Research Paper Volume 16, Issue 19 pp 12850—12865

Figure 6. USP4 enhances PTC proliferation by stabilizing LDHA. (A, B) Western blot analysis to detect LDHA following USP4 knockdown in B-CPAP and TPC-1 cells or USP4 overexpression in K1 and KTC-1 cells; (C, D) B-CPAP cells were lysed with RIPA buffer. The lysates were immunoprecipitated with anti-IgG, anti-USP4, or anti-LDHA antibodies, followed by Western blot analysis with the specified antibodies; (E) USP4-knockdown B-CPAP cells were treated with MG132 (10 μM) for 4 hours, followed by Western blot analysis using the specified antibodies to detect proteins; (F, G) ATP/ADP ratio assay revealed a reduction in the ATP/ADP ratio in B-CPAP and TPC-1 cells following USP4 knockdown; (H) Western blot assays were conducted to evaluate changes in ERK, phosphorylated ERK, AKT, and phosphorylated AKT levels in B-CPAP and TPC-1 cells following USP4 knockdown. A similar analysis was performed on B-CPAP and TPC-1 cells with USP4 knockdown subjected to ATP treatment (1 mM); (I, J) CCK-8 assay was utilized to evaluate the proliferative capacity of B-CPAP and TPC-1 cells following USP4 knockdown and subsequent ATP treatment; (K, L) Colony formation assays were conducted to investigate the proliferation potential of B-CPAP and TPC-1 cells following USP4 knockdown and subsequent ATP treatment (1 mM). All ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.