Fig 1: Ablation of BRIX1 activates the nucleolar stress‐p53 pathway to suppress the growth of cancer. A‐D) Knockdown of RPL5 or RPL11 impairs the activation of p53 upon BRIX1 depletion. CAL‐51 (A, B) and HCT116 p53+/+ C, D) cells were transfected with siRNAs as indicated for 48 h, followed by IB analysis. E, F) Knockdown of BRIX1 increases the interactions of MDM2 with RPL5 and RPL11, respectively. Cells stably expressing shNC or shBRIX1 were used for co‐IP‐IB analysis with antibodies as indicated. The proteasome inhibitor MG132 was supplemented into the medium for 5 h before cell harvest. G) Knockdown of BRIX1 diminishes p53 ubiquitination induced by MDM2. HCT116 p53−/− cells stably expressing control or BRIX1 shRNA were transfected with plasmids as indicated for 48 h and treated with MG132 for 5 h, followed by in vivo ubiquitination assay and IB analysis. H) Knockdown of BRIX1 extends the half‐life of p53 protein. Cells were transfected with control or BRIX1 siRNA. Cycloheximide (CHX) (100 mg mL−1) was supplemented into the medium for the indicated time before cells were harvested for IB analysis. I, J) Knockdown of BRIX1 inhibits the proliferation of breast cancer cells with wild‐type p53. CAL‐51 (I) and MCF‐7 (J) cells were transfected with control or BRIX1 siRNAs for 6–12 h and seeded in 96‐well plates for a cell viability assay. K, L) Knockdown of BRIX1 impedes the colony‐forming ability of wild‐type p53‐harboring cancer cells. CAL‐51 (K) and MCF‐7 (L) cells were transfected with control or BRIX1 siRNAs for 6–12 h and were seeded in 6‐well plates for about 14 days. Colonies were fixed with methanol, and visualized by crystal violet staining. M, N) Knockdown of BRIX1 induces G1‐cell cycle arrest in breast cancer cells harboring wild‐type p53. CAL‐51 (M) and MCF‐7 (N) cells were transfected with control or BRIX1 siRNAs for 48 h, followed by flow cytometric analysis. O, P) Knockdown of BRIX1 represses the migration of wild‐type p53‐harboring cancer cells. CAL‐51 (O) and MCF‐7 (P) cells were transfected with control or BRIX1 siRNAs for 6–12 h, followed by a cell migration assay. ***p < 0.001.
Fig 2: Ablation of endogenous SBDS induces RPL5- and RPL11-dependent p53 stabilization.a, b Knockdown of SBDS prolongs p53 protein half-life. HCT116p53+/+ cells were transfected with control or SBDS siRNA for 48–72 hr. CHX was supplemented into the medium for the indicated time before the cells were harvested for IB analysis a. Quantification of the p53/β-actin ratios is shown in the b. c RPL5 and RPL11 are required for SBDS depletion-induced p53 activation. HCT116p53+/+ cells were transfected with the indicated siRNAs for 48–72 hr and harvested for IB analysis using the antibodies as indicated. d Knockdown of SBDS enhances the RPL11-MDM2 interaction. HCT116p53+/+ cells were transfected with control or SBDS siRNA for ~48 hr. The proteasome inhibitor MG132 was supplemented into the medium for 4 hr before the cells were harvested for co-IP-IB assays using antibodies as indicated. e Knockdown of SBDS enhances the RPL5-MDM2 interaction. The same experiments were conducted as described in d, except that the anti-RPL5 antibody was used.
Fig 3: Ablation of UTP11 stabilizes p53 through RPL5/RPL11 inhibition of MDM2. (A–D) Knockdown of RPL5 or RPL11 compromises the induction of p53 by UTP11 depletion. CAL-51 (A, B) and HCT116 p53+/+ cells (C, D) were transfected with control, UTP11 siRNA, RPL5 siRNA, and RPL11 siRNA as indicated. Cell lysates were subjected to IB analysis with indicated antibodies. (E, F) RPL5-MDM2 and RPL11-MDM2 interactions are increased by depletion of UTP11. CAL-51 cells were transfected with control or UTP11 siRNA, followed by co-IP-IB assays using antibodies as indicated. The proteasome inhibitor MG132 was supplemented into medium for 5 h before cell harvest. (G) Knockdown of UTP11 diminishes MDM2-induced p53 ubiquitination. HCT116 p53−/− cells stably expressing control or UTP11 shRNA were transfected with plasmids encoding p53, His-Ub, and HA-MDM2 as indicated and treated with MG132 for 5 h, followed by in vivo ubiquitination assay and IB analysis. (H) UPT11 knockdown extends the half-life of p53 protein. CAL-51 cells were transfected with control or UTP11 siRNA. CHX (100 mg/ml) was supplemented into medium for the indicated time before cells were harvested for IB analysis. ***p < 0.001.
Fig 4: MiR-101-3p suppresses RPL11 deubiquitination by USP47. (a) HEK293T cells were transfected with HA-RPL11 alone or in combination with Flag-USP47. The interaction between Flag-USP47 and HA-RPL11 was detected by immunoblotting after immunoprecipitation with anti-Flag magnetic beads. (b) HEK293T cells were transfected with His-ubiquitin alone or together with HA-RPL11 and Flag-USP47, and then treated with proteasomal inhibitor MG132 (10 μM) for 4 h. RPL11 ubiquitination was observed using a Ni-NTA pulldown assay. (c) HEK293T cells were transfected with His-ubiquitin alone or together with HA-RPL11, miR-101-3p mimic, Flag-USP47, or Flag-USP47C109S, and then treated with proteasomal inhibitor MG132 (10 μM) for 4 h. RPL11 ubiquitination was observed using a Ni-NTA pulldown assay. (d) A594 cells were transfected with miR-101-3p mimic or Flag-USP47 alone, or together. Western blot analysis was performed to detect the USP47 and RPL11 protein levels. IP, immunoprecipitation; TCL, total cell lysates.
Fig 5: Schematic models for miR-101-3p function in lung cancer cells. (a) In cancer cells, increased USP47 deubiquitinates RPL11 and places it in the nucleolus. As a result, free-MDM2 can ubiquitinate p53 and induce its proteasomal degradation, which in turn promotes cell proliferation. (b) When miR-101-3p is overexpressed, USP47 levels are reduced by miR-101-3p and the ubiquitinated RPL11 shifts to the nucleoplasm, binds to MDM2, and inhibits p53 degradation by MDM2.
Supplier Page from Abcam for Anti-RPL11 antibody