Journal: Cell Communication and Signaling : CCS

Article Title: SUMO3 modification by PIAS1 modulates androgen receptor cellular distribution and stability

doi: 10.1186/s12964-019-0457-9

Figure Lengend Snippet: Subcellular localization of AR in the presence of PIAS and SUMO. ( a/b ) DU145 cells in a 12-well plate were cotransfected with indicated plasmids for 48 h. Cells were then fixed and stained with anti-AR rabbit polyclonal antibody, then PI-conjugated anti-rabbit IgG antibody (red). Nuclei were visualized by DAPI staining. Representative images of transfected cells were acquired using immunofluorescence microscope

Article Snippet: The following primary antibodies were used in this study: SUMO3, Ubiquitin and AR rabbit polyclonal antibodies (Santa Cruz), PIAS1 rabbit polyclonal antibody (Cell signaling), MDM2 rabbit polyclonal antibody (Bioss), Flag tag and myc tag mouse monoclonal antibodies (TransGene Biotech).

Techniques: Staining, Transfection, Immunofluorescence, Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: SUMO3 modification by PIAS1 modulates androgen receptor cellular distribution and stability

doi: 10.1186/s12964-019-0457-9

Figure Lengend Snippet: MDM2 is recruited by SUMO3 modificd PIAS1 and required for degradation of AR. ( a ) DU145 cells were transiently co-transfected with flag-AR, GFP-SUMO3 and myc-PIAS1 or myc-PIAS1 (K117 L) for 72 h. Whole-cell lysates were immunoblotted with anti-AR, anti-myc and anti-actin antibodies. ( b ) DU145 cells were transiently co-transfected as described in A for 48 h. Whole-cell lysates were immunoprepapited with anti-myc antibodies. The immunoprepapite was then detected by indicated antibodies against myc, MDM2 and ChIP. Whole-cell lysates (Input) were also immunoblotted with anti-myc, anti-MDM2, anti-ChIP and anti-actin antibodies. ( c ) DU145 cells were transiently co-transfected with PIAS1, empty vector or GFP-SUMO3 or GFP-SUMO3 and myc-PIAS1. Whole-cell lysates were immunoprepapited with anti-myc antibody. The immunoprepapite was then detected by indicated antibodies against myc, SUMO3, MDM2 and ChIP. Whole-cell lysates (Input) were also immunoblotted with anti-myc, anti-MDM2, anti-ChIP and anti-actin antibodies. ( d ) DU145 cells were transiently co-transfected with flag-AR, myc-PIAS1 and empty vector or GFP-SUMO3 or GFP-SUMO3 and MDM2 shRNA for 72 h. Whole-cell lysates were immunoblotted with anti-AR, anti-myc and anti-actin antibodies

Article Snippet: The following primary antibodies were used in this study: SUMO3, Ubiquitin and AR rabbit polyclonal antibodies (Santa Cruz), PIAS1 rabbit polyclonal antibody (Cell signaling), MDM2 rabbit polyclonal antibody (Bioss), Flag tag and myc tag mouse monoclonal antibodies (TransGene Biotech).

Techniques: Transfection, Plasmid Preparation, shRNA

Journal: Cell Communication and Signaling : CCS

Article Title: SUMO3 modification by PIAS1 modulates androgen receptor cellular distribution and stability

doi: 10.1186/s12964-019-0457-9

Figure Lengend Snippet: MDM2 is not required for SUMO3 modified PIAS1 induced AR nuclear export. DU145 cells were transiently co-transfected with flag-AR, SUMO3, myc-PIAS1 and control shRNA or MDM2 shRNA for 48 h. Cells were then fixed and stained with anti-MDM2 (green) and anti-flag (red) or anti-myc mouse monoclonal antibody (red). Nuclei were visualized by DAPI staining. Representative images of transfected cells were shown

Article Snippet: The following primary antibodies were used in this study: SUMO3, Ubiquitin and AR rabbit polyclonal antibodies (Santa Cruz), PIAS1 rabbit polyclonal antibody (Cell signaling), MDM2 rabbit polyclonal antibody (Bioss), Flag tag and myc tag mouse monoclonal antibodies (TransGene Biotech).

