Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 1 Depletion of p54nrb lowers the viability of tumor cells. P54nrb expression level in healthy tissue versus tumor tissue A from cervix carcinoma, B from colon carcinoma, and C from melanoma patients. Values were obtained from Oncomine database. To test significance Student´s t test was performed. ****p < 0.0001. Immunoblot of p54nrb-level in shRNA-control and shRNA-p54nrb#1 knockdown cells D from DLD-1 and E from SK-MEL. Counts of colonies of 3D soft agar tumor growth assay of shRNA-control and shRNA-p54nrb#1 cells F from DLD-1 and G from SK-MEL. 1000 cells/well were seeded and grown for 3 weeks and stained with 0.1% crystal-violet. Student´s t test was performed. *p < 0.05, n = 3 (G). Representative wells of 3D soft agar tumor growth assay of shRNA-control and shRNA-p54nrb#1 cells H from DLD-1 and I from SK-MEL cells. J Schematic representation of protein domains of p54nrb (also: NoNO): H and Q rich domain (HQ), RNA recognition motif (RRM), NonA/paraspeckle domain (NOPS), coiled-coil domain (COIL), and proline-rich region (P). Brackets indicate the region for RNA, DNA, and protein binding.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: Expressing, Western Blot, shRNA, Control, Knockdown, Growth Assay, Staining, Protein Binding

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 2 Depletion of p54nrb leads to increased cell death susceptibility. A Immunoblot of p54nrb-level in shRNA-control and shRNA- p54nrb#3 knock down HeLa cells. B Flow cytometry of HeLa shRNA-control and shRNA-p54nrb#3 cells at 24 h after treatment with 10 µM etoposide (Eto) and 240 ng/ml human recombinant TRAIL (TRAIL). Cell viability was measured by detection and totaling of Annexin-V single positive and Annexin-V and propidium-iodide- double positive cells. The cells not falling to either of these categories were considered as viable cells. Significance was calculated with Student´s t test, *p < 0.05, n = 3. C Flow cytometry of HeLa shRNA-control and shRNA-p54nrb#3 cells at 24 h after treatment with 10 µg/ml Doxorubicin (DOX). Cell viability was measured by detection and totaling of Annexin-V single positive and Annexin-V and Sytox Blue- double-positive cells. The cells not falling to either of these categories were considered as viable cells. Significance was calculated with Student´s t test, **p < 0.01, n = 3.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: Western Blot, shRNA, Control, Knockdown, Flow Cytometry, Recombinant

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 3 Loss of p54nrb leads to an altered expressional pattern of carcinogenesis relevant genes. A Volcano plot diagram of LC/MS data derived from the analysis of HeLa shRNA-control versus shRNA-p54nrb cells. (n = 3). Arrowheads depict significantly (p < 0.01) upregulated (arrow up, red) and downregulated (arrow down, blue) proteins, and the total numbers of both categories are indicated on the diagram. B Up- and C downregulated proteins (p < 0.01, Log2 ratio either >0.5 or < −0.5) of HeLa-shRNA-p54nrb cells compared to shRNA control cells, which exert tumor regulatory and/or apoptosis regulatory properties. Differently colored oval diagrams represent various functional subcategories of these hits. D Immunoblot of HeLa shRNA-control and shRNA-p54nrb cells. Detection of p54nrb, gelsolin, cathepsin-Z, NQO1, and TPD52 levels. E Quantitative evaluation of immunoblots of p54nrb, gelsolin, cathepsin-Z, NQO1, TPD52, and CDKN2A from HeLa shRNA-control and shRNA-p54nrb#1 and #3 cells. The bands were normalized to the shRNA-control samples. Significance was calculated with one sample t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: Liquid Chromatography with Mass Spectroscopy, Derivative Assay, shRNA, Control, Functional Assay, Western Blot

