Fig. 5: Pharmacologically targeting HSP70 degrades N-Myc expression via STUB1.

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 Source Data file.