Journal: Advanced Science

Article Title: The Crosstalk Between CRL5 and APC/C E3 Ligases Regulates Metastasis and Chemosensitivity of Cancer Cells

doi: 10.1002/advs.202512652

Figure Lengend Snippet: APC11 selectively interacts with CUL5 among cullin family members. a) Silver staining pattern of CUL5‐interacting proteins. MIA PaCa‐2 cells stably overexpressing SBP‐S‐CUL5 or mock vector pLVX were harvested for tandem affinity purification, followed by SDS‐PAGE and silver staining (top). APC11 was a candidate CUL5‐binding protein identified by LC‐MS/MS analysis (bottom, scatter plot of CUL5‐binding proteins). b) Co‐immunoprecipitaion (Co‐IP) of exogenous FLAG‐CUL5 and endogenous APC11 in MIA PaCa‐2 cells. c) Co‐IP of exogenous FLAG‐tagged cullin proteins and endogenous APC11 or SAG in HEK293 cells. d) The interaction of exogenous FLAG‐APC11 with endogenous CUL5 in HEK293 cells. e) Co‐IP of endogenous CUL5 and APC11 in MIA PaCa‐2, A549, Hep3B, and PLC/PRF/5 cells. f) CUL5 binds to APC11 in vitro. Recombinant HIS‐APC11 was incubated with purified FLAG‐CUL5 or vector in vitro, followed by FLAG‐pull‐down and then IB with anti‐APC11 and FLAG Abs. WCE, whole cell extracts.

Article Snippet: [ ] The following antibodies were used: ACTIN (Sigma–Aldrich, A5441), FLAG (Sigma–Aldrich, F1804), FLAG (Sigma–Aldrich, F7425), GFP (Santa Cruz, sc‐9996), MYC (Santa Cruz, sc‐789), APC2 (Cell Signaling Technology, 12301), APC8 (Cell Signaling Technology, 15100), APC10 (Cell Signaling Technology, 14807), APC11 (Cell Signaling Technology, 14090), APC11 (Santa Cruz, sc‐517142), SAG (Proteintech, 11905‐1‐AP), RBX1 (Proteintech, 14895‐1‐AP), CUL1 (Santa Cruz, sc‐11384), CUL5 (Abcam, ab184177), CUL5 (Santa Cruz, sc‐373822), CAND1 (Abcam, ab183748), integrin β1 (Cell Signaling Technology, 34 971), p‐SRC (Y416) (Cell Signaling Technology, 6943), ATM (Cell Signaling Technology, 2873), Cyclin B1 (Cell Signaling Technology, 4138), RPB1 (Cell Signaling Technology, 14958), EGFR (Cell Signaling Technology, 4267), DEPTOR (Cell Signaling Technology, 11 816), NOXA (Millipore, OP180), UBE2F (Proteintech, 17056‐1‐AP), COPS5 (Cell Signaling Technology, 6895), SOCS3 (Proteintech, 14025‐1‐AP), LC3 (Cell Signaling Technology, 2775), securin (Cell Signaling Technology, 13445), ITCH (Proteintech, 20920‐1‐AP), Cyclin E1 (Cell Signaling Technology, 20808), p‐H3 (Cell Signaling Technology, 3377), and p‐Aurora A/B/C (Cell Signaling Technology, 2914), Aurora A (Cell Signaling Technology, 14 475), Aurora B (Abcam, ab2254), and NEDD8 (Abcam, ab81264).

Techniques: Silver Staining, Stable Transfection, Plasmid Preparation, Affinity Purification, SDS Page, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, In Vitro, Recombinant, Incubation, Purification

Journal: Advanced Science

Article Title: The Crosstalk Between CRL5 and APC/C E3 Ligases Regulates Metastasis and Chemosensitivity of Cancer Cells

doi: 10.1002/advs.202512652

Figure Lengend Snippet: APC11 binds to CUL5 in a manner distinct from SAG. a,b) Co‐IP of transfected FLAG‐APC11 (a) or FLAG‐SAG (b) and endogenous CUL5 in HEK293 cells with 1 µM MLN4924 treatment for 12 h. c) The interaction of endogenous CUL5 with APC11 or SAG upon SAG or APC11 knockdown. A549 and PLC/PRF/5 cells were transfected with siRNA oligos targeting APC11, SAG or scrambled control siRNA for 72 h and then immunoprecipitated with IgG or anti‐CUL5 Ab. d) Co‐IP of endogenous APC11, SAG or CAND1 and FLAG‐tagged CUL5 truncations including wild‐type CUL5 (aa 1–780), CUL5‐N (aa 1–400), CUL5‐C (aa 385–780), and CUL5‐ΔSAG (deletion of aa 565‒582) in HEK293 cells. e) A structural modeling illustration of docking APC11 (red) to CUL5 (blue). The putative CUL5‐interaction sites in APC11 are shown in the enlarged image. f) Co‐IP of transfected FLAG‐APC11 wild‐type or mutants in N terminus (including V3A, I5A, W8A, and W14A) and endogenous CUL5 or APC2, in HEK293 cells. g) Schematic representation of the key APC11 residues involved in binding to full‐length CUL5.

