Fig 2: Simvastatin reduced MCM7 and RB expressions via activating cell autophagy and ER stress signaling pathway. (a) Hep3B cells were treated with 5 or 10 µM Simvastatin for 24 or 72 h. The mRNA expression levels of MCM2, MCM4, MCM7 and RB in the resulting cells were analyzed by RT-PCR. (b) MCM7 and RB expressions were decreased after Simvastatin treatment. Hep3B cells were treated with 5 or 10 µM Simvastatin for 24 or 72 h, and then analyzed by immunoblotting with MCM7 or RB antibodies. (c) Hep3B cells were treated with 2 µM MG132 and 10 µM Simvastatin for 48 h, then harvested and analyzed by immunoblotting with MCM7 and RB antibodies. (d and e) Hep3B cells were cultured with 10% FBS or 0.01% FBS for 24 h and subjected to DNA content analysis using a FACS flow cytometer; or analyzed the expression of MCM7 and RB by RT-PCR and immunoblotting. (f) Hep3B cells were treated with 5 or 10 µM Simvastatin for 24 or 72 h, and then cell extracts were analyzed by immunoblotting with AMPK cell signaling or cell autophagy-related antibodies. (g) Hep3B cells were treated with serum starvation or 5 µM Simvastatin for 72 h. Cell monolayers were fixed and stained with LC3A/B, and immunofluorescence images were presented (Scale bars, 10 µm). (h) Hep3B cells were treated for 48 h with 5 µM compound C (AMPK inhibitor; Sigma-Aldrich, St. Louis, MO, USA) or 10 µM Simvastatin, then harvested and analyzed by immunoblotting with p-AMPK, MCM7 or RB antibodies. (i) Hep3B cells were treated with 5 or 10 µM Simvastatin for 24 h or 72 h, and then cell extracts were analyzed by immunoblotting with PERK, p-PERK and p-eIF2a antibodies. (j) Hep3B cells were treated for 48 h with 2 µM GSK2606414 (PERK inhibitor; Millipore, Bedford, MA, USA) or 10 µM Simvastatin, then harvested and analyzed by immunoblotting with p-PERK, MCM7 or RB antibodies

Fig 3: A model for p97Ufd1-Npl4-Ubxn7 assembly on ubiquitylated CMG.a Ubxn7 binds weakly and transiently to ubiquitylated CMG-CRL2Lrr1 prior to p97Ufd1-Npl4 binding. b Multi-valent and cooperative interactions stabilize p97Ufd1-Npl4-Ubxn7 binding to ubiquitylated CMG-CRL2Lrr1, allowing initiation of ubiquitin chain unfolding. c Ubiquitylated Mcm7 is extracted from CMG and threaded through the central pore of p97.

Fig 4: RB-deficient or inactive tumor cells were more sensitive to Simvastatin than RB-proficient cells. (a) The expression status of RB in different cells. Extracts of U2OS/SaOS2/SiHa/C33A/LNCaP/Du145 cells were analyzed by immunoblotting with MCM7 or RB antibodies. (b) The cell survival rates of U2OS, SaOS2, SiHa, C33A, LNCaP and Du145 cells after treatment with Simvastatin for 24, 48 or 72 h. The viable cells were harvested and counted three times for each treatment. (c) U2OS and SaOS2 cells were treated with 5 µM Simvastatin for 72 h, incubated with annexin V-FITC and PI, and then examined by FACS. (d) U2OS/SaOS2 and SiHa/C33A were treated with Simvastatin (2.5 or 5 µM) for 24 or 72 h, and then cell extracts were analyzed by immunoblotting with apoptosis-related antibodies

Fig 5: Acute depletion of MCM7 expression induced ?-H2AX expression and apoptosis in RB inactive or deficient tumor cells. (a) RB expression in human osteosarcoma cell lines U2OS/SaOS2 and human cervical cancer cell lines SiHa/C33A. (b) SiHa cells and C33A cells were transfected with siMCM7 or sicontrol for 72 h. The resulting cells were analyzed by immunoblotting with antibodies MCM7, RB and ?-H2AX. (c) U2OS cells and SiHa cells were transfected with siMCM7 or sicontrol for 24 h, then infected with HPV E7 adenovirus or control adenovirus to deplete RB expression for 48 h. The resulting cells were analyzed by immunoblotting with antibodies MCM7, RB or ?-H2AX. (d) SaOS2 cells and C33A cells were transfected with siMCM7 and sicontrol for 24 h, then infected with Adpsm-RB adenovirus or control adenovirus to activate RB for 48 h. The resulting cells were analyzed as described above. (e and f) U2OS cells, SiHa cells, SaOS2 cells and C33A cells were treated as above. The resulting cells were then incubated with annexin V-FITC and PI, then examined by FACS. (g and h) Proteins involved in DNA damage repair were analyzed by western blotting. U2OS, SiHa, SaOS2 and C33A cells were treated with siRNA and adenovirus as described above. Equal protein loading was evaluated by ß-actin. Data were represented as mean±S.D of the three independent expressions