Fig. 2: Dysregulated USP21 accelerates ESCC progression through its deubiquitinase activity in vitro and in vivo.

A KYSE-150 cells were transfected with si-NC, si-USP21#1, or si-USP21#2. Western blot analysis was used to detect the protein levels of USP21, PCNA, E-Cadherin, and N-Cadherin. Plate colony formation assays (B) were applied to assess the proliferation ability of KYSE-150 cells transfected with si-NC, si-USP21#1, or si-USP21#2, while transwell assays (C) were used to evaluate cell migration and invasion capability. Representative staining and quantification as indicated. D Western blot analysis for verifying USP21 protein levels in stable KYSE-150 cells with sh-USP21 or sh-NC expression. The image of xenografts (E), tumor volume curve (F), and quantification for tumor weights (G) from BALB/c-nude mice (n = 7) subcutaneously xenografted with KYSE-150 cells expressing sh-USP21 or sh-NC for 19 days. H, I KYSE-150 cells with sh-USP21 or sh-NC expression were injected into the tail vein of BALB/c-nude mice (n = 4) for 6 weeks. Representative images (H) and statistical analysis (I) of metastatic nodules in mice lungs. J Western blot analysis was applied to determine the indicated protein levels in KYSE-150 cells with Vector, Flag-USP21^WT, or Flag-USP21^C221A expression. The Vector-, USP21^WT-, or USP21^C221A-expressing KYSE-150 cells growth, migration, and invasion were evaluated by plate colony formation (K) and transwell assays (L). Representative images and quantification as shown. M–O KYSE-150 cells with Vector-, USP21^WT-, or USP21^C221A-expression were subcutaneously xenografted into BALB/c-nude mice (n = 6) for 16 days. The representative xenograft image (M), volume curve for tumor growth (N), and statistical analysis for tumor weights (O) as indicated. P, Q The Vector-, USP21^WT-, or USP21^C221A-expressing KYSE-150 cells were injected into BALB/c-nude mice (n = 5) through a tail vein for 6 weeks. Representative images (P) and statistical analysis (Q) for pulmonary metastatic nodules as shown. Scale bars (red line) in (C) and (L) are 100 μm. The data in (B, C, F, G, I, K, L, N, O, Q) are presented as means ± SD. An unpaired t-test was performed to determine the statistical significance in (B, C, G, I, K, L, O, Q) while a two-way ANOVA was used for (F, N). The P values are displayed in the corresponding panels, respectively.