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

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

Journal: Bioengineering & Translational Medicine

Article Title: E3 ligase activity of Carboxyl terminus of Hsc70 interacting protein ( CHIP) in Wharton's jelly derived mesenchymal stem cells improves their persistence under hyperglycemic stress and promotes the prophylactic effects against diabetic cardiac damages

doi: 10.1002/btm2.10234

Figure Lengend Snippet: CHIP targets HG induced‐PTEN for ubiquitin‐mediated proteasomal degradation cooperated by HSP70 under HG conditions. (a–c) Cells transfected with HA‐vector or HA‐CHIP in the presence and absence of MG‐132 for 6 h were subjected to HG challenge for 24 h. Whole cell lysate was immunoprecipitated with the anti‐HA, anti‐CHIP, and anti‐PTEN antibody, and subsequently immunoblotted with the primary antibodies, including anti‐HA, anti‐PTEN, and anti‐ubiquitin. (d, e) Cells transfected with HA‐vector, HA‐CHIP, and CHIP mutants (K30A, an H260Q) were treated with or without MG‐132 for 6 h in the presence of HG for 24 h. Whole cell lysate was immunoprecipitated with the anti‐HA and anti‐PTEN antibody followed by immunoblotting with the anti‐HA, anti‐PTEN, and anti‐ubiquitin antibody. (f) Cells were transfected with increasing concentrations of siHSP70 (10, 20, 30 nM) after challenged with HG for 24 h, and the expression level of PTEN and HSP70 was measured employing Western blot analysis. (g) Following cotransfection of GFP‐vector or GFP‐CHIP with increasing concentration of siHSP70 in WJMSCs were challenged with HG for 24 h, and the protein expression was measured via immunoblotting. (h) WJMSCs transfected with sicontrol, CHIP siRNA, or siHSP70 were subjected to HG challenge for 24 h, and the total cell extract was immunoblotted with CHIP, PTEN, and HSP70. β‐actin served as a loading control. (i) Docking studies demonstrating the molecular interaction of HSP70 with PTEN forming a heteromer complex (HSP70 and PTEN shown in quaternary structure with helices and sheets in complex). Values shown are mean ± SD. Quantification of the results are shown ( n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 indicates the significant difference. CHIP, carboxyl terminus of Hsc70 interacting protein; HG, high glucose; PTEN, phosphatase and tensin homolog; WJMSCs, Wharton's jelly derived mesenchymal stem cells

Article Snippet: Lentiviral GFP‐CHIP was purchased from the Sino biological (RG83573‐ACGLN), while the lentiviral expressing small hairpin RNAs (shRNAs), including shcontrol (pLAS.Void), shCHIP (TRCN0000007528 NM_005861), shPTEN (TRCN0000002746 NM_000314), and lentiviral packaging plasmids (pCMVΔR8.91 and pMD.G), were obtained from the national RNAi core facility (Academia Sinica).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Cotransfection, Concentration Assay, Derivative Assay

Journal: Bioengineering & Translational Medicine

Article Title: E3 ligase activity of Carboxyl terminus of Hsc70 interacting protein ( CHIP) in Wharton's jelly derived mesenchymal stem cells improves their persistence under hyperglycemic stress and promotes the prophylactic effects against diabetic cardiac damages

doi: 10.1002/btm2.10234

Figure Lengend Snippet: CHIP overexpressed WJMSCs rescued hyperglycemic effects under diabetic conditions. (a) Schematic illustration of STZ‐induced diabetes, and WJMSCs administration expressing different plasmids, including GFP‐CHIP, shCHIP, and shPTEN. (b) The oral glucose tolerance test (OGTT) was performed after 6 weeks treatment for the indicated time points (0, 30, 60, 90, and 120 min) in various experimental groups, including control, STZ‐induced diabetes (STZ), STZ‐induced diabetes administered with WJMSCs alone (STZ + WJMSCs), STZ‐induced diabetes injected with CHIP overexpressed WJMSCs (STZ + CHIP‐WJMSCs), STZ‐induced diabetes transplanted with CHIP knockdown WJMSCs (STZ + shCHIP‐WJMSCs), and STZ‐induced diabetic rats infused with PTEN knockdown WJMSCs (STZ + shPTEN‐WJMSCs) after the rats were fasted for 14 h. (c) Morphological assessment of cardiac tissues in different experimental groups. (d) Echocardiographic evaluation of cardiac function in different experimental groups (control, STZ, STZ + WJMSCs, STZ + CHIP‐WJMSCs, STZ + shCHIP‐WJMSCs, and STZ + shPTEN‐WJMSCs). (e) Total cell lysate from the left ventricle was quantified and measured using Western blot. Protein expression levels of the apoptosis (p‐AKT, Bax, and Cyt‐c) and oxidative stress markers (catalase, SOD2, and gp91 PHOX ) were assessed. GAPDH act as a loading control. Values shown are mean ± SD. Quantification of the results are shown ( n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 shows the significance. CHIP, carboxyl terminus of Hsc70 interacting protein; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; PTEN, phosphatase and tensin homolog; STZ, streptozotocin; WJMSCs, Wharton's jelly derived mesenchymal stem cells

Article Snippet: Lentiviral GFP‐CHIP was purchased from the Sino biological (RG83573‐ACGLN), while the lentiviral expressing small hairpin RNAs (shRNAs), including shcontrol (pLAS.Void), shCHIP (TRCN0000007528 NM_005861), shPTEN (TRCN0000002746 NM_000314), and lentiviral packaging plasmids (pCMVΔR8.91 and pMD.G), were obtained from the national RNAi core facility (Academia Sinica).

Techniques: Expressing, Injection, Western Blot, Derivative Assay

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Comparison of As 2 O 3 and As 4 O 6 in the Detection of SiHa Cervical Cancer Cell Growth Inhibition Pathway

doi: 10.4143/crt.2004.36.4.255

Figure Lengend Snippet: Confirmations of cDNA chip experiment by Northern blot analysis. Total RNA obtained from As2O3 (lane 2) and As4O6 (lane 3) treated cells subjected to Northern analysis, as described in 'Materials and methods'.

Article Snippet: These were centrifuged for 10 min at 14,000 rpm, and the supernatant containing probes added drop wise to a 384 cDNA chip (Macrogen, Seoul, Korea).

Techniques: Northern Blot