Fig. 4: DHODH is a substrate of USP24 in TNBC cells.

A, B Cellular proteins in the indicated MDA-MB-231 and MDA-MB-468 cell lines was measured by Western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. C, D Western blotting analysis of DHODH proteins in MDA-MB-231 and MDA-MB-468 cells treated with the indicated doses of WP1130 for 24 h. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001. E, F MDA-MB-231 cells were stably transfected with two individual USP9X shRNAs or control shRNA. USP9X expression in cells was measured by Western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, **P < 0.01. ns no significance. G, H Western blotting analysis of cellular proteins in the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with cycloheximide (CHX, 100 μg/ml for MDA-MB-231 cells; 30 μg/ml for MDA-MB-468 cells) for the indicated time periods. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ns no significance. I, J MDA-MB-231 cells were treated with MG132 (10 μM) or chloroquine (CQ, 30 μM) for the indicated time periods. The protein levels of DHODH were measured by Western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05,, ***P < 0.001. ns no significance. K, L The indicated MDA-MB-231 and MDA-MB-468 cell lines were treated with or without MG132 (10 μM) for 12 h. The expression of DHODH and USP24 proteins was assessed by Western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. ns no significance.