Fig 2: c-Jun inhibits Akt phosphorylation via Rictor downregulation and inhibition of Torc2 complex formation(A) Western blots of signaling molecules showing dysregulation in the AKT pathway by decreased p-Akt-S473 and p-Foxo-1 in c-Jun wild-type mice fed high-fat diet. In contrast, the c-Jun knockout samples showed that p-Akt-S473 and p-Foxo-1 signals increased.(B, left) Western Blots of signaling molecules showing similar dysregulation in the AKT pathway by decreased p-AKT-S473 and p-FOXO-1 in c-JUN overexpressed HepG2 samples. (B, right) c-JUN downregulates the RICTOR levels in mTORC2 complex. Western blot analysis shows that RICTOR levels were decreased in c-JUN overexpressed HepG2 cells, whereas the other components of mTORC2 (MTOR, SIN-1) complex were the same.(C) Real-time RT-PCR analysis also showed that RICTOR mRNA expression was significantly reduced in c-JUN overexpressed HepG2 cells, whereas the expression of the components remained the same in all the samples. Data are represented as mean ± SD. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001.(D, left) Immunoprecipitation analysis revealed that there was no complex formation between SIN-1 and RICTOR. (D, right) Reverse IP-western blot was performed to show if c-JUN overexpression inhibited RICTOR-mTLR1-SIN1 complex formation. c-JUN expression inhibited the interactions between mTOR1 and SIN1.(E) (left) Immunohistochemistry of c-Jun wild-type and c-Jun knockout HCFD-fed mice showed increased Rictor expression in the knockout samples. These liver tissue sections were collected from HCFD-fed Alb-Cre;Junfl/fl or Alb-Cre;Junfl/fl;NS5A Tg mice. (Right) Immunoreactivity score was calculated based on both staining frequency and intensity. Scale bars represent 10 µm. Data are represented as mean ± SD. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001.(F) Dysregulation of AKT pathway disrupts downstream pathways. Activation of FOXO-1 activates the gluconeogenic genes. Real-time RT-PCR analysis revealed that PEPCK and G6PASE are increased in c-Jun overexpressed HepG2 cells. Data are represented as mean ± SD. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. (F, the 3rd to the left) Real-time RT-PCR analysis also showed that the expression of glucokinases decreased in c-Jun overexpressed HepG2 cells. Data are represented as mean ± SD. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. (F, right) Western blot analysis showed that GSK3ß was activated as it is hypophosphorylated in c-JUN overexpressed samples; it also showed that activation of GSK3ß inactivates glycogen synthase, as it was hyperphosphorylated in c-Jun overexpressed HepG2 cells. N = 5. Data are represented as mean ± SD. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: c-Jun silencing reduces self-renewal, tumor-initiation property whereas RICTOR silencing promotes self-renewal ability(A) (Top) Colony formation assay. To determine if c-Jun inhibition reduced oncogenicity, colony formation was employed using loss-of-function approach by use of lentivirus expressing shRNA targeting c-Jun. Silencing c-Jun significantly reduced colony numbers in soft agar assays. (Bottom) c-Jun silencing reduced colony numbers. N = 3. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean +/- SD.(B) CD133+ TICs had higher levels of c-Jun expression than those of non-TIC population. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean +/- SD.(C) Validation of RICTOR silencing in HepG2 cells.(D) (Top) Representative images of spheroids of Huh7 cells transduced with lentivirus expressing shRNA targeting c-JUN, GLUT2 or scrambled shRNAs. (Bottom) RICTOR silencing promoted spheroid formation in Huh7 cells especially in Huh7 cells that are cultured in low-glucose media. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean ± SD.(E) Overexpression of c-JUN promoted CD133 protein levels.(F) CD133(+)/CD49F(+) cells have elevated levels of CD133, CD49F, c-Jun, Nanong, and Oct4 proteins.(G) (Left) c-Jun silencing reduced Nanog expression, but induced Rictor expression. The c-Jun silencing promoted Rictor expression in TICs, indicating that c-Jun was required to maintain stemness and oncogenicity of TICs and partly depended on Rictor suppression. (Right) Xenograft tumors with shRNA targeting c-JUN showed significantly smaller tumor sizes, indicating that c-JUN played a key role for self-renewal and tumor-initiation property. N = 8 mice per groups. Student’s T-Test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Data are represented as mean ± SD.(H) Xenograft tumors with c-JUN overexpression in TIC population (CD133+/CD49F+) showed significantly larger tumor sizes with HCFD feeding. Overexpression of c-JUN further promoted tumor growth. N = 6 mice per groups. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean ± SD.(I) Xenograft tumors with c-JUN overexpression in non-TIC population (CD133-/CD49F-) showed significantly larger tumor sizes with HCFD feeding, indicating that c-JUN and HCFD feeding effects played key roles for self-renewal and tumor-initiation property. These results indicated that c-Jun overexpression promoted the self-renewal tumor-initiation property of non-TIC population induced by HCFD feeding. N = 7 mice per groups. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean ± SD.(J) AC133 promoter has five major promoter regions (P1–P5).(K) The P1 promoter region of AC133 promoter had the highest levels of transactivation activity in Huh7 HCC cell line. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean ± SD.(L) Truncation promoter analyses showed that the region of AC133 promoter between -341 and +50 had the highest levels of transactivation ability. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean ± SD.(M) To test if c-JUN/AP1 and/or NANOG binding transactivate the AC133 promoter, in vitro mutagenesis abrogated the AP1 binding sites and NANOG promoter binding sites. These AP1 and/or NANOG binding sites reduced AC133 promoter activity, indicating that AP1 and NANOG proteins are required to transactivate AC133 promoter. Student’s t test was used to calculate statistical significance. Asterisk (*) indicates statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean ± SD.