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 4 P54nrb is cleaved upon apoptosis stimulation depending on the presence of caspase-2. A Immunoblot of p54nrb cleavage in HeLa cells at 24 h after treatment with DMSO or 20 µM Z-VDVAD-fmk (-1 h) and 250 nM staurosporine (STS). B Immunoblot of p54nrb cleavage and caspase expression in HeLa cells. HeLa cells were transfected either with 300 or 1000 ng/ml pcDNA3-caspase-2-Flag or pcDNA3-caspase-3- myc, and 24 h later were treated either with DMSO as vehicle control or 250 nM STS. The samples were harvested for Western blot at 24 h. C Immunoblot of p54nrb cleavage and caspase-2 in DLD-1 CRISPR-control or DLD-1 CRISPR–caspase-2 cells at 24 h after treatment with DMSO, 10 µM or 50 µM Eto. D Immunoblot of p54nrb cleavage and caspase-2 in HeLa shRNA-control or HeLa shRNA-caspase-2 cells at 24 h after treatment with DMSO, 250 nM STS, 1 µM STS or 10 µM Eto. E Immunoblot of p54nrb cleavage and caspase-2 in HeLa shRNA-control or HeLa shRNA-caspase-2 cells at 1, 3, 6, and 10 h after treatment with 1 µM STS. DMSO was used as vehicle control. F Immunoblot of p54nrb and caspase-2 of nuclear and cytoplasmic fraction of HeLa cells. Lamin-A was detected as nuclear marker.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: Western Blot, Expressing, Transfection, Control, CRISPR, shRNA, Marker

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 5 Caspase-2 directly interacts with p54nrb and cleaves p54nrb at D422. A Immunoblot of p54nrb caspase-2 and caspase-3 after endogenous immunoprecipitation (IP) of p54nrb from HeLa cells. B Immunoblot of p54nrb and caspase-2 after in vitro caspase cleavage assay with 0.2 µg or 2 µg recombinant p54nrb and 10 U human recombinant active caspase-2 after 3 h incubation at 37 °C. C Immunoblot of p54nrb cleavage and caspase-2 from HeLa cells at 48 h after ectopic expression of 1 µg/ml empty vector pcDNA3-Flag, pcDNA3-caspase-2-Flag, and pcDNA3-caspase-2-C303A-Flag (inactive). D Graphical illustration of p54nrb cleavage by activated caspase-2 via either overexpression, apoptotic induction, or recombinant caspase-2 protein. The active site of caspase-2 dimer is marked with yellow stars. Location of the putative cleavage sites in the p54nrb protein domains are marked with arrows, and its anticipated cleavage fragments are shown. E Immunoblot of p54nrb cleavage in HeLa shRNA-p54nrb#1 cells after ectopic expression of pcDNA3-Flag, pFlag-p54nrb, pFlag-p54nrb-D58N and pFlag- p54nrb-D422A (1 µg/ml plasmid) (+24 h) and 24 h treatment with 50 µM Eto. F Endogenous ribonucleoprotein immunoprecipitation of p54nrb in HeLa cells and subsequent isolation of coprecipitated DNA or RNA coupled with cDNA-synthesis. Validation of potential co- precipitate by PCR and subsequent agarose gel-electrophoretic detection. G In vitro p54nrb/DNA binding assay. 0.5 µg human recombinant p54nrb (Origene, Rockville, MD USA) and 100 ng plasmid encoding the sequence of gelsolin (Ch-gelsoln) [#37262] [Addgene]) were incubated at 37 °C for 18 h. Immunoprecipitation (IP) was performed by employing either p54nrb antibody or the same amount and species of IgG as control. Next, the potentially binding DNA was isolated as described in Methods (see ChIP). P54nrb was detected by western blot and the binding of specific DNA was confirmed by PCR, employing gelsolin-specific primers.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: Western Blot, Immunoprecipitation, In Vitro, Cleavage Assay, Recombinant, Incubation, Expressing, Plasmid Preparation, Over Expression, shRNA, Isolation, cDNA Synthesis, Biomarker Discovery, Agarose Gel Electrophoresis, DNA Binding Assay, Sequencing, Control, Binding Assay