Techniques: Modification, Transfection, shRNA, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: SUMO3 modification by PIAS1 modulates androgen receptor cellular distribution and stability

doi: 10.1186/s12964-019-0457-9

Figure Lengend Snippet: Model for the regulation of AR subcellular localization and turnover by sumoylation and ubiquitination systems. In castration-resistant prostate cancer cells, the binding of androgen contained in the serum makes AR to be released from the cytoplasmic associated heat shock proteins (HSP) and translocate to the nucleus; likewise, the overexpressed PIAS1 and SUMO3 are also gathered in nucleus. SUMO3 can be conjugated to the 117th lysine of PIAS1 which is a SUMO E3 ligase itself ( a ), and then the sumoylated PIAS1 recruit the MDM2 protein( b ) and also interact with AR through its 386th and 845th lysines, which may block the AR dimer formation ( c ), further resulting in the nuclear export of AR and its binding partners. The MDM2 cooperating with ubiquitin E1 and E2 promotes the polyubiquitination of AR and its subsequent proteasome-mediated degradation. The SUMO3 modification of partial AR is also accompanied in this process ( d )

Article Snippet: The following primary antibodies were used in this study: SUMO3, Ubiquitin and AR rabbit polyclonal antibodies (Santa Cruz), PIAS1 rabbit polyclonal antibody (Cell signaling), MDM2 rabbit polyclonal antibody (Bioss), Flag tag and myc tag mouse monoclonal antibodies (TransGene Biotech).

Techniques: Binding Assay, Blocking Assay, Modification

Journal: Cell death & disease

Article Title: Erythropoietin inhibits chemotherapy-induced cell death and promotes a senescence-like state in leukemia cells.

doi: 10.1038/s41419-018-1274-6

Figure Lengend Snippet: Fig. 5 EPO increases Mdm2 expression after DNR or nutlin-3a treatment and suppresses nutlin-3a-induced apoptosis. Western blot showing p53 and Mdm2 protein levels in cells treated with DNR (a, b) or with nutlin-3a (c, d) in the presence or absence of EPO. In (a) and (b), DA3/EPOR cells were cultured in the absence of EPO for 1 h followed by 10 h of treatment with DNR (0.25 µM) with or without EPO (1 U/ml). In (c) and (d), cells were cultured in the absence of EPO for 0.5 h followed by 24 h of treatment with nutlin-3a (10 µM) with or without EPO (1 U/ml). Protein expression levels were determined by western blot analysis using the indicated antibodies. The HDM2-323 antibody recognizes full-length MDM2 and the SMP14 antibody recognizes the MDM2 p60 cleavage product. (e) Cells were treated as in (c, d) and the proportion of cells undergoing apoptosis (cells having <2 N DNA content) was measured by flow cytometry after staining with propidium iodide

Article Snippet: Antibodies to Mdm2 included: SMP14 (sc-965; Santa Cruz Biotechnology), p-MDM2 (Ser166) (#3521; Cell Signaling Technology), and HDM2-323 (sc-56154; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Cell Culture, Cytometry, Staining

Journal: Annals of Translational Medicine

Article Title: Pathological prognostic factors of retroperitoneal liposarcoma: comprehensive clinicopathological analysis of 124 cases

doi: 10.21037/atm-21-972

Figure Lengend Snippet: Typical histopathological features of 124 RLS patients in the study. (A) Well-differentiated liposarcoma fat vacuoles vary in size [hematoxylin-eosin (HE), 400×]; (B) A variable number of lipoblasts can be seen in WDL (HE 400×); (C) Abundant lymphocytes and scattered bizarre stromal cells are present in the field of inflammatory subtype of WDL (HE 200×). (D) Dedifferentiated component was fibrosarcoma (HE 400×). (E) Dedifferentiated liposarcoma may feature a distinctive osteosarcomatous area or osteogenic differentiation (HE 400×). (F) This field shows PLPS -like morphology that feature pleomorphic tumour cell with bizarre cell nucleus (HE 400×). (G) Sclerosing liposarcoma in the lymph nodes (HE 200×). (H) Tumor emboli can be seen in the vascular (HE 400×). (I) Liposarcoma cells invading the nerve (HE 400×). (J) Diffuse nuclear and cytoplasm expression of P16 is consistently observed in dedifferentiated liposarcoma (EnVision method 400×). (K) Diffuse nuclear expression of CDK4 is consistently observed in dedifferentiated liposarcoma (EnVision method 400×). (L) MDM2 gene amplification (fluorescence in-situ hybridization method 1,000×).

