysis of Six1 in gastric cancer with 631 public available microarray data. (F) Survival analysis of Six1 in 422 samples with lymphatic metastasis. (G) Survival analysis of Six1 in 121 transcriptome date with poor differentiation. The low and high Six1 subgroups were divided according to the median value of Six1 levels. The default parameters were used for analysis. Logrank p < 0.05 was considered to be statistically.

Fig 4: Six1 silence can trigger apoptosis especially mitochondrial pathway apoptosis. (A) Six1 knock-down promoted apoptosis in SGC-7901 cells. Apoptotic cells were stained with Annexin V and 7-AAD. (B) Histogram showed the percent of apoptotic cells in NC and Six1 knock-down groups. (C) Six1 silence decreased the mitochondrial membrane potential (??m) and triggered mitochondrial apoptosis detected by JC-1 assay. JC-1 displays red fluorescence in cells with high ??m, whereas JC-1 exhibits green fluorescence in cells with low ??m. (D) Flow cytometry showed the distribution ratio of JC-1 red and JC-1 green cell population. (E) Histogram calculated the relative ratio of red against green fluorescence. *, p < 0.05.

Fig 5: Six1 can mediate the nuclear translocation of Eya1 in the absence of Six2. (A,B) Whole-mount in situ hybridization for Six1 (A) and Eya1 (B) in the kidney rudiments of wild-type, Eya1 Six1/+ , Eya1 Six1/+ ;Six2 -/- and Six2 -/- embryos at E11.5. (C) Anti-Eya1 immunostaining on kidney sections of E11.5 wild-type, Eya1 Six1/+ , Eya1 Six1/+ ;Six2 -/- and Six2 -/- embryos. Lower panels are higher magnification of the boxed areas. Scale bars: 50 µm.