Journal: Advanced Science

Article Title: The FBXW7‐RPAP2 Axis Controls the Growth of Hepatocellular Carcinoma Cells and Determines the Fate of Liver Cell Differentiation

doi: 10.1002/advs.202404718

Figure Lengend Snippet: HSP90 inhibitor induces RPAP2 degradation mediated by FBXW7 and CUL5. A,B) Degradation of RPAP2 in paired FBXW7 +/+ and FBXW7 −/− DLD‐1 (A), as well as in Huh7 cells upon FBXW7 knockdown (B) following 17‐AAG treatment. DLD‐1 FBXW7 +/+ and FBXW7 −/‐ cells (A) or Huh7 cells transfected with indicated siRNA oligos (B) were pretreated with 1 µ m 17‐AAG for 12 h and then co‐treated with 100 µg mL −1 CHX for the indicated time periods before being harvested for IB analysis. C) Ubiquitylation of RPAP2 in HEK293 cells upon FBXW7 knockdown following 17‐AAG treatment. HEK293 cells were co‐transfected with indicated plasmids and siRNA oligos and then treated with 1 µ m 17‐AAG for 12 h before being harvested for purification with Ni‐NTA. Pull‐downs (top) and WCE (bottom) were subjected to IB analysis with indicated Abs, respectively. D) Degradation of wild‐type RPAP2 and its S562A/T565A mutant in Huh7 cells upon 17‐AAG treatment. Huh7 cells transfected with indicated plasmids were pretreated with 1 µ m 17‐AAG for 12 h and then co‐treated with 100 µg mL −1 CHX for the indicated time periods before being harvested for IB analysis. E) Degradation of RPAP2 in PLC/PRF/5 cells upon CUL5 knockdown following 17‐AAG treatment. PLC/PRF/5 cells infected with indicated lentiviral shRNA viruses were pretreated with 0.5 µ m 17‐AAG for 3 h and then co‐treated with 100 µg mL −1 CHX for the indicated time periods before being harvested for IB analysis. Densitometry quantifications were performed with ImageJ, and the decay curves are shown (right) (A,B,D, and E). F) Co‐IP of transfected FLAG‐RPAP2 and endogenous CUL1, CUL5, HSP90, or CDC37 in HEK293 cells treated with 1 µ m 17‐AAG for 8 h. G,H) The correlation between RPAP2 protein levels and HCC cell sensitivity to HSP90 inhibitor. The CCK8 assays were employed to assess the sensitivity of multiple HCC cells with varying RPAP2 levels (G), as well as the sensitivity of SUN‐423 cells with or without ectopic expression of FLAG‐RPAP2 (H), to 17‐AAG treatment. Data are presented as mean ± SEM from three independent experiments (G,H). I) SNU‐423 cells were infected with indicated lenti‐viruses and then treated with 17‐AAG (10 µ m ) for the indicated time periods, followed by IB analysis.

Article Snippet: The following antibodies were used in western blotting: RPAP2 (17401‐1‐AP, Proteintech), FBXW7 (A301‐720A, Bethyl Laboratories), GAPDH (2118, Cell Signaling Technology); ACTIN (A5441, Sigma), FLAG (F1804, Sigma), FLAG (F7425, Sigma), p‐S6K1 (9234, Cell Signaling Technology), t‐S6K1 (sc‐230, Santa Cruz), p‐AKT (4060, Cell Signaling Technology), t‐AKT (4691, Cell Signaling Technology), p‐ERK1/2 (9101, Cell Signaling Technology), c‐MYC (565, Cell Signaling Technology), p‐GSK3 (9331, Cell Signaling Technology), GSK3 (5676, Cell Signaling Technology), p‐p38 (4511, Cell Signaling Technology), p38 (8690, Cell Signaling Technology), p‐Ser/Thr‐Pro (05‐368, Upstate), CUL1 (sc‐11384, Santa Cruz), CUL4A (2699, Cell Signaling Technology), CUL5 (ab184177, Abcam), HSP90 (4874, Cell Signaling Technology), CDC37 (10218‐1‐AP, Proteintech), PARP (9542, Cell Signaling Technology), Caspase‐3 (9665, Cell Signaling Technology), USP7 (4833, Cell Signaling Technology), RPB1 (14 958, Cell Signaling Technology), MDM2 (OP46, Calbiochem), c‐MET (25869‐1‐AP, Proteintech), IGF‐IRβ (9750, Cell Signaling Technology), SRC (2109, Cell Signaling Technology), Cyclin D1 (55 506, Cell Signaling Technology) and CDK4 (12 790, Cell Signaling Technology).

Techniques: Knockdown, Transfection, Purification, Mutagenesis, Infection, shRNA, Co-Immunoprecipitation Assay, Expressing