Article Snippet: IHC antibodies included Ki-67 (OriGene, Clone UMAB107) and P53 (OriGene, clone DO7) and CDK4 (cyclin-dependent kinase 4) (ZSGB Bio Co., Ltd., Clone EP180) and MDM2 (OriGene, Clone 1E6&17B3) and P16 (ZSGB Bio Co., Ltd., Clone G175-405).

Techniques: Expressing, Amplification, Fluorescence, In Situ Hybridization

Journal: International Journal of Molecular Sciences

Article Title: Proximal Tubular Lats2 Ablation Exacerbates Ischemia/Reperfusion Injury (IRI)-Induced Renal Maladaptive Repair through the Upregulation of P53

doi: 10.3390/ijms242015258

Figure Lengend Snippet: The effect of renal proximal tubule-specific Lats2 ablation on p53 and its target gene expression after I/R injury. ( A ) Representative dual fluorescence and enlarged images of p53 and p-p53 (Ser 15) staining in sham and IRI-injured kidneys. Scale bars, 100 μm. ( B ) Western blotting analysis of p53, p-p53, p-MDM2 (Ser186), p21, Bax, cleaved caspase-3, Bcl-xL, and Bcl-2 in sham and IRI 14d mice and ( C , D ) quantified and normalized to β-actin expression. Data expressed as means ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 defined as significant.

Article Snippet: For immunochemistry, α-SMA, fibronectin, and collagen I were detected using monoclonal rabbit anti-α-smooth muscle actin (Cat# 19245S, CST), polyclonal rabbit anti-fibronectin (Cat# 15613-1-AP, Proteintech), polyclonal rabbit anti-collagen I (Cat# 14695-1-AP, Proteintech), and polyclonal rabbit anti-phospho-MDM2 (Ser186) (Cat# bs-5471R, Bioss).

Techniques: Targeted Gene Expression, Fluorescence, Staining, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Proximal Tubular Lats2 Ablation Exacerbates Ischemia/Reperfusion Injury (IRI)-Induced Renal Maladaptive Repair through the Upregulation of P53

doi: 10.3390/ijms242015258

Figure Lengend Snippet: The influence of Lats2 overexpression on p53 in TEC cells in response to hypoxia and reoxygenation (H/R) injury. ( A ) Vector map of the lentivirus incorporating LATS2 recombinant plasmid. ( B ) Representative images of GFP expression in TEC cells with lentivirus incorporating Lats2 recombinant plasmid or empty plasmid. Scale bars, 20 μm. ( C ) Western blotting analysis of GFP, LATS2, p53, p-p53, p-MDM2 (Ser186), p21, Bax and cleaved caspase-3 in TEC cells subjected to H/R injury and ( D ) quantified and normalized to GAPDH expression. Data expressed as means ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 defined as significant. ns, not statistically significant.

Article Snippet: For immunochemistry, α-SMA, fibronectin, and collagen I were detected using monoclonal rabbit anti-α-smooth muscle actin (Cat# 19245S, CST), polyclonal rabbit anti-fibronectin (Cat# 15613-1-AP, Proteintech), polyclonal rabbit anti-collagen I (Cat# 14695-1-AP, Proteintech), and polyclonal rabbit anti-phospho-MDM2 (Ser186) (Cat# bs-5471R, Bioss).

Techniques: Over Expression, Plasmid Preparation, Recombinant, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Cellular sensitivity to UV-irradiation is mediated by RNA polymerase I transcription

doi: 10.1371/journal.pone.0179843

Figure Lengend Snippet:

Article Snippet: α HDM2 , Acris Antibodies GmbH, Herford, Germany / AM00224PU-N/Western blot.

Techniques: