Fig 2: (20S) G-Rh2-induced interaction disruption of HSP90A and Cdc37, degradation of CDKs, cell cycle arrest, and proliferation inhibition of HepG2 cells. (A) Immunoprecipitation was performed with whole-cell lysate under treatment with different concentrations of (20S) G-Rh2 for 24 h, and the interaction was analyzed by an immunoblot. (B) Immunoblotting analysis of the cell cycle-related protein levels in HepG2 cells treated with different concentrations of (20S) G-Rh2 for 24 h. (C, Left) Cell cycle distribution was determined by PI staining of HepG2 cells after treatment with the indicated concentrations of (20S) G-Rh2 for 24 h. (Right) The cell cycle distribution is represented as a graphic histogram. (D) HepG2 cells were treated with the indicated concentrations of (20S) G-Rh2 for 48 h, and the cell viability was determined by MTT assay. All data are shown as the mean ± SD of experiments performed in triplicate. A two-tail Student’s t-test was used for statistical analyses (* p < 0.01, ** p < 0.001).

Fig 3: HSALR1 acts as a scaffold and stabilizes HSP90AB1‐Akt1 complex. (A) RNA pulldown assay using lncRNA HSALR1 sense and antisense RNAs in HBF cells, followed by silver staining (n = 3 biological replicates). (B) Schematic representation of the PPI network of overlapping genes occurring twice or more in MS. (C) The bindings of HSALR1 to HSP90AB1 and CDC37 predicted via RNAInter. (D) The interaction between HSALR1 and HSP90AB1 versus HSALR1 and CDC37 detected by Western blotting with the extract of the RNA pulldown assay. (E) RIP‐qPCR analysis with anti‐HSP90AB1 antibody and anti‐CDC37 antibody in HBF cells (n = 3 biological replicates, Student's t‐test). (F) RIP‐qPCR analysis with anti‐HSP90AB1 antibody and anti‐CDC37 antibody after HSALR1 overexpression in HBF cells (n = 3 biological replicates, Student's t‐test). (G) Co‐IP analysis of lnc9‐overexpressed HBFs using anti‐HSP90AB1 antibody. Western blot was used to verify the Co‐IP results with anti‐p‐AKT and anti‐CDC37 antibodies. (H) Co‐IP analysis of lnc9‐overexpressed HBFs using anti‐p‐AKT antibody. Western blot was performed to verify the Co‐IP results with anti‐HSP90AB1 and anti‐CDC37 antibody. (I) Co‐IP analysis of lnc9‐overexpressed HBFs using anti‐HSP90AB1 antibody. Western blot was conducted to verify the Co‐IP results with anti‐AKT, anti‐p‐MAPK and anti‐MAPK antibodies. (J) Western blot and (K) qRT‐PCR analysis of HSP90AB1 expression after transfection with three siRNAs (siHSP90AB1‐1, si‐HSP90AB1‐2 and siHSP0AB1‐3) in HBF cells; and (L) qRT‐PCR analysis of HSALR1 expression after transfection with above siRNAs (n = 3 biological replicates, one‐way ANOVA). (M) 48 h after overexpression of HSALR1, the HBFs were incubated with cycloheximide (CHX, 40 μg/mL) and negative control (DMSO), then harvested at 0, 3, 6, 9 h (n = 5 biological replicates, one‐way ANOVA). (N) 48 h after overexpression of HSALR1, the HBFs were incubated with MG‐132 (5 μm) and negative control (DMSO), then harvested at 12 h (n = 5 biological replicates, Student's t‐test). Data information: Error bars represent means ± SD. *p < .05, **p < .01 and ***p < .05.