Fig. 5

CircSHKBP1 directly interacts with HSP90 and inhibits its degradation. a. Silver staining of circSHKBP1 pulldown. Arrows show different bands between the sense and antisense lanes. b. List of the top 10 differentially expressed proteins identified by mass spectrometry. c. Level of circSHKBP1 detected by qRT-PCR after RIP for HSP90 in BGC823 cells. d. Expression of HSP90 in GC tumors (n = 408) and normal tissues (n = 211) obtained from TCGA database. e Levels of HSP90A and HSP90B in paired GC tumors and normal tissues detected by qRT-PCR (n = 34). f Levels of HSP90 in paired GC tumors and normal tissues detected by ELISA (n = 34). g Correlation of HSP90 protein and circSHKBP1 expression in GC tissues (R² = 0.2881, P = 0.011). h Western blot with grey value columns showing the expression of HSP90 in BGC823 cells transfected with control vector or circSHKBP1 plasmid after treatment with CHX (100 μg/ml) for 0 h, 4 h, 8 h, 12 h (tubulin as an internal control). i Western blot with grey value columns showing the expression of HSP90 in BGC823 cells treated with or without MG132 after treatment with CHX (100 μg/ml) for 0 h, 4 h, 8 h, 12 h (tubulin as an internal control). j and k STUB1 immunoprecipitated using an anti-HSP90 antibody. STUB1 was reduced in the immunoprecipitate of BGC823 and HGC27 cells transfected with circSHKBP1. l Assessment of the proliferation of BGC823 and HGC27 cells treated with or without HSP90 inhibitor NMS-E973 by CCK8 assay. m and n Assessment of the migration and invasion of BGC823 and HGC27 cells treated with or without the HSP90 inhibitor NMS-E973 by Transwell assay. Quantitative data from three independent experiments are shown as the mean ± SD (error bars). *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test)