Article Snippet: [ ] The following antibodies were used: ACTIN (Sigma–Aldrich, A5441), FLAG (Sigma–Aldrich, F1804), FLAG (Sigma–Aldrich, F7425), GFP (Santa Cruz, sc‐9996), MYC (Santa Cruz, sc‐789), APC2 (Cell Signaling Technology, 12301), APC8 (Cell Signaling Technology, 15100), APC10 (Cell Signaling Technology, 14807), APC11 (Cell Signaling Technology, 14090), APC11 (Santa Cruz, sc‐517142), SAG (Proteintech, 11905‐1‐AP), RBX1 (Proteintech, 14895‐1‐AP), CUL1 (Santa Cruz, sc‐11384), CUL5 (Abcam, ab184177), CUL5 (Santa Cruz, sc‐373822), CAND1 (Abcam, ab183748), integrin β1 (Cell Signaling Technology, 34 971), p‐SRC (Y416) (Cell Signaling Technology, 6943), ATM (Cell Signaling Technology, 2873), Cyclin B1 (Cell Signaling Technology, 4138), RPB1 (Cell Signaling Technology, 14958), EGFR (Cell Signaling Technology, 4267), DEPTOR (Cell Signaling Technology, 11 816), NOXA (Millipore, OP180), UBE2F (Proteintech, 17056‐1‐AP), COPS5 (Cell Signaling Technology, 6895), SOCS3 (Proteintech, 14025‐1‐AP), LC3 (Cell Signaling Technology, 2775), securin (Cell Signaling Technology, 13445), ITCH (Proteintech, 20920‐1‐AP), Cyclin E1 (Cell Signaling Technology, 20808), p‐H3 (Cell Signaling Technology, 3377), and p‐Aurora A/B/C (Cell Signaling Technology, 2914), Aurora A (Cell Signaling Technology, 14 475), Aurora B (Abcam, ab2254), and NEDD8 (Abcam, ab81264).

Techniques: Co-Immunoprecipitation Assay, Transfection, Knockdown, Control, Immunoprecipitation, Binding Assay

Journal: Advanced Science

Article Title: The Crosstalk Between CRL5 and APC/C E3 Ligases Regulates Metastasis and Chemosensitivity of Cancer Cells

doi: 10.1002/advs.202512652

Figure Lengend Snippet: APC11 depletion promotes CUL5 neddylation but stabilizes integrin β1. a) Immunoblot of indicated proteins in PLC/PRF/5, Hep3B, and PANC‐1 cells transfected with siRNA targeting APC11 or with a scrambled control siRNA. b) Co‐IP of transfected FLAG‐CUL5 and endogenous CAND1, COPS5, UBE2F or SAG in HEK293 cells transfected with indicated siRNA oligos. c) Co‐IP of endogenous CUL5 with UBE2F or APC11 in A549 cells transfected with indicated siRNA oligos. d) Immunoblot of CUL5 in A549 and PLC/PRF/5 cells transfected with indicated siRNA oligos. e) The stability of SOCS3 and integrin β1 in A549 and MIA PaCa‐2 cells upon APC11 knockdown. A549 and MIA PaCa‐2 cells were transfected with indicated siRNA oligos for 72 h and then treated with 100 µg mL −1 CHX for the indicated time periods, followed by IB analysis. Densitometry quantifications were performed with ImageJ, and the decay curves are shown (right). Data are presented as mean ± SEM, n = 3. For statistical analysis, significances were determined by Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. f) Diagram illustrating how APC11 depletion promotes CUL5 neddylation and leads to integrin β1 accumulation.

