Fig 1: Cephalomannine pharmacologically inhibits UBE2S and blocks the lymphatic metastasis of BCa cells.A UBE2S protein levels in BCa cells treated with various concentrations of cephalomannine for 48 h, as determined by western blot assays. Representative images and quantitative analyses of transwell migration (B) and invasion (C) assays of BCa cells treated with cephalomannine at the indicated concentrations. D Schematic illustration of the intraperitoneal injection of PBS or cephalomannine in a UM-UC-3 popliteal lymphatic metastasis model. Representative bioluminescence images (E) and quantitative analysis (F) of popliteal metastatic LNs from nude mice. LNs lymph nodes. Representative images of popliteal LNs (G) and quantitative analysis of their volumes (H). I Representative images of H&E staining in popliteal LNs from each group. Scale bars: black, 500 μm; blue, 50 μm. J Percentages of lymphatic metastasis in each group. K Representative images of IHC staining showing UBE2S, LPP, N-cadherin, Vimentin, E-cadherin and Ki-67 expression in footpad tumors of the indicated groups. Scale bars: black, 50 μm. L Representative images of H&E staining in the major organs treated with the indicated concentrations of cephalomannine. Scale bars: black, 50 μm. **P < 0.01.
Fig 2: LPP reverses the metastasis-promoting effects of UBE2S.A Representative images of UBE2S and LPP immunohistochemistry staining in BCa tissues. Scale bars: black, 200 μm; red, 50 μm. B Pearson’s correlation analysis of UBE2S and LPP protein expression in Cohort 2, which consisted of 59 paired BCa tissues. C LPP protein levels in Cohort 2. NAT normal adjacent tissues; LN− CA, BCa tissues without lymphatic metastasis; LN+ CA, BCa tissues with lymphatic metastasis. D Relative LPP mRNA expression in NAT and BCa tissues from the TCGA database. Kaplan‒Meier curves showing the overall survival (E) and disease-free survival (F) of BCa patients with high LPP expression and low LPP expression in cohort 2. G LPP protein levels in LPP-silenced BCa cells, as detected by western blot assays. Representative images and quantitative analysis of wound healing (H), transwell migration (I) and invasion (J) assays using LPP-silenced BCa cells. Representative images and quantitative analyses of wound healing (K), transwell migration (L) and invasion (M) assays of UBE2S-silenced BCa cells combined with LPP knockdown. *P < 0.05 and **P < 0.01.
Fig 3: UBE2S promotes EMT in BCa cells in an LPP-dependent manner.Protein levels of EMT markers (N-cadherin, Vimentin and E-cadherin) in UBE2S-silenced and UBE2S-overexpressed (A) or LPP-silenced (B) BCa cells, as detected by western blot assays. C Representative images of immunofluorescence staining showing N-cadherin expression in UBE2S-silenced and UBE2S-overexpressing T24 cells. Scale bars: white, 20 μm. DAPI was used to label the cell nucleus. D Representative images of immunohistochemistry staining showing UBE2S, LPP, N-cadherin, Vimentin, E-cadherin and Ki-67 expression in footpad tumors. Scale bars: black, 50 μm. E The protein levels of EMT markers and LPP in UBE2S-silenced BCa cells combined with LPP knockdown, as detected using western blot assays.
Fig 4: UBE2S interacts with TRIM21 to degrade LPP by inducing K11-linked ubiquitination.A LPP protein levels in different groups of BCa cells after treatment with MG132 (10 μM, 12 h) or DMSO. The total (B, C) and K11-linked (D, E) ubiquitination levels of LPP in UBE2S-knockdown or TRIM21-knockdown HEK293T cells transfected with corresponding plasmids (48 h) and treated with MG132 (10 μM, 12 h). WT wild type. The total (F) and K11-linked (G) ubiquitination levels of LPP in UBE2S- and TRIM21-overexpressing HEK293T cells transfected with corresponding plasmids (48 h) and treated with MG132 (10 μM, 12 h). H An in vitro ubiquitination assay was performed in the presence of LPP, ubiquitin (Ub), Ub-K11R, UBE1, UBE2S and TRIM21. The reaction was analyzed by western blotting with an anti-LPP antibody (25045-1-AP, Proteintech). I Schematic diagram of TRIM21 and its truncated mutant. The total (J) and K11-linked (K) ubiquitination levels of LPP in full-length TRIM21 and TRIM21-ΔRING HEK293T cells transfected with the corresponding plasmids (48 h) and treated with MG132 (10 μM, 12 h).
Fig 5: UBE2S interacts with TRIM21 and LPP in both the nucleus and cytoplasm of BCa.A The endogenous interaction among UBE2S, TRIM21 and LPP in BCa cells was determined by performing co-IP and western blot assays. Anti-UBE2S (14115-1-AP, Proteintech), anti-TRIM21 (12108-1-AP, Proteintech) and anti-LPP (25045-1-AP, Proteintech) were used for Co-IP (each 2 μg). B Representative immunofluorescence images of UBE2S, TRIM21 and LPP colocalization in the nucleus and cytoplasm of BCa cells. Scale bars: white, 25 μm. DAPI was used to label the cell nucleus. C UBE2S, TRIM21 and LPP protein levels in UBE2S-silenced or UBE2S-overexpressing BCa cells. D TRIM21 and LPP protein levels in TRIM21-silenced BCa cells.
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