Role of deubiquitinase JOSD2 in the pathogenesis of esophageal squamous cell carcinoma

Figure 1 Expression level, survival analysis, and enrichment analyses of JOSD2 in esophageal squamous cell carcinoma tissues. A: Boxplots of JOSD2 expression in esophageal squamous cell carcinoma (ESCC) tissues and normal esophageal tissues based on University of Alabama at Birmingham CANcer database; B: Kaplan-Meier plot showing the survival difference between ESCC patients with high vs low JOSD2 expression based on the Kaplan-Meier Plotter database; C: Nomogram of JOSD2 expression predicting 1-, 3- and 5-year survival probability of ESCC patients; D-F: Gene Ontology (D), Kyoto Encyclopedia of Genes and Genomes (E) and Gene Set Enrichment Analysis (F) enrichment analyses of JOSD2. ^bP < 0.001; ^cP < 0.0001.

Figure 2 Single-cell sequencing data of esophageal squamous cell carcinoma in GSE160269. A: Uniform manifold approximation and projection plots showing the grouping of different cell types (left) and the expression profile of JOSD2 (right) in different cell types; B: Violin plots showing JOSD2 expression in different cell types.

Figure 3 JOSD2 expression in esophageal squamous cell carcinoma cell lines. Real-time fluorescence quantitative polymerase chain reaction and western blotting results showing JOSD2 mRNA (A) and protein (B) expression in esophageal squamous cell carcinoma cell lines and a normal esophageal epithelial cell line. A: JOSD2 mRNA; B: Protein.

Figure 4 JOSD2 knockdown significantly suppresses the activity of esophageal squamous cell carcinoma cells. A: JOSD2 was successfully knocked down using shRNA1/2 directed against JOSD2 in KYSE150 cells; B: JOSD2 knockdown significantly inhibited cell proliferation; C: JOSD2 knockdown significantly inhibited the ability of the cells to form colonies. ^cP < 0.0001.

Figure 5 JOSD2 contributes to the proliferation, drug resistance, and metastatic capability of esophageal squamous cell carcinoma cells. A: JOSD2-knockdown KYSE150 cells exhibited increased sensitivity to 48-h treatment with various concentrations of cisplatin; B: JOSD2-knockdown KYSE150 cells exhibited significantly decreased migration and invasion in Transwell migration and invasion assays; C: JOSD2-overexpressing KYSE30 cells exhibited significantly increased cell growth in cell proliferation assays; D: JOSD2-overexpressing KYSE30 cells exhibited increased resistance to 48-h treatment with various concentrations of cisplatin; E: JOSD2-overexpressing KYSE30 cells exhibited significantly increased migration and invasion in Transwell migration and invasion assays. ^aP < 0.01; ^bP < 0.001; ^cP < 0.0001.

Figure 6 JOSD2 promotes in vivo esophageal squamous cell carcinoma cell proliferation and cisplatin resistance. A and B: JOSD2-knockdown cisplatin-treated KYSE150 cells exhibited significantly decreased tumor growth in both volume (A) and weight (B) compared to cisplatin-treated control cells; C and D: JOSD2-overexpressing cisplatin-treated KYSE30 cells exhibited significantly increased tumor growth in both volume (C) and weight (D) compared to cisplatin-treated control cells. ^bP < 0.001; ^cP < 0.0001.

Figure 7 JOSD2 enhances the activation of phosphorylation pathways in esophageal squamous cell carcinoma. A: Western blotting showing that JOSD2 knockdown in esophageal squamous cell carcinoma (ESCC) cells decreased the phosphorylation of ERK1/2 and AKT; B: Western blotting showing that JOSD2 overexpression in ESCC cells activated the MAPK/ERK and PI3K/AKT signaling pathways.

Figure 8 Mass spectrometry analysis of proteins that bind to JOSD2 protein. A: Protein bands after SDS-PAGE gel silver staining; B: Proteins that potentially interact with JOSD2.