Fig. 6: Inhibition of ubiquitination-associated proteins leads to reduced cell viability and transformation of TFG-RET expressing cells.

a HUWE1 was transiently knocked down using siRNA in Nthy-TFG-RET cells. The cells were seeded in 6-well and 96-well plates and subjected to EdU, cell counting and MTT assay after 48 and 72 h, respectively. Knockdown of HUWE1 resulted in a significant reduction of cell viability (MTT), proliferation (EdU) and cell number (cell counting). Error bars represent ± SEM (n = 4). Paired t-test, two-tailed, p-values: *<0.05, **<0.01 and ***<0.0001. For FACS analysis, the gating strategy is depicted in Supplementary Fig. 3A. For MTT assays, the percentages of viable cells are shown. For EdU assays, the percentages of cells in S phase are depicted. In the cell-counting experiments, the absolute viable cell numbers are shown. b HUWE1 was transiently knocked down using siRNA in Nthy-TFG-RET cells and cultured in soft agar for 2 weeks followed by staining with crystal violet. HUWE1 knockdown resulted in a significant reduction in the number of colonies. Error bars represent ± SEM. Three independent experiments with technical duplicates were performed (n = 3). Paired t-test, two-tailed, p-values: *<0.05, ***<0.0001. c Tyrosine kinase, DUB and HUWE1 inhibitors reduce oncogenic growth in TFG-RET expressing cells—soft agar colony formation assay. Nthy-ori 3-1 cells stably expressing TFG-RET were treated with inhibitors with indicated concentrations. Error bars represent ± SEM (n = 3). Paired t-test, two-tailed, p-value: *<0.05, **<0.01, ***<0.0001, ns not significant. In b and c the fold changes in the number of viable colonies from the analyzed samples are presented.