Fig 1: UCHL1 and cyclin B1 colocalize and interact in vitro. (A) Co-immunoprecipitation of ARK1 protein lysate was performed using antibodies against UCHL1 and cyclin B1, with normal mouse IgG as a control. (B) Immunofluorescence staining and the Duolink proximity ligation assay was performed on ARK1 cells. Following the Duolink assay, red fluorescent dots indicate presence of protein-protein interaction. As controls, ARK1 cells were also stained without primary antibody, with anti-UCHL1 antibody plus control IgG, and with anti-cyclin B1 antibody plus control IgG. Representative experiment (3 biological replicates). Scale bar, 50 µm. Results in the dot plot show the number of red signals per cell in three independent experiments. Statistical significance was determined by the Kruskal-Wallis test. Error bars represent mean ± SD (p < 0.0001). The uncropped blots and molecular weight markers are shown in Supplementary Materials.
Fig 2: UCHL1 is upregulated in USC and correlates with poorer overall survival. (A–D) Analysis of the endometrial cancer TCGA data set. (A) Heat map of the genes differentially expressed between USC and low grade EEC, with a significant association with overall survival in USC patients. (B) UCHL1 RNA expression across histological subtypes of endometrial cancer (Normal, n = 21; EEC, n = 348; Mixed EEC/USC, n = 15; USC, n = 91). (C) UCHL1 RNA expression across EEC grades (G1, n = 87; G2, n = 100; G3, n = 161). Statistical significance for (B,C) was determined by the Kruskal-Wallis test. Error bars represent median with interquartile range. * p < 0.05, *** p < 0.001. (D) Kaplan-Meier analysis of UCHL1 expression and overall survival in USC patients (n = 91). Statistical significance was determined by the log-rank test (p = 0.006). (E,F) Validation of UCHL1 upregulation in an independent cohort of paraffin-embedded tumor samples. (E) Representative slides for UCHL1 staining in various gynecological tissues. Scale bar, 100 µm. (F) UCHL1 staining intensity across tissue types (Normal endometrium, n = 11; EEC, n = 34; Mixed EEC/USC, n = 27; Pure USC, n = 53). Statistical significance was determined by the Kruskal-Wallis test. Error bars represent median with interquartile range. *** p < 0.001.
Fig 3: SARS-CoV-2 infection-derived factors promote apoptotic cell death of undifferentiated spermatogonia.(A) Representative TUNEL staining in STC exposed to supernatant from mock (HAE Sup-Mock) and infected HAE cells (HAE Sup-Inf), and to control and COVID-19 plasma for 24 hrs. The green fluorescence depicts TUNEL+ cells. (B) Quantification of percent TUNEL positive cells and mean fluorescence intensity in each group. Data represents the average of at least six fields per coverslip from 3 independent experiments captured using Image J. Error bars represent ±SEM. (C) STC exposed to UV-inactivated HAE supernatant and COVID-19 plasma were co-stained with TUNEL and UCHL1, a marker for undifferentiated spermatogonia including spermatogonia stem cells (SSC). Co-localization was evaluated by merging TUNEL and UCHL1 and white arrows indicate overlapping green and red staining (yellow). ***p<0.001; ****p<0.0001
Fig 4: Importance of the deubiquitinating activity of UCHL1 in the upregulation of HIF-1 activity in murine breast cancer EMT6 cells. (a–h) EMT6 cells were transfected with either pcDNA4/UCHL1 (UCHL1) or its empty vector (EV), cultured under the indicated oxygen conditions, and subjected to both the luciferase assay using a co-transfected reporter plasmid, p5HRE-luc (a,g), pGL3/HIF-1a-5'UTR-Luc (b), or pGL3/ODD-luc (d,h), and Western blotting using the indicated antibodies (a–h). The relative intensity of the bands in Fig. 1e (HIF-1a vs. ß-actin) was quantified using ImageJ (f). (i) Cells transiently transfected with both pMT132 and either pcDNA4/myc-His A (EV) or pcDNA4A/HIF-1a-myc were additionally transfected with pcDNA4/myc-His A (EV) or pcDNA4/UCHL1 (UCHL1), and cultured with MG132 (10 µM) for 6 h. Ubiquitinated HIF-1a was immunoprecipitated using an anti-myc antibody and detected with an anti-HA antibody (left). One-twentieth of the whole cell lysate was subjected to Western blotting with the indicated antibodies as input controls (right). (j) EMT6 cells were cotransfected with four kinds of plasmids: pG5H1bLuc containing five Gal4-binding sites upstream of an adenovirus E1b promoter and firefly luciferase CDS33, 50, pRL-SV40 as an internal control for normalization, pcDNA6/Gal4 DBD-HIF-1aTAD P564A, and either pcDNA4/UCHL1 (UCHL1) or its EV. Cells were cultured under normoxic or hypoxic conditions and subjected to the luciferase assay to evaluate the transactivation activity of HIF-1a TADs. All results (except for c and e) are presented as means ± s.d. n = 3. *P < 0.05. **P < 0.01. NS, not significant (Student’s t-test).
Fig 5: UCHL1 silencing reduces USC cell proliferation in vitro. (A) qRT-PCR quantification of UCHL1 RNA expression in endometrial cancer cell lines (Type I, n = 8; Type II, n = 5). (B) Western blot analysis of UCHL1 protein expression in endometrial cancer cell lines (Type I, n = 8; Type II, n = 5). (C,D) Cell proliferation of ARK1 and ARK2 cells after UCHL1 knockdown (C), and of ARK2 and HEC-50 cells stably overexpressing UCHL1 (D), as measured by the MTT assay. Statistical significance was determined by the T-Test (two-tailed, equal variance). Error bars represent mean ± SD (3 biological replicates). * p < 0.05, ** p < 0.01, *** p < 0.001. (E,F) Luciferase-labelled ARK1 cells transduced with doxycycline-inducible control shRNA (n = 8) or anti-UCHL1 shRNA (n = 9) were injected intraperitoneally into nude mice; the mice were then switched to a doxycycline diet the following day. (E) Representative images of in vivo bioluminescence imaging at week 10. (F) Fold change increase in in vivo bioluminescence of intraperitoneal tumors. Statistical significance was determined by the Mann-Whitney test. Error bars represent mean ± SD. * p < 0.05. (G–I) Luciferase-labelled ARK1 cells were injected intraperitoneally into nude mice; after 2 weeks, mice were treated thrice weekly by intraperitoneal injection of LDN-57444 (n = 21) or PBS control (n = 23). (G) Representative images of in vivo bioluminescence imaging at week 6. (H) Fold change increase in in vivo bioluminescence of intraperitoneal tumors. Statistical significance was determined by the Mann-Whitney test. Error bars represent mean ± SD. * p < 0.05. (I) Kaplan-Meier survival curves of the LDN-57444 treatment group (n = 21) and control group (n = 23). Statistical significance was determined by the log-rank test (p = 0.032). The uncropped blots and molecular weight markers are shown in Supplementary Materials.
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