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 6 Cleavage by caspase-2 disrupts the gene regulatory function of p54nrb. A Schematic illustration of p54nrb’s gene regulatory function in the presence or absence of active caspase-2 and its subsequent effect on tumor cell survival.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques:

Journal: Cell Death & Disease

Article Title: DNA damage-induced paraspeckle formation enhances DNA repair and tumor radioresistance by recruiting ribosomal protein P0

doi: 10.1038/s41419-022-05092-1

Figure Lengend Snippet: A Screening of IR-induced NONO-binding protein. HEK 293T cells were transfected with indicated plasmid and treated with IR (5 Gy). After recovery (30 min), the protein interacting with NONO was assessed by CoIP, separated with SDS-PAGE, and identified with LC/MS assay. GFP was used as a negative control. B The differentially expressed band at 36 kDa in Fig. 1A was analyzed by LC/MS. C The sample in Fig. 1A was analyzed with western blotting. D Flag-RPLP0 and Myc-NONO were interacted with each other in HEK 293T cells. Twenty-four hours after transfection with Flag-RPLP0 and Myc-NONO plasmid, cells were subjected to CoIP assay with anti-Myc or anti-Flag antibodies. E endogenous RPLP0 and NONO are associated in HCT116 cells. NONO or RPLP0-binding protein was assessed by CoIP assay and analyzed using western blotting. F IR enhances the interaction of NONO and RPLP0. HCT116 or U2OS cells were treated with IR (10 Gy) and recovered for indicated time before CoIP assay. G Duolink PLA assay was performed to analyze radiation-induced interaction between NONO and RPLP0. Cells were irradiated with 10 Gy X-ray and cultured for 1 h before PLA assay. n = 130 (0 Gy), n = 90 (10 Gy, antibodies), n = 109 (10 Gy, IgG). ***, P < 0.001.

Article Snippet: Primary antibodies used in this assay: anti-RPLP0 (A5557, Abclonal) and anti-NONO (611279, BD Bioscience), anti-MRE11 (ab214, Abcam), and anti-RAD50 (3427S, CST).

Techniques: Binding Assay, Transfection, Plasmid Preparation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Negative Control, Western Blot, Co-Immunoprecipitation Assay, Irradiation, Cell Culture

Journal: Cell Death & Disease

Article Title: DNA damage-induced paraspeckle formation enhances DNA repair and tumor radioresistance by recruiting ribosomal protein P0

doi: 10.1038/s41419-022-05092-1

Figure Lengend Snippet: A NONO was not detected in ribosome. Ribosomal protein of HCT116 cells with or without IR was fractionated with sucrose density gradient ultracentrifugation. B The colocalization between NONO and RPLP0 was analyzed with immunofluorescence. C Z-stacking imaging confirmed the association between RPLP0 and NONO. D RPLP0-tdTomato and NONO-GFP colocalize in HEK 293T cells. Twenty-four hours after transfection with RPLP0-tdTomato and NONO-GFP plasmids, HEK 293T live cells were imaged using confocal microscopy. E RPLP0 and NONO interact in nucleus. Cytoplasmic and nuclear proteins were fractionated and subjected to CoIP assay with anti-NONO antibody. GAPDH and Lamin A/C served as negative controls that did not interact with NONO for cytoplasmic and nuclear extracts, respectively. F RRM1 and RRM2 domains of NONO associate with RPLP0. HEK 293T cells were transfected with different mutants of Myc-NONO and Flag-RPLP0 before CoIP assay.

Article Snippet: Primary antibodies used in this assay: anti-RPLP0 (A5557, Abclonal) and anti-NONO (611279, BD Bioscience), anti-MRE11 (ab214, Abcam), and anti-RAD50 (3427S, CST).

Techniques: Immunofluorescence, Imaging, Transfection, Confocal Microscopy, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: DNA damage-induced paraspeckle formation enhances DNA repair and tumor radioresistance by recruiting ribosomal protein P0

doi: 10.1038/s41419-022-05092-1

Figure Lengend Snippet: A Purified NONO-GFP protein formed condensates in vitro. Ten μM NONO protein and 100 ng/ul total RNA extracted from HEK 293T cells were used. Scale bars, 5 μm. B NONO-GFP form condensates with high rate of FRAP in vivo. n = 3 biological replicates. Scale bars, 5 μm. C NONO condensates recruit RPLP0 protein in vitro. Purified NONO-GFP protein formed condensates in vitro, which were then incubated with nuclear proteins of U2OS cells for 10 min. NONO condensates were separated by centrifugation and subjected to western blotting analysis. D 1,6-Hexanediol disrupts the association between NONO and RPLP0. Cells were irradiated (10 Gy) and cultured with complete medium containing 1.5% 1,6-Hexanediol for 30 min before Duolink PLA assay. n = 74 (Ctrl), n = 78 (1,6-Hex). Scale bars, 5 μm. E IR induces paraspeckle formation. U2OS cells were treated with radiation (5 Gy) and allowed to recover for indicated time before IF assay. n = 3 biological replicates. Scale bars, 10 μm. F RNA-immunoprecipitation assay showed that RPLP0 was associated with NEAT1. 28s rRNA and U6 were used as the positive and negative control, respectively. G RNase A disrupts the association between NONO and RPLP0. RNase A (0.1 μg/μl) was added to U2OS cell lysate before CoIP assay. To verify the RNase A-mediated removing of RNA, total RNA was extracted from 10% of flow through lysate and analyzed with gel electrophoresis. H siNEAT1 reduced the interaction between NONO and RPLP0. HCT116 cells were transfected with siNEAT1 (pool of siNEAT1-1 and siNEAT1-2) for 48 h before CoIP ( right panel ) or RT-PCR ( left panel ) analysis. ***, P < 0.001.

Article Snippet: Primary antibodies used in this assay: anti-RPLP0 (A5557, Abclonal) and anti-NONO (611279, BD Bioscience), anti-MRE11 (ab214, Abcam), and anti-RAD50 (3427S, CST).

Techniques: Purification, In Vitro, In Vivo, Incubation, Centrifugation, Western Blot, Irradiation, Cell Culture, Immunoprecipitation, Negative Control, Co-Immunoprecipitation Assay, Nucleic Acid Electrophoresis, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: DNA damage-induced paraspeckle formation enhances DNA repair and tumor radioresistance by recruiting ribosomal protein P0

doi: 10.1038/s41419-022-05092-1

Figure Lengend Snippet: A RPLP0 colocalizes with γ-H2A.X after irradiation. U2OS cells were irradiated (2 Gy) and cultured for 30 min before analysis. Scale bars, 10 μm. B NONO and RPLP0 load into chromosome upon DNA damage. Twelve hours after seeding, cells were treated with radiation (10 Gy) and cultured for indicated time before chromosome fractionation. C 4-OHT induces DNA damage by facilitating the nuclear import of endonuclease ER-AsiSI. Cells were treated with 300 nM 4-OHT for 4 h before IF assay. Scale bars, 10 μm. D The binding of NONO and RPLP0 to damaged DNA was examined with ChIP assay. Four hours after 4-OHT or DMSO treatment, ER-AsiSI-expressing U2OS cells were analyzed with ChIP assay. E , F NONO depletion or RPLP0 silencing inhibits the phosphorylation of DNA-PK at T2609. U2OS cells were irradiated (10 Gy) and cultured for indicated time before nuclei isolation, which were further analyzed by western blotting. *, unspecific band. G The construction of NONO-RPLP0 fusion protein. (GGGGS) 3 , triplication of GGGGS; P2A, GSGATNFSLLKQAGDVEENPGP. H NONO-RPLP0 fusion protein promotes DNA repair. n (Vector) = 178 nuclei, n (NO-P0) = 199 nuclei, n (P2A) = 179 nuclei. Scale bars, 10 μm. ***; P < 0.001; ns no significance.

