Fig 2: Working model. Solid lines: intrinsic apoptosis. Dashed lines: extrinsic apoptosis. When hepatoma carcinoma cells were treated with cisplatin, ATM was activated and combined with RMP, activating p65 to translocate into nucleus and promote the transcription of Bcl-xl. RMP tipped the balance of the ratio of pro-apoptotic and anti-apoptotic proteins and ultimately suppressed the intrinsic apoptosis response of HCC cells toward chemotherapy. However, the RMP repression of the extrinsic apoptosis induced by TRAIL was not dependent on NF-κB activation in which P53 played an important role. RMP triggered the expression of death receptors and consequently sensitized HCC cells toward extrinsic apoptosis via the p53 family members as transactivators. Besides, P53, as an important transcription factor, could also directly regulate the expression of Bcl-xl and finally control the intrinsic apoptosis. It should be noted that the intrinsic and extrinsic apoptosis are not entirely separable and these two pathways of apoptosis usually coordinated together to contribute to the regulation of apoptosis.

Fig 3: URI1 induces autophagy via FOXO3. (A) FOXO3 induces autophagy in HepG2 cells. HepG2 cells were transiently transfected with FOXO3 overexpression plasmid. After 36 h, the cells were treated with HCQ (50μM) for 12 h. MAP1LC3B-II was analyzed by western blot. GAPDH was used as a loading control. (B) Depletion of URI1 inhibits FOXO3-induced autophagy. HepG2 cells were transfected with FOXO3 overexpression plasmid followed by 12 h treatment with HCQ (50μM) in the presence or absence of URI1i plasmid and lysed directly after the treatment. URI1, FOXO3 and MAP1LC3B-II were analyzed by western blot. GAPDH was used as a loading control. (C) URI1 increases the expression of FOXO3 in both mRNA and protein levels in HepG2 cells. Left panel: HepG2 cells were transfected with URI1 overexpression (URI1o) or URI1 interference (URI1i) plasmid and FOXO3 was analyzed by western blot. GAPDH was used as a loading control. Right panel: HepG2 cells were transfected with URI1o or URI1i plasmid and the mRNA level of FOXO3 was analyzed by qRT-PCR. Data of 3 independent experiments are presented as mean ± SEM. **p<0.005, ****P<0.0001. (D) FOXO3 decreases irradiation-induced ROS in HepG2 cells. Left panel: HepG2 cells transfected with FOXO3 overexpression plasmid and then exposed to irradiation for 2 h. ROS levels were analyzed by microplate reader. Values are representative results of 3 independent experiments. ****p<0.0001. Right panel: Western blot showed FOXO3 expression level after HepG2 cells were transfected with FOXO3 overexpression plasmid.

Fig 4: RMP inhibited the TRAIL-induced exogenous apoptosis pathway of HCC cells. (A) Stably over-expressing or depleted of RMP HepG2 cells were treated with 100ng/ml TRAIL for 24h. Then cells were harvested and applied for flow cytometric analysis. Results showed that RMP silencing significantly raised the apoptosis rate of HepG2 cells compared with empty control vector, whereas the apoptosis rate decreased in RMP over-expressing HepG2 cells. (B) After incubation with increasing concentrations of TRAIL (0, 20, 40, 60, 80, 100ng/ml), cell viability was analyzed by CCK-8 analysis. With the increasing concentration of TRAIL, there exhibited a gradual decline of OD values. These values increased in RMP over-expressing HepG2 cells, but decreased in RMP depleted cells. (C&D) HepG2 cells that stably over-expressing or depleted of RMP were treated with 100ng/ml TRAIL for 3h, total mRNA was extracted and real-time RT-PCR was used to assess the relative mRNA expression of DR4 (C) and caspase8 (D) respectively.

Fig 5: URI1 induces autophagy by activating AMPK. (A) Activation of AMPK induces autophagy in HepG2 cells. HepG2 cells were treated with AICAR (1mM) for 6 h and HCQ (50µM) for 12 h. MAP1LC3-II was analyzed by western blot. GAPDH was used as a loading control. AICAR was used as an activator of AMPK. (B) Activation of AMPK induces phosphorylation of FOXO3 at the Ser253 residue. HepG2 cells were treated with AICAR (1mM) or DOX (10µM) for 6 h. FOXO3 was analyzed by western blot for p-FOXO3 (Ser253) and p-FOXO3 (Ser318/321). GAPDH was used as a loading control. AICAR was used as an activator of AMPK. DOX was used as an inhibitor of AMPK. (C) URI1 induces phosphorylation of AMPK at the Thr172 residue and phosphorylation of FOXO3 at the Ser253 residue. HepG2 cells with stable interference (URI1i) or overexpression (URI1o) of URI1 were treated with AICAR (1mM) for 6 h. AMPK was analyzed by western blot for p-AMPKa (Thr172). FOXO3 was analyzed by western blot for p-FOXO3 (Ser253) and p-FOXO3 (Ser318/321). GAPDH was used as a loading control. (D) The ratio of p-AMPKa (Thr172) to AMPKa and the ratio of p-FOXO3(Ser253) to FOXO3 were quantified by using Image J 1.80 software. Representative results of 3 independent experiments are shown. (E) Depletion of URI1 decreases the phosphorylation of AMPK and FOXO3 under the condition of irradiation. HepG2 cells were separately treated with URI1i plasmid or AICAR or DOX and then irradiation for 2 h. AMPK was analyzed by western blot for p-AMPKa (Thr172). FOXO3 was analyzed by western blot for p-FOXO3 (Ser253). GAPDH was used as a loading control. (F) Activation of AMPK decreases irradiation-induced ROS. HepG2 cells were treated with AICAR (1mM) for 6h followed by irradiation for 2 h. Right panel: ROS were analyzed by microplate reader. Values are representative results of 3 independent experiments. ****p<0.0001. Left panel: Western blot showed the phosphorylation of AMPK.