Fig. 1

UCHL1 stabilizes KLF5 via deubiquitination in TNBC cells. (A) Predicted interactions between UCHL1 and its potential deubiquitinase-substrates, using UbiBrowser 2.0. (B) Immunofluorescence staining was used to detect the subcellular location of UCHL1 and KLF5. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the DNA. Scale bar, 10 µM. (C) Co-immunoprecipitation was performed to detect endogenous interactions between UCHL1 and KLF5 proteins in MDA-MB-468 and SUM149 cells. Endogenous proteins were immunoprecipitated with the rabbit anti-KLF5 or anti-UCHL1 antibody. Rabbit IgG serves as the negative control. The presence of immunoprecipitated proteins were detected using mouse anti-UCHL1 or anti-KLF5 antibody respectively. The experiment was repeated three times, and a representative result is shown. (D) Mapping the KLF5 domain that interacts with UCHL1. FLAG-tagged full-length or mutants of KLF5 (a schematic diagram is shown below the panel) and Myc-UCHL1 were co-transfected into MDA-MB-468 cells. Immunoprecipitation was performed with FLAG-M2 beads. The experiment was repeated three times, and a representative result is shown. (E) The expression of UCHL1 and KLF5 protein in MDA-MB-468 and SUM149 cells with UCHL1 knockdown, with or without the presence of MG132 (10 µM for 4 h). F-G. UCHL1 positively modulates the half-life of KLF5. MDA-MB-468 or SUM149 cells were infected for UCHL1 knockdown (F, top two panels) or overexpression (F, bottom two panels) and treated with cycloheximide (CHX) for the indicated time points. Western blotting was used to measure the endogenous KLF5 protein level. G. The graphs showing the quantitative results of KLF5 levels in panel F by ImageJ. Error bars represent S.D. The experiment was repeated three times, and a representative result is shown. *, comparison between shNC and shUCHL1#1 or between vector and UCHL1; #, comparison between shNC and shUCHL1#2. H. UCHL1 modulates endogenous KLF5 protein ubiquitination in TNBC cells. MDA-MB-468 (left) and SUM149 (right) cells were infected for UCHL1 knockdown or overexpression (wild-type or C90S) for 48 h. Scramble shRNA or vector were used as the negative control. After the cells were treated with MG132 (10 µM for 4 h), cells were lysed for immunoprecipitation using anti-KLF5. Immunoblotting was used to detect the ubiquitinated KLF5 protein levels. The experiment was repeated three times, and a representative result is shown. IP, immunoprecipitation; IB, immunoblotting. DSI: deubiquitinase-substrate interactions. LCR: low complexity region. ** and ^##, p < 0.01; *** and ^###, p < 0.001