Article Snippet: [ ] The following antibodies were used: ACTIN (Sigma–Aldrich, A5441), FLAG (Sigma–Aldrich, F1804), FLAG (Sigma–Aldrich, F7425), GFP (Santa Cruz, sc‐9996), MYC (Santa Cruz, sc‐789), APC2 (Cell Signaling Technology, 12301), APC8 (Cell Signaling Technology, 15100), APC10 (Cell Signaling Technology, 14807), APC11 (Cell Signaling Technology, 14090), APC11 (Santa Cruz, sc‐517142), SAG (Proteintech, 11905‐1‐AP), RBX1 (Proteintech, 14895‐1‐AP), CUL1 (Santa Cruz, sc‐11384), CUL5 (Abcam, ab184177), CUL5 (Santa Cruz, sc‐373822), CAND1 (Abcam, ab183748), integrin β1 (Cell Signaling Technology, 34 971), p‐SRC (Y416) (Cell Signaling Technology, 6943), ATM (Cell Signaling Technology, 2873), Cyclin B1 (Cell Signaling Technology, 4138), RPB1 (Cell Signaling Technology, 14958), EGFR (Cell Signaling Technology, 4267), DEPTOR (Cell Signaling Technology, 11 816), NOXA (Millipore, OP180), UBE2F (Proteintech, 17056‐1‐AP), COPS5 (Cell Signaling Technology, 6895), SOCS3 (Proteintech, 14025‐1‐AP), LC3 (Cell Signaling Technology, 2775), securin (Cell Signaling Technology, 13445), ITCH (Proteintech, 20920‐1‐AP), Cyclin E1 (Cell Signaling Technology, 20808), p‐H3 (Cell Signaling Technology, 3377), and p‐Aurora A/B/C (Cell Signaling Technology, 2914), Aurora A (Cell Signaling Technology, 14 475), Aurora B (Abcam, ab2254), and NEDD8 (Abcam, ab81264).

Techniques: Western Blot, Transfection, Control, Co-Immunoprecipitation Assay, Knockdown

Journal: Cell reports

Article Title: The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation

doi: 10.1016/j.celrep.2024.115101

Figure Lengend Snippet: (A) Ablation of RBX1 diminishes amino acid-induced mTORC1 activation in EMSCs. Three gRNAs designed to target distinct exon regions of RBX1 were used. Cells were starved in amino acid-deprived medium for 50 min and treated with 1× amino acids for 10 min. gRNA targeting GFP was used as a control. Note that RBX1–3 gRNA failed to ablate RBX1. (B) Ablation of RBX1, but not RBX2, diminishes amino acid-induced mTORC1 activation in EMSCs. Cells were treated as in (A). Levels of phosphorylated S6K1 in the indicated cells (amino acid-replete conditions) were quantified and expressed as the ratio of pS6K/S6K1. * p < 0.05, ** p < 0.01, mean ± SD, n = 4. (C) Ablation of RBX1 diminishes CQ/MG-induced mTORC1 activation in Ragulator-deficient cells. RBX1 is ablated in p18/LAMTOR1 knockout (KO) EMSCs. Cells were treated with CQ (50 μM) or/and MG (20 μM) in amino acid-free medium for 45 min. (D) Ablation of RBX1 diminishes Rheb ubiquitination. RBX1 KO EMSCs were lysed under amino acid-replete conditions in RIPA buffer containing the deubiquitinase inhibitor (100 mM) N-ethylmaleimide (NEM). The lysates were subjected to SDS-PAGE, and levels of Rheb and Ub-Rheb were monitored with a Rheb antibody. Non-Ub-Rheb and Ub-Rheb are indicated. (E) MLN4924 specifically inhibits mTORC1 activity induced by amino acids in EMSCs. Cells were treated with the indicated concentrations of MLN4924 for 16 h. Cells were amino acid starved for 50 min and re-stimulated with 1× amino acids for 10 min. Levels of phosphorylated S6K1 in the indicated cells (amino acid-replete conditions) were quantified and expressed as the ratio of pS6K/S6K1. **** p < 0.0001, mean ± SD, n = 4. (F) MLN4924 inhibits mTORC1 activity induced by amino acids in both control and DEPDC5 KO HEK293T cells. Cells were treated with MLN4924 (1 μM) as in (E). Cells were amino acid starved for 50 min and re-stimulated with 1× amino acids for 10 min. The effect of DEPDC5 ablation was confirmed by the high levels of mTORC1 activity resistant to amino acid starvation. (G) MLN4924 diminishes levels of Ub-Rheb in HEK293T cells. Cells were treated with MLN4924 (1 μM) for 16 h and lysed as in (D), and the lysates were subjected to immunoblotting with a Rheb antibody.

Article Snippet: RBX2 polyclonal antibody , Proteintech , Cat# 11905-1-AP; RRID:AB_10697836.

Techniques: Activation Assay, Control, Knock-Out, Ubiquitin Proteomics, SDS Page, Activity Assay, Western Blot

Journal: Cell reports

Article Title: The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation

doi: 10.1016/j.celrep.2024.115101

Figure Lengend Snippet:

Article Snippet: RBX2 polyclonal antibody , Proteintech , Cat# 11905-1-AP; RRID:AB_10697836.

Techniques: Virus, Recombinant, Magnetic Beads, Control, Negative Control, Ubiquitin Proteomics, shRNA, Plasmid Preparation, Immunofluorescence, Software

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Anti-SAG antibody , Proteintech , Cat #11905-1-AP; RRID: AB_10697836.

Techniques: Mutagenesis, Recombinant, CCK-8 Assay, Software