Article Snippet: Primary antibodies used in this assay: anti-RPLP0 (A5557, Abclonal) and anti-NONO (611279, BD Bioscience), anti-MRE11 (ab214, Abcam), and anti-RAD50 (3427S, CST).

Techniques: Irradiation, Cell Culture, Fractionation, Binding Assay, Expressing, Isolation, Western Blot, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: DNA damage-induced paraspeckle formation enhances DNA repair and tumor radioresistance by recruiting ribosomal protein P0

doi: 10.1038/s41419-022-05092-1

Figure Lengend Snippet: Knocking out of NONO sensitized tumor cells to irradiation. Twenty-four hours after seeding, HCT116 (5000 cells) ( A ) and U2OS (3000 cells) ( B ) were irradiated with indicated dose and cultured for 10 days. Cells were fixed and stained with 0.2% crystal violet. n = 3 independent experiments. C Knocking out of NONO sensitized xenografts to radiation in vivo. Ten days after MC38 cells (2 × 10 5 ) injection, tumors were irradiated with or without 10 Gy (day 0) X-ray and observed for 13 days. RT., Radiotherapy; Ctrl., Control. n = 6 xenografts . D The NONO and RPLP0 levels in biopsy were analyzed by IHC. n = 15 (TRG0), 11 (TRG1), 49 (TRG2), or 22 (TRG3) specimens of rectal cancer. E The NONO and RPLP0 levels in operation specimens were analyzed by IHC. n = 45 (TRG0&1) or 37 (TRG2&3) specimens of rectal cancer. F , G The mRNA and protein level of NONO and RPLP0 were examined in rectal cancer tissues. The mRNA and protein level of NONO and RPLP0 in 30 pairs of normal and rectal cancer tissues were examined by qPCR and western blotting analysis. n = 30 pairs of normal and rectal cancer tissues. H The association of NONO and RPLP0 was examined with Duolink PLA assay in rectal cancer biopsy species. IgGs was used as a negative control. (Top) Scale bars, 25 μm; (Bottom) Scale bars, 10 μm. antibodies: anti-NONO and anti-RPLP0 antibodies; IgGs: immunoglobulin G from mouse and rabbit. I NONO-RPLP0 fusion protein promotes radioresistance. n = 3 biological replicates. *, P < 0.05; **; P < 0.01, ***; P < 0.001.

Article Snippet: Primary antibodies used in this assay: anti-RPLP0 (A5557, Abclonal) and anti-NONO (611279, BD Bioscience), anti-MRE11 (ab214, Abcam), and anti-RAD50 (3427S, CST).

Techniques: Irradiation, Cell Culture, Staining, In Vivo, Injection, Western Blot, Negative Control

Journal: Theranostics

Article Title: The mitochondria‒paraspeckle axis regulates the survival of transplanted stem cells under oxidative stress conditions

doi: 10.7150/thno.88764

Figure Lengend Snippet: Loss of NEAT1 impaired paraspeckle formation and DNA repair machinery in MSCs. (A) Representative images and quantification of the colocalization of NEAT1-1 and NEAT1-2 by FISH in hMSCs upon siTFAM or siNEAT1 treatment (scale = 2 µm) (n = 3; ***p < 0.001 vs. NC group). (B) Western blot analysis and quantification of the levels of the PSF, NONO and PSPC1 proteins in hMSCs treated with siNEAT1 (n = 3; ***p < 0.001 vs. NC group). (C) Double-IF staining and colocalization of PSF (green), PSPC1 (red) or PSF (green), NONO (red) in hMSCs treated with siNEAT1 (scale = 10 µm) (n = 3; **p < 0.01 vs. NC group; ***p < 0.001 vs. NC group). (D) Western blot analysis and quantification of the levels of the RPA32 and TFAM proteins in hMSCs treated with siNEAT1(n = 3; ***p < 0.001 vs. NC group). (E)