Fig 2: Protein level of YTHDF2 is regulated by HSP90β and STUB1 in HCC. A) Western blot assay for YTHDF2 and STUB1 in HepG2 and Hep3B cells exposed to STUB1 siRNAs or control siRNAs for 72 h. Quantification was shown below the images. B) Western blot assay for YTHDF2 and STUB1 in HepG2 cells exposed to HA‐STUB1 or control plasmids for 48 h. Quantification was shown below the images. C) Western blot assay for YTHDF2 in HepG2 and Hep3B cells exposed to NVP‐AUY922 (NVP) for 48 h. Quantification was shown below the images. D) Western blot assay for YTHDF2 and STUB1 in HepG2 cells exposed to STUB1 or control siRNAs for 48 h, followed by cycloheximide treatment (CHX, 100 µg mL−1) for 12, 24, and 36 h. Quantification was shown on the right. E) Western blot assay for YTHDF2 was performed in HepG2 cells exposed to NVP‐AUY922 (0.5 × 10−6 m) for 24 h, followed by the treatment of CHX for 12, 24, and 36 h. Quantification was shown on the right. F) RT‐qPCR assays for YTHDF2 and STUB1 were performed in HepG2 cells exposed to STUB1 siRNAs or control siRNAs for 36 h. G) RT‐qPCR assays for YTHDF2 were performed in HepG2 cells exposed to NVP for 12 h. H) Western blot assay for YTHDF2 in HepG2 cells exposed to NVP for 24 h, followed by bortezomib (BTZ) treatment for 24 h. Quantification was shown on the lower side. I) Western blot assay for YTHDF2 and STUB1 in HepG2 cells exposed to HA‐STUB1 or control plasmids for 24 h, followed by the treatment of bortezomib (BTZ) for 24 h. Quantification was shown on the lower side. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns represents not significant.

Fig 3: Clinical relationship of YTHDF2, HSP90β, and STUB1 in HCC tissues. A) IHC assay was performed in paraffin‐embedded HCC tissues and adjacent normal tissues using YTHDF2, HSP90β, or STUB1 antibodies. Representative images were shown at 400×. Scale bar, 50 µm. B–D) Quantification of YTHDF2, HSP90β, and STUB1 in (A) was shown. Data were analyzed with paired t‐tests. E–G) Correlation analysis of YTHDF2 with HSP90β or STUB1 protein levels, and STUB1 with HSP90β protein levels based on B using Pearson r assay. Tumor tissues and adjacent normal tissues were included in the statistics.

Fig 4: Overall survival analysis of HCC patients using Kaplan–Meier curves. A,B) Analysis of HSP90β mRNA expression in HCC tissues based on cancer stages and tumor grades by analyzing the TCGA and UALCAN databases. *p < 0.05, & p < 0.01, &&& p < 0.0001. C,D) Kaplan–Meier curves from HCC patients expressing low and high HSP90β/STUB1 from the tissue microarray. Overall survival data were shown. E,F) Kaplan–Meier curves from HCC patients (in stage 2+3 or stage 3+4) expressing low and high STUB1 from the tissue microarray. G) A proposed model of HSP90β/STUB1 in the regulation of YTHDF2 in HCC. *p < 0.05, ** p < 0.01, **** p < 0.0001.

Fig 5: Inhibition of HSP90β sensitizes HCC cells to the treatment of sorafenib. A) Cell viability analyses were performed using MTS assay in HepG2 and Hep3B cells exposed to NVP for 24 and 48 h. B) HepG2 and Hep3B cells were treated with NVP or vehicle for 24 h. Plate colony formation assay was performed post‐treatment. Images were shown on the left, while the quantitative data were shown on the right. C) Cell viability analyses were performed using MTS assay in HepG2 and Hep3B cells exposed to sorafenib with or without NVP for 24 h. D) HepG2 and Hep3B cells were treated with sorafenib with or without NVP for 24 h. Plate colony formation assay was performed post‐treatment. Images were shown on the upper side, while the quantitative data were shown on the lower side. E) Annexin V‐FITC/PI staining assays were performed in HepG2 cells treated with sorafenib with or without NVP for 24 h. Green indicates FITC positive, and red indicates PI positive. Scale bar, 100 µm. Quantification was shown below the images. F) HepG2 xenografts were established and grown in BALB/c nude mice. The mice were divided into four groups and treated with NVP (i.p. 25 mg kg−1/2 days), sorafenib (p.o. 20 mg kg−1/2 days), the combination of NVP and sorafenib, or vehicle for 3 weeks. Images of the xenografts. G) Tumor volume was recorded every 3 days. The curves of tumor volume. H) Tumor weight and I) body weight of mice. J) Cell viability analyses were performed in HepG2 and Hep3B cells exposed to sorafenib treated with two pairs of YTHDF2/HSP90β siRNAs or control siRNA for 48 h. K) Co‐IP assay was performed using YTHDF2 antibodies in lysates from HepG2 cells exposed to NVP for 24 h, subjected to the immunoblotting for YTHDF2 and STUB1. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.