WDR68 is required for normal levels of DYRK1A in C2C12 cells.

A) C2C12 NT1 and Δwdr68-9 sublines in growth medium (GM, lanes 1–2) versus differentiation medium (DM, lanes 3–4). WDR68 panel: Lanes 1 and 3, WDR68 was detected in NT1 cells and induced higher in DM. Lanes 2 and 4, WDR68 was not detected above background in Δwdr68-9 cells. DYRK1A panel: Lanes 1 and 3, DYRK1A was detected in NT1 cells and induced higher in DM. Lanes 2 and 4, reduced DYRK1A expression in Δwdr68-9. β-tubulin panel: β-tubulin controls indicated similar loading in each lane. A’) Quantitative analysis confirmed significantly reduced levels of WDR68 in the Δwdr68-9 subline. Three independent biological replicates were analyzed. A”) Quantitative analysis confirmed significantly reduced DYRK1A expression in the Δwdr68-9 subline. Four independent biological replicates were analyzed. B) C2C12 NT1 and Δdyrk1a sublines in GM (lanes 1–3) versus DM (lanes 4–6). DYRK1A panel: Lanes 1 and 4, DYRK1A was detected in NT1 cells and induced higher in DM. Lanes 2 and 5, reduced DYRK1A level in Δdyrk1a-12 cells. Lanes 3 and 6, reduced DYRK1A level in Δdyrk1a-2 cells. WDR68 panel: Lanes 1 to 6, WDR68 was detected in NT1, Δdyrk1a-12, and Δdyrk1a-2 cells. β-tubulin panel: β-tubulin controls indicated similar loading in each lane. B’) Quantitative analysis confirmed significantly reduced DYRK1A level in the Δdyrk1a sublines. Three independent biological replicates were analyzed. B”) Quantitative analysis revealed significant induction of WDR68 in NT1 cells cultured in DM versus GM, but no significant differences in WDR68 levels between NT1, Δdyrk1a-12, and Δdyrk1a-2 cells in DM. Four independent biological replicates were analyzed. C) C2C12 NT1 and siWdr68-4650-1 cells in DM. WDR68 panel: Lane 1, WDR68 was detected in NT1 cells. Lane 2, reduced WDR68 level in siWdr68-4650-1 cells. DYRK1A panel: Lane 1, DYRK1A was detected in NT1 cells. Lane 2, reduced DYRK1A level in siWdr68-4650-1 cells. β-tubulin panel: β-tubulin controls indicated similar loading in each lane. C’). Quantitative analysis confirmed significantly reduced WDR68 level in the siWdr68-4650-1 subline. Three independent biological replicates were analyzed. C”). Quantitative analysis confirmed significantly reduced DYRK1A in the siWdr68-4650-1 subline. Three independent biological replicates were analyzed. All plots show means +/- standard deviations.