Fig 2: USP39 shRNA knockdown decreases xenograft growth of ESCC cells. (A) Representative photographs of tumor xenografts removed from nude mice at 28 days post-injection with ECA109 cells transduced with sh-control or sh-USP39-1. (B, C) Caliper-based measurements of tumor size measured at the indicated intervals (B) and final tumor weights (C). The results are shown as mean ± S.D. of three independent experiments. Student’s t-test: *** p < 0.001.

Fig 3: Ectopic expression of USP39 and TAZ restores malignant properties of glioma cells with knockdown of USP39 in vitro and in vivo. a Western blotting analysis of lysates prepared from modified U87MG and P3 cells. GAPDH was used for normalization. b OD from CCK8 assays plotted as a function of time in hours for indicated cells to evaluate cell viability. c Graphic representation of relative luciferase activity from luciferase reporter constructs regulated by Hippo signaling in U87MG cells from indicated groups. d qRT-PCR analysis of the expression of TAZ target genes from indicated cells. GAPDH was used for normalization. e, f Representative images of luciferase bioluminescence at the indicated days after injection of U87MG and P3 cells into the brains of nude mice. Graphic representation of the total flux at the indicated days for the indicated cell types. Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001

Fig 4: Knockdown of USP39 decreases proliferation, invasion, and migration of glioma cells in vivo. a Representative images of luciferase bioluminescence at the indicated days after injection of luciferase-expressing U87MG and P3 cells into the brains of nude mice. b Graphic representation of the total flux at the indicated days for the indicated cell types. c Representative images of HE staining of orthotopic xenografts derived from the indicated cell types, U87MG- and P3-sh-USP39-1 cells and controls. Scale bars, 1000 and 200 µm. d Kaplan–Meier analysis of survival for tumor bearing mice implanted with U87MG- and P3-sh-USP39-1 cells and controls. Student’s t-test: n.s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Log-rank test: p < 0.01

Fig 5: USP39 is involved in Rictor pre-mRNA splicing and maturation. (A) Schematic illustrating the qPCR primer pairs used to measure pre-mRNA and mature mRNA species based on the Rictor gene sequence in the Ensembl database. (B, C) qRT-PCR analyses showing relative mRNA levels of spliced and unspliced Rictor RNA transcripts in ECA109 and KYSE30 cells with or without USP39 shRNA knockdown (B). Splicing efficiency is expressed as the ratio between mature/pre-mRNA levels (C). GAPDH was used for normalization. (D) RNA immunoprecipitations (RIP) performed using antibodies against USP39 or an IgG control. qRT-PCR were used to measure relative Rictor mRNA levels recovered in the immunoprecipitated complex. The results are shown as mean ± S.D. of three independent experiments. Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.001.