Journal: Nature Communications

Article Title: Proteostasis perturbation of N-Myc leveraging HSP70 mediated protein turnover improves treatment of neuroendocrine prostate cancer

doi: 10.1038/s41467-024-50459-x

Figure Lengend Snippet: a Cell proliferation and N-Myc expression were assessed in H660 cells transfected with control and N-Myc siRNA (n = 3; statistical significance determined by one-way ANOVA with a Tukey multiple-comparison test). b H660 and CWR22Rv1 cells were cultured with MG132 for different times. N-Myc expression was examined by western blotting. c N-Myc binding proteins were determined by the immunoprecipitation pull-down assay followed by proteomic profiling. d The representative HSPA1B peptide pull-down with N-Myc was listed. e , f HEK293 cells were co-transfected with N-Myc with or without Flag-HSP70 or Flag-STUB1, and the cell lysates were immunoprecipitated with the anti-Flag antibody. g , h H660 and CWR22Rv1 cells were transfected with control and HSP70 siRNA, or then treated with or without MG132 for 6 h. Whole cell lysates were separated by electrophoresis and blotted for N-Myc and HSP70. i, j The protein expression of N-Myc was determined after H660 and CWR22Rv1 cells were transfected with Flag-STUB1, or then treated with or without MG132 for 6 h. k In vitro ubiquitination assays were conducted to detect the ubiquitination of N-Myc. l HEK293 cells were transfected with HA-N-Myc or with Flag-STUB1 and treated with or without MG132 for 6 h. Immunoprecipitation was performed with the anti-HA antibody. m C4-2B cells overexpressing N-Myc were transfected with control or Flag-STUB1 and treated with cycloheximide for different times. N-Myc expression was analyzed by western blotting to calculate its half-life (n = 3 independent experiments; data are presented as mean ± S.D.). n Similar to ( m ), but CWR22Rv1 cells were transfected with STUB1 siRNA or control before cycloheximide treatment (n = 3 independent experiments; data are presented as mean ± S.D.). o Lysates of HEK293 cells co-transfected with HA-N-Myc, Flag-STUB1, and His-HSP70 were immunoprecipitated with the anti-Flag antibody. p HEK293 cells were co-transfected with HA-N-Myc and Flag-STUB1 with or without His-HSP70, and the interaction of N-Myc and STUB1 was determined by Proximity Ligation Assay (PLA). His-HSP70 was stained by fluorescein isothiocyanate (FITC). Scale bar represents 40 microns. q HEK293 cells were co-transfected with His-N-Myc and HA-Ubiquitin with or without Flag-HSP70, and the whole cell lysates were immunoprecipitated with the anti-His antibody. Results are the mean of three independent experiments ( ± S.D.). Source data are provided as a file.

Article Snippet: LNCaP (American Type Culture Collection, ATCC, CRL-2505), C4-2B (a kind gift from Dr. Leland Chung), C4-2B MDVR (C4-2B enzalutamide resistant), PC3 (ATCC, CRL-1435), DU145 (ATCC, HTB-81), and CWR22Rv1 (ATCC, CRL-2505) cells were maintained in RPMI1640, whereas VCaP, UCDCaP (derived from an 84-year-old male Gleason 10 patient), UCDCaP-CR (UCDCaP castration resistant) and HEK293 (ATCC, CRL-1573.3) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin.

Techniques: Expressing, Transfection, Control, Comparison, Cell Culture, Western Blot, Binding Assay, Immunoprecipitation, Pull Down Assay, Electrophoresis, In Vitro, Proximity Ligation Assay, Staining

Journal: Nature Communications

Article Title: Proteostasis perturbation of N-Myc leveraging HSP70 mediated protein turnover improves treatment of neuroendocrine prostate cancer

doi: 10.1038/s41467-024-50459-x

Figure Lengend Snippet: a Schematic representation of the N-Myc deletion mutants used for domain mapping. Myc boxes: I, II, III, IV; BR, basic region; HLH, helix-loop-helix; LZ, leucine zipper. b HEK293 cells were co-transfected with Flag-STUB1 and wild-type or indicated HA-N-Myc mutant constructs. The cell lysates were immunoprecipitated with the anti-Flag antibody. The red arrows mark the expected positions of the full-length or truncated N-Myc pulled down by STUB1. c HEK293 cells were co-transfected with Flag-HSP70 and wild-type or indicated HA-N-Myc mutant constructs. The cell lysates were immunoprecipitated with the anti-Flag antibody. The red arrows mark the expected positions of the full-length or truncated N-Myc pulled down by HSP70. d Alignment of the potential binding sites in N-Myc in different species to HSP70. e The Sanger Sequence chromatogram of the wild-type (WT) N-Myc plasmid and the corresponding deletion in N-Myc-ΔLILKR. f HEK293 cells co-transfected with/without His-HSP70, Flag-STUB1, and HA-WT-N-Myc or HA-N-Myc-ΔLILKR. Whole cell lysates were harvested and immunoprecipitated with the anti-HA antibody. g HEK293 cells were co-transfected with HA-WT-N-Myc or HA-N-Myc-ΔLILKR, with Flag-HSP70 or Flag-STUB1 for 3 days, and the interaction of N-Myc and HSP70 or STUB1 was determined by Proximity Ligation Assay (PLA). Scale bar represents 20 microns. h HEK293 cells were transfected with HA-WT-N-Myc or HA-N-Myc-ΔLILKR and treated with 50 μg/ml cycloheximide for 0, 30, 60, and 120 min. Whole cell lysates were separated by electrophoresis and blotted with the anti-HA antibody, and the half-life of the full-length and deleted N-Myc molecules was calculated (n = 3 independent experiments and data presented as mean ± S.D.). i HEK293 cells were transfected with HA-WT-N-Myc, HA-N-Myc-ΔLILKR, or HA-N-Myc-CLPQS, with or without Flag-STUB1 for 3 days. Total cell lysates were collected for western blotting to detect the expression of N-Myc. j HEK293 cells co-transfected with His-HSP70, HA-WT-N-Myc, HA-N-Myc-ΔLILKR, or HA-N-Myc-CLPQS and Flag-STUB1 plasmids. Whole cell lysates were immunoprecipitated with the anti-N-Myc antibody. Source data are provided as a file.

Article Snippet: LNCaP (American Type Culture Collection, ATCC, CRL-2505), C4-2B (a kind gift from Dr. Leland Chung), C4-2B MDVR (C4-2B enzalutamide resistant), PC3 (ATCC, CRL-1435), DU145 (ATCC, HTB-81), and CWR22Rv1 (ATCC, CRL-2505) cells were maintained in RPMI1640, whereas VCaP, UCDCaP (derived from an 84-year-old male Gleason 10 patient), UCDCaP-CR (UCDCaP castration resistant) and HEK293 (ATCC, CRL-1573.3) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin.

Techniques: Transfection, Mutagenesis, Construct, Immunoprecipitation, Binding Assay, Sequencing, Plasmid Preparation, Proximity Ligation Assay, Electrophoresis, Western Blot, Expressing

Journal: Nature Communications

Article Title: Proteostasis perturbation of N-Myc leveraging HSP70 mediated protein turnover improves treatment of neuroendocrine prostate cancer

doi: 10.1038/s41467-024-50459-x

Figure Lengend Snippet: a HEK293 cells were co-transfected with wild-type (WT) STUB1 or mutants and HA-N-Myc. The expression of N-Myc was determined by western blotting. b HEK293 cells were co-transfected with Flag-WT-STUB1 or mutants and HA-N-Myc. Whole cell lysates were immunoprecipitated with the anti-HA antibody and blotted with anti-ubiquitin, Flag, or HA antibodies. c HEK293 cells were co-transfected with HA-WT-N-Myc or mutants in the absence or presence of the Flag-STUB1 construct. The expression of N-Myc was determined by western blotting. d HEK293 cells were co-transfected with HA-WT-N-Myc or Δ382-464-N-Myc and Flag-STUB1 plasmids. Whole cell lysates were immunoprecipitated with the anti-HA antibody and blotted with Ubiquitin, Flag, or HA antibodies. e HEK293 cells were transfected with HA-WT-N-Myc or Δ382-464-N-Myc for 3 days and then treated with 50 µg/mL cycloheximide. Total cell lysates were collected at 0, 5, 15, 30, 45, and 60 min after the treatment and subjected to western blotting. Half-lives of WT and truncated N-Myc were calculated (n = 3 independent experiments, data presented as mean ± S.D.). f Schematic representation of potential ubiquitination modification sites within the residues 382–464 in N-Myc. g HEK293 cells were co-transfected with HA-N-Myc WT or mutants and Flag-STUB1 plasmids. The expression of N-Myc was determined by western blotting. h HEK293 cells were co-transfected with HA-WT-N-Myc or selected mutants and Flag-STUB1 plasmids. Whole cell lysates were immunoprecipitated with the anti-N-Myc antibody and blotted with anti-ubiquitin, Flag, or N-Myc antibodies. i HEK293 cells were co-transfected with His-N-Myc, HA-WT-Ubiquitin, or Ub mutants and Flag-STUB1. Whole cell lysates were immunoprecipitated with the His-Tag Dynabeads™ and blotted with the HA or N-Myc antibodies. j HEK293 cells were co-transfected with His-N-Myc, HA-Ubiquitin (WT, K0, or K11) and Flag-STUB1 plasmids. Cell lysates were immunoprecipitated with the His-Tag Dynabeads™ and probed for HA or N-Myc. k HEK293 cells were co-transfected with His-N-Myc, HA-Ubiquitin (WT, K11, or K63), and Flag-STUB1 plasmids with or without 5 µM MG132. Cell lysates were immunoprecipitated with the His-Tag Dynabeads™ and probed for HA or N-Myc. Source data are provided as a file.

Article Snippet: LNCaP (American Type Culture Collection, ATCC, CRL-2505), C4-2B (a kind gift from Dr. Leland Chung), C4-2B MDVR (C4-2B enzalutamide resistant), PC3 (ATCC, CRL-1435), DU145 (ATCC, HTB-81), and CWR22Rv1 (ATCC, CRL-2505) cells were maintained in RPMI1640, whereas VCaP, UCDCaP (derived from an 84-year-old male Gleason 10 patient), UCDCaP-CR (UCDCaP castration resistant) and HEK293 (ATCC, CRL-1573.3) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin.

Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation, Construct, Modification

Journal: Nature Communications

Article Title: Proteostasis perturbation of N-Myc leveraging HSP70 mediated protein turnover improves treatment of neuroendocrine prostate cancer

doi: 10.1038/s41467-024-50459-x

Figure Lengend Snippet: a CWR22Rv1, H660, and UCDCaP-CR cells were treated with JG231, and N-Myc expression was determined by western blotting. b Similar to a, but HEK293 cells were transfected with HA-N-Myc for 2 days before JG231 treatment. c CWR22Rv1 and UCDCaP-CR cells were treated with JG231 (2.5 µM) and MG132. The expression of N-Myc was determined by western blotting. d Similar to c, but C4-2B cells were transfected with N-Myc before JG231 treatment. e CWR22Rv1 cells were treated with cycloheximide in the absence or presence of JG231 (10 µM). N-Myc expression was analyzed by western blotting to calculate its half-life (n = 3 independent experiments, data presented as mean ± S.D.). f CWR22Rv1 cells were treated with JG231 (5 µM) overnight and then treated with MG132, and immunoprecipitation was performed with N-Myc antibody. g CWR22Rv1 cells were transiently transfected with STUB1 siRNA, followed by treatment with JG231. The cell lysates were collected and subjected to western blotting. h, i HEK293 cells were co-transfected with HA-N-Myc and Flag-STUB1, followed by treatment with JG231 (2.5 µM) and MG132. The localization and interaction of N-Myc and STUB1 were analyzed by immunofluorescence and Proximity Ligation Assay (PLA), respectively. Scale bars represent 50 and 20 microns, respectively. j CWR22Rv1 cells were treated with JG231 (2.5 µM) and then treated with MG132. The nuclear protein was immunoprecipitated with N-Myc antibody. k HEK293 cells were transfected with WT-N-Myc, N-Myc-K416R, or N-Myc-K419R, and then treated with JG231 (2.5 µM). Immunoprecipitation was performed with HA antibody. l HEK293 cells were co-transfected with His-N-Myc, HA-Ubiquitin (WT, K0, K11, or K63), and then treated with JG231 (5 µM). Immunoprecipitation was performed with His-tag Dynabeads™. m The volcano plot shows the nuclear proteins from C4-2B N-Myc cells treated with JG231 for 4 h by proteomic profiling. Blue dots represent down-regulated, and yellow dots represent up-regulated (1.3-fold and p < 0.05) proteins. n , o Gene Ontology and pathway analyses demonstrate the enrichment of functional annotations in down-regulated proteins in nuclear lysates from C4-2B N-Myc cells treated with 5 µM JG231. Source data are provided as a file.

Article Snippet: LNCaP (American Type Culture Collection, ATCC, CRL-2505), C4-2B (a kind gift from Dr. Leland Chung), C4-2B MDVR (C4-2B enzalutamide resistant), PC3 (ATCC, CRL-1435), DU145 (ATCC, HTB-81), and CWR22Rv1 (ATCC, CRL-2505) cells were maintained in RPMI1640, whereas VCaP, UCDCaP (derived from an 84-year-old male Gleason 10 patient), UCDCaP-CR (UCDCaP castration resistant) and HEK293 (ATCC, CRL-1573.3) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin.

Techniques: Expressing, Western Blot, Transfection, Immunoprecipitation, Immunofluorescence, Proximity Ligation Assay, Functional Assay