Fig 2: Sal-miR-1 and 3 abrogate OTUD7B upregulation by thrombin via targeting the OTUD7B 3'UTR. (A-B) VSMCs were treated with the different doses of thrombin for 24 h. OTUD7B expression was examined by Western blotting (A) and qRT-PCR (B). (C-D) VSMCs were treated with 1 U/mL thrombin for the indicated times. Western blotting (C) and qRT-PCR (D) detected OTUD7B expression. (E) Immunofluorescence staining detected OTUD7B expression in VSMCs transfected with Sal-miR-1+3 and then treated or not with 1 U/mL thrombin for 24 h. Red and blue staining indicates OTUD7B and the nuclei, respectively. Scale bar=25 µm. (F) VSMCs were transfected with Sal-miR-1+3 and then treated or not with 1 U/mL thrombin for 24 h. The expression of OTUD7B, KLF4, VCAM-1, SM a-actin, and NMHC IIA was determined by Western blotting. (G) The carotid arteries injured by ligation were in situ-transfected with Sal-miR-1+3. The expression of OTUD7B, KLF4, VCAM-1, SM a-actin, and NMHC IIA was determined by Western blotting. (H) Immunofluorescent staining for SM a-actin and OTUD7B was performed on sections from the carotid arteries transfected or not with Sal-miR-1+3. Red, green and blue staining indicates SM a-actin, OTUD7B and the nuclei, respectively. Scale bars=25 µm. (I-K) Luciferase reporter assays were performed in HEK293A cells transfected with OTUD7B 3'UTR reporter vector containing the wild-type or mutated Sal-miR-1, Sal-miR-3 or Sal-miR-1+3 binding site in the presence or absence of Sal-miR-1, Sal-miR-3 or Sal-miR-1+3. The pmirGLO vector was used as negative control. The Sal-miR-1 (I), Sal-miR-3 (J) and Sal-miR-1+3 (K) binding sites in the 3'UTR of the mouse OTUD7B mRNA are showed in red; the mutated sites are shown in green. (L) Representative hematoxylin and eosin staining of cross-sections from unligated and ligated carotid arteries transfected with miR-Ctl+si-Ctl (n=5), miR-Ctl+si-OTUD7B (n=5), si-Ctl+Sal-miR-1+3 (n=5), or Sal-miR-1+3+si-OTUD7B (n=5). Scale bars=100 µm. (M) VSMCs were transfected with si-OTUD7B, Sal-miR-1+3 or Sal-miR-1+3+si-OTUD7B and then treated or not with 1 U/mL thrombin for 24 h. The expression of SMMHC, ICAM-1, VCAM-1, SM a-actin, calponin and SM22a was determined by Western blotting. (N) VSMCs were treated as in (M). The cells were fixed, stained for F-actin with TRITC-phalloidin and with DAPI to visualize nuclei, and imaged with a laser scanning confocal microscope. Scale bars=7.5 µm. (O) VSMCs were treated as in (M), a scratch wound assay was performed and photomicrographed. Relative migration rate was measured by Image J. n=5 fields, Scale bars=200 µm. (P) Macrophage adhesion to VSMCs treated as in (M) was evaluated by staining macrophage marker with anti-MAC-2. Scale bars=10 µm. The numbers of macrophages adhered to VSMCs per field were measured by Image J. n=5 fields. (Q) VSMCs were transfected with Sal-miR-1+3 and/or pcDNA3.1-OTUD7B for 24 h. A scratch wound assay was performed and photomicrographed. Relative migration rate was measured by Image J. n=5 fields. All experiments were performed for three independent experiments. Student's t-test, one-way ANOVA test or repeated Measures ANOVA: *P<0.05, **P<0.01, ***P<0.001 versus the corresponding control.

Fig 3: OTUD7B promotes metastasis via deubiquitinating LSD1. A) Parental or C) LSD1 reconstituting LM2 cells infected with the indicated lentiviruses were injected into the mammary fat pads of nude mice. Representative bioluminescent images of mice with spontaneous lung metastasis (left panel), quantification (right panel), and (B) representative H&E staining analysis of lung metastasis is shown. Results represent mean ± SEM. n = 8 mice per group. Scale bars: 40 µm. ***P < 0.001. P values were calculated by two-tailed unpaired t test. D) Representative IHC staining of LSD1 and OTUD7B in normal breast tissues (n = 26) and breast carcinomas (n = 379) (histological I, II, and III). Scale bars: 100 µm for low magnification (10×) and 25 µm for high magnification (40×). E) Quantification of LSD1 and OTUD7B intensity in (D). P values were calculated by two-tailed unpaired t test. * P < 0.05, ** P < 0.01, and *** P < 0.001. F) Representative IHC staining of LSD1 and OTUD7B in normal tissue (n = 22) and the indicated breast carcinoma subtypes (luminal = 76, HER2+ = 49, and basal-like = 130). Scale bars: 100 µm. G) Quantification of OTUD7B and LSD1 intensity in (F). *** P < 0.001. P values were calculated by two-tailed unpaired t test. H) TCGA DNA sequencing results showing that the OTUD7B gene is amplified at higher frequencies in basal-like subtype (27%) compared to other subtypes (19%). I) Kaplan–Meier plots analysis of distant metastasis free survival rates (DMFS) in basal-like breast cancer patient with high or low OTUD7B mRNA levels. Patient number at risk at different times of analysis is shown at the bottom of the plots. J) The working model of OTUD7B-mediated LSD1 deubiquitination in coordinating LSD1 turnover, LSD1/CoREST corepressor complex assembly, and the subsequent impact on cell cycle and cancer metastasis.

Fig 4: OTUD7B is a bona fide LSD1 deubiquitinase. A) IB analysis of WCL and Flag immunoprecipitate derived from HEK293T cells transfected with the indicated constructs and treated with BTZ for 5 h before harvesting. B) HEK293T cells stably expressing OTUD7B sgRNAs were transfected with Myc-tagged Ub, followed by IP and IB analysis as indicated. C) HEK293T cells stably expressing OTUD7B sgRNAs were co-transfected with the indicated Ub constructs, followed by IP and IB analysis as indicated. D) HEK293T cells were co-transfected with Flag-tagged LSD1, HA-tagged ubiquitin, and Myc-tagged OTUD7B. Cell lysates were subjected to IP and mass spectrometry analysis to identify ubiquitination sites. The recovered LSD1 peptide and the ubiquitination sites were highlighted in red (upper panel). Lower panel: A schematic diagram showing two evolutionarily conserved lysine residues (K226 and K277) within the SWIRM domain of LSD1. E) HEK293T cells were co-transfected with the indicated plasmids, followed by IP and IB analysis as indicated. F) LSD1WT- or LSD12KR-reconstituted MDA-MB-231 cells were treated with CHX for the indicated times. Lysates were subjected to IB analysis as indicated (top panel). The graph shows the quantification of protein levels (bottom panel). G) IB analysis of WCL derived from the indicated LSD1 reconstituting cells expressing OTUD7B sgRNA lentiviruses. H) LSD1-reconstituted cells stably expressing the indicated sgRNAs were transfected with K63-only Ub plasmid, followed by IP and IB analysis as indicated. Data were presented as mean ± SEM of three independent experiments.

Fig 5: K63-linked polyubiquitination of LSD1 facilitates its proteasomal degradation via promoting interaction with p62. MDA-MB-231 cells stably expressing A) CoREST shRNA or B) OTUD7B sgRNAs were infected with lentiviruses encoding p62 shRNA, followed by western blotting. C) MDA-MB-231 cells stably expressing CoREST shRNA or OTUD7B sgRNAs were treated with Baf A (200 × 10-9 m) for 6 h followed by IB analysis. D) IP and IB analysis of MDA-MB-231 cells infected with the indicated lentiviral constructs. E) p62 reconstituting MDA-MB-231 cells expressing the indicated lentiviruses were subjected to IB analysis. F) HEK293T cells transfected with the indicated plasmids were collected for IP with anti-Flag antibody, followed by IB analysis as indicated. G) LSD1 reconstituting cells infected with the indicated lentiviral constructs were collected for IP and IB analysis. H) MDA-MB-231 cells stably expressing the indicated lentiviruses were subjected to IP and IB analysis. I) p62 reconstituting cells infected with the indicated lentiviruses were collected for IP and IB analysis. Data were presented as mean ± SEM of three independent experiments.