Fig. 2: The UPS inhibitor CBK77 causes global impairment of the UPS.

A Representative confocal images of MelJuSo cells stably expressing the indicated fluorescent UPS reporters. Cells were treated for 16 h with either CBK77 or CBK07 (10 µM) or bortezomib (PI proteasome inhibitor, 25 nM) as a positive control. DMSO at 0.1% was used as negative control. Cells were fixed and nuclei were counterstained with Hoechst. Scale bars = 20 µm. B Cells were treated for 16 h with CBK77 or CBK07 (10 µM for all reporter cell lines besides YFP-CL1, treated at 5 µM). The proteasome inhibitor bortezomib (25 nM) and DMSO 0.1% were included as positive and negative controls, respectively. After nuclei counterstaining with Hoechst, cells were imaged in an automated manner with a widefield fluorescent microscope. Frequency and distribution of the reporter (YFP) intensity per cell are shown as violin plots from a representative experiment (of two independent experiments). Data are normalized to percentages were 0% = minimum value in the DMSO sample; 100% = maximum value in the proteasome inhibitor sample. Black lines within each distribution represent the median; colored lines represent the upper and lower interquartile range limits. Ub-YFP: n = 591, n = 283, n = 514, n = 337; Ub-R-GFP: n = 391, n = 190, n = 201, n = 208; YFP-CL1: n = 666, n = 470, n = 623, n = 397; CD3δ-YFP: n = 717, n = 335, n = 505, n = 447 in DMSO, CBK77, CBK07, and bortezomib-treated cells, respectively. C MelJuSo Ub-YFP cells were treated with either CBK77, CBK07 (10 µM) or epoxomicin (PI = proteasome inhibitor, 100 nM) and harvested at the indicated timepoints. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Representative blots from one out of three independent experiments are shown. D MelJuSo Ub-YFP cells were pretreated for 3 h with the reversible proteasome inhibitor bortezomib (BTZ, 25 nM) to increase the levels of Ub-YFP and p53 before the chase. Samples were taken directly after pretreatment (t = 0). The remaining wells were co-treated with cycloheximide (CHX, 50 µg/ml) and the indicated compounds and harvested after 4 h (CHX 4 h). Cell lysates were analyzed by immunoblotting with the indicated antibodies. Representative blots from one of two independent experiments are shown.