hsp70 expression  (Molecular Instruments)

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: Cells were transiently transfected with plasmids expressing HA-N-Myc (Addgene, Catalog#74163), His-N-Myc (SinoBiological, Catalog# HG17471-CH), Flag-STUB1 (Sino Biological, Catalog# HG12496-NF), HSP70 (OriGene, Catalog# SC116767), Flag-HSP70 (SinoBiological, Catalog# HG11660-NF), His-HSP70 (GenScript, CloneID#OHu15193), Flag-Max (GenScript, CloneID#OHu16927), Flag-AURKA (GenScript, CloneID#OHu23690), pRK5-HA-Ub K11R (Addgene, Catalog#121154), pRK5-HA-Ub K48R (Addgene, Catalog#17604), pRK5-HA-Ub K0 (Addgene, Catalog#17603) or pRK5-HA-Ubiquitin (Addgene, Catalog#17608), using Lipofectamine 2000 (Invitrogen, # 11668019).

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: Cells were transiently transfected with plasmids expressing HA-N-Myc (Addgene, Catalog#74163), His-N-Myc (SinoBiological, Catalog# HG17471-CH), Flag-STUB1 (Sino Biological, Catalog# HG12496-NF), HSP70 (OriGene, Catalog# SC116767), Flag-HSP70 (SinoBiological, Catalog# HG11660-NF), His-HSP70 (GenScript, CloneID#OHu15193), Flag-Max (GenScript, CloneID#OHu16927), Flag-AURKA (GenScript, CloneID#OHu23690), pRK5-HA-Ub K11R (Addgene, Catalog#121154), pRK5-HA-Ub K48R (Addgene, Catalog#17604), pRK5-HA-Ub K0 (Addgene, Catalog#17603) or pRK5-HA-Ubiquitin (Addgene, Catalog#17608), using Lipofectamine 2000 (Invitrogen, # 11668019).

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 Cell proliferation was measured in CWR22Rv1 cells transfected with siRNA against negative control, HSP70, AURKA, or in combination for 5 days (n = 3 samples). b , c CWR22Rv1 cells were transfected with siRNA targeting AURKA or negative control and then treated with JG231. Cell proliferation ( b ) and N-Myc expression ( c ) were determined by cell counting or western blotting, respectively (n = 3 samples). d CWR22Rv1, H660, and UCDCaP-CR cells were treated with increasing doses of alisertib for 3 days, and the viable cells were counted. e The expression of N-Myc was detected in CWR22Rv1 cells treated with alisertib alone or combined with JG231. f CWR22Rv1, H660, and UCDCaP-CR cells were treated with alisertib (0.025 µM for CWR22Rv1, 0.1 µM for other cell lines), JG231 (0.1 µM) or the combination for 7-10 days, and the viable cells were counted (n = 3 samples). g CWR22Rv1 cells were treated with JG231 alone or with alisertib in the clonogenic assay. h Organoids from LuCaP93 PDX were treated with JG231 (0.1 µM), alisertib (0.25 µM) alone or in combination for 7 days. Cell viability was assayed by the CellTiter-Glo Luminescent assay and the live-and-dead cells were visualized by immunofluorescence (n = 3 samples). Scale bar represents 100 microns. i Mice bearing H660 xenografts were treated with vehicle control, JG231 (2 mg/Kg i.p), alisertib (10 mg/Kg p.o), or JG231 plus alisertib for 30 days (n = 6). Tumor volumes were measured twice weekly. Tumors were photographed and weighed. j , k Drug toxicity tests were conducted by measuring the blood urea nitrogen (BUN) and alanine aminotransferase (ALT) levels in the serum samples collected from animals in each group (n = 4 samples). l IHC staining of N-Myc and Ki67 in each group was performed. Scale bar represents 20 microns. For ( a , f , and h – k ), statistical significance was determined by one-way ANOVA with a Tukey multiple-comparison test. For ( b ), statistical significance was determined by an unpaired two-sided t-test. Results are the mean of three independent experiments ( ± S.D.). Source data are provided as a file.

Article Snippet: Cells were transiently transfected with plasmids expressing HA-N-Myc (Addgene, Catalog#74163), His-N-Myc (SinoBiological, Catalog# HG17471-CH), Flag-STUB1 (Sino Biological, Catalog# HG12496-NF), HSP70 (OriGene, Catalog# SC116767), Flag-HSP70 (SinoBiological, Catalog# HG11660-NF), His-HSP70 (GenScript, CloneID#OHu15193), Flag-Max (GenScript, CloneID#OHu16927), Flag-AURKA (GenScript, CloneID#OHu23690), pRK5-HA-Ub K11R (Addgene, Catalog#121154), pRK5-HA-Ub K48R (Addgene, Catalog#17604), pRK5-HA-Ub K0 (Addgene, Catalog#17603) or pRK5-HA-Ubiquitin (Addgene, Catalog#17608), using Lipofectamine 2000 (Invitrogen, # 11668019).

Techniques: Transfection, Negative Control, Expressing, Cell Counting, Western Blot, Clonogenic Assay, Luminescence Assay, Immunofluorescence, Control, Immunohistochemistry, Comparison

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: The HSP70/STUB1 complex translocates from the cytoplasm to the nucleus. The substrate binding domain (SBD) of HSP70 occupies the “SELILKR” binding motif on native N-Myc protein, preventing the access of STUB1 and slowing down the turnover of N-Myc protein. However, in the presence of the HSP70 allosteric inhibitor JG231, HSP70 pulls STUB1 closer through binding between the EEVD domain of HSP70 and K30 of the TRP domain of STUB1. This allows the Ubox of STUB1 to recruit E2 and promote N-Myc ubiquitination at K416 and K419 sites, forming a K11 and K63-linked polyubiquitination chain for degradation in the nucleus and dissociating N-Myc/MAX binding. Dual targeting of AURKA (alisertib) and HSP70 (JG231) causes synergistic degradation of N-Myc and suppresses NEPC tumor growth. Created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: Cells were transiently transfected with plasmids expressing HA-N-Myc (Addgene, Catalog#74163), His-N-Myc (SinoBiological, Catalog# HG17471-CH), Flag-STUB1 (Sino Biological, Catalog# HG12496-NF), HSP70 (OriGene, Catalog# SC116767), Flag-HSP70 (SinoBiological, Catalog# HG11660-NF), His-HSP70 (GenScript, CloneID#OHu15193), Flag-Max (GenScript, CloneID#OHu16927), Flag-AURKA (GenScript, CloneID#OHu23690), pRK5-HA-Ub K11R (Addgene, Catalog#121154), pRK5-HA-Ub K48R (Addgene, Catalog#17604), pRK5-HA-Ub K0 (Addgene, Catalog#17603) or pRK5-HA-Ubiquitin (Addgene, Catalog#17608), using Lipofectamine 2000 (Invitrogen, # 11668019).

Techniques: Binding Assay