Fig 2: USP39 promotes chemoresistance in ESCC cells. (A) Cell apoptosis detected by the Annexin V-PI assay in USP39-overexpressed KYSE30 and control cells with or without DDP (14 µg/mL) treatment. n = 3. (B) Cell apoptosis detected by the Annexin V-PI assay after USP39 knockdown in KYSE450 cells with or without DDP (7 µg/mL) treatment. n = 3. (C) Representative immunoblotting analysis of the cleaved PARP and Caspase3 expression in USP39-overexpressed KYSE30 cells with DDP treatment. The blot was analyzed by Image J. Relative protein levels were normalized to ß-actin. Data in A, B, and C represent mean ± S.D and were analyzed by unpaired two-tailed Student’s t-test. ns = no significant, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Fig 3: USP39 is highly expressed in human glioma. (A) USP39 copy number in Oncomine database samples grouped by different glioma types. The data are means ± SEM. Significance calculated using Student’s t-test. ***P < 0.001. (B) Kaplan–Meier curve showing the 17-year survival rate of TCGA samples classified by low-expressed (n = 338) or high-expressed (n = 338) USP39. The P-value was calculated using the log-rank test. (C) Representative images of IHC staining in human glioma TMA with USP39 antibody. Scale bar: 100 µm. (D) Graphical representation of the H-score of USP39 in different glioma types from TMA (Normal sample, n = 4; Astrocytoma, n = 7; GBM, n = 21; Oligodendroglioma, n = 12; Metastatic glioma, n = 23). The data are means ± SEM. Significance calculated using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Knockdown expression of USP39 inhibits the growth and invasion of U251 cells in vivo. The U251 cells expressing USP39 shRNA or negative control shRNA were intracranially implanted into BALB/c nude mice. (A, B) Hematoxylin-eosin (HE) staining of orthotopic xenografts derived from the indicated U251-shUSP39 cells and control in nude mice (n = 3/group). Representative images are shown. Scale bar: 2000, 20 or 100 µm (as indicated in the picture). (C) Kaplan–Meier analysis of survival for tumor-bearing mice implanted with U251-shUSP39 cells and control (n = 6/group). Log-rank test: P < 0.001. (D) Western blotting analysis of USP39, ADAM9 and integrin ß1 protein levels in tumor tissues. (E) IHC staining of USP39, ADAM9 and integrin ß1 levels in xenograft sections from U251-shNC and U251-shUSP39 groups. Representative images are shown. Scale bar: 20 or 100 µm (as indicated in the picture). Representative data are from three independent experiments.

Fig 5: USP39 interacts with several spliceosome components. (A) Overexpression efficiency of USP39 in KYSE30 cells was determined by Western blot. (B) The USP39 stable expression clone was established. Proteins that interacted with USP39 were purified from KYSE30 cells expressing Flag-tagged USP39 or vector control. (C) GO enrichment analysis of proteins pulled down by Flag antibody from KYSE30 cells expressing Flag-tagged USP39. (D) KEGG pathway analysis of proteins pulled down by Flag antibody from KYSE30 cells expressing Flag-tagged USP39. (E) The interaction between USP39 and core spliceosome factors was validated by Co-IP in KYSE30 cells.