Figure Legends

FIGURE 1 CAR activation induces hepatomegaly and increase the liver-to-body weight ratio. (A) Mice were treated by TCPOBOP for 5 days or 10 days. (B) The liver-to-body weight ratio significantly increased in TCPOBOP-treated group (n = 5 per group). (C) Liver morphology also showed a liver hypertrophy. (D) H&E staining suggested that there was no liver injury (n = 5 per group). Scale bar =50 µm. (E) CTNNB1 staining was performed to measure the cell size, which indicated an increase of cell size around CV area (n = 5 per group). Scale bar =50 µm. (F) Quantification of CTNNB1 staining in TCPOBOP-treated mice. (G) The expression of KI67 was measured by KI67 staining, the number of KI67 positive cells was increased in PV area after treated by TCPOBOP (n = 5 per group). Scale bar =50 µm. (H) Quantification of KI67 staining in TCPOBOP-treated mice. Data are presented as mean ±S.D . *P < 0.05, significantly different from Vehicle 5 d; #P < 0.05, significantly different from Vehicle 10 d; Student’s t test.
FIGURE 2 CAR activation promotes liver regeneration and facilitates the restore of liver weight after partial hepatectomy. (A) Mice after partial hepatectomy were treated with TCPOBOP for 2 days or 5 days. (B) The liver-to-body weight ratio was much higher in TCPOBOP-treated group (n = 5 per group). (C) Liver morphology suggested an increase of liver size. (D) H&E staining suggested that no liver injury occurred (n = 5 per group). Scale bar =50 µm. (E) Hepatocytes size was quantified by CTNNB1 staining which showed hepatocytes hypertrophy around CV area (n = 5 per group). Scale bar =50 µm. (F) Quantification of CTNNB1 staining in TCPOBOP-treated mice after PHx. (G) The number of KI67 positive cells significantly was increased around PV area which indicated an increase of proliferative response (n = 5 per group). Scale bar =50 µm. (H) Quantification of KI67 staining in TCPOBOP-treated mice after PHx. Data are presented as mean ± S.D. *P < 0.05, significantly different from PHx 2d+Vehicle; #P < 0.05, significantly different from PHx 5d+Vehicle; Student’s ttest.
FIGURE 3 YAP participates in CAR-induced hepatomegaly and liver regeneration. (A) Total YAP, nuclear YAP and YAP downstream targets were all upregulated, while the cytoplasmic p-YAP was downregulated, which indicated the activation of YAP pathway. Proliferation-related proteins were also upregulated after treated by TCPOBOP (n = 5 per group). (B) In PHx model, total YAP, nuclear YAP and YAP downstream targets CTGF, ANKRD1 were upregulated, cytoplasmic p-YAP was downregulated. Meanwhile proliferation-related proteins such as CCND1 and CCNA1 were upregulated (n = 5 per group). (C) The intensity of nuclear YAP was much higher after treated by CITCO in HepG2 cells, which indicated that CAR activation promotes the nuclear translocation of YAP (n= 6 per group). (D) Co-immunoprecipitation results suggested the potential protein-protein interaction between CAR and YAP (n = 6 per group). Scale bar =20 µm.
FIGURE 4 CAR-induced hepatomegaly is partially via YAP pathway. (A) Wildtype or Yap-/- mice were treated by TCPOBOP for 10 days. (B) The liver-to-body weight ratio in TCPOBOP-treated Yap-/- mice was lower than TCPOBOP-treated wildtype mice, but was still much higher than vehicle group of Yap-/- mice (n = 5 per group). (C) Liver morphology suggested that liver size was increased even inYap-/- mice. (D) H&E staining suggested that no liver injury occurred (n = 5 per group). Scale bar =50 µm. (E) CTNNB1 staining showed that there was still a slight increase of hepatocytes size around CV area in Yap-/- mice (n = 5 per group). Scale bar =50 µm. (F) Quantification of CTNNB1 staining in wildtype or Yap-/- mice. (G) The number of KI67 positive cells still was increased inYap-/- mice around PV area (n = 5 per group). Scale bar =50 µm. (H) Quantification of KI67 staining in wildtype or Yap-/- mice. Data are presented as mean ± S.D. *P < 0.05, significantly different from Yap+/+ +Vehicle;#P < 0.05, significantly different from Yap-/- +Vehicle;§P < 0.05, significantly different from Yap+/+ +TCPOBOP; Student’s t test.
FIGURE 5 Other factors are involved in CAR-induced hepatomegaly. (A) The mRNA level of other related genes in wildtype mice treated by TCPOBOP or corn oil for 5 days or 10 days (n = 5 per group). (B) The mRNA level of other related genes in wildtype orYap-/- mice (n = 5 per group). (C) The protein levels of C-Myc, FOXM1 and MET were upregulated after treated by TCPOBOP for 5 days or 10 days (n = 5 per group). (D) The protein levels of C-Myc, FOXM1, CTNNB1 and MET were upregulated after treated by TCPOBOP in Yap-/- mice (n = 5 per group). Data are presented as mean ± S.D . *P < 0.05, significantly different from Vehicle 5 d;#P < 0.05, significantly different from Vehicle 10 d; §P < 0.05, significantly different from Yap+/+ +Vehicle;$ P < 0.05, significantly different from Yap-/- +Vehicle; Student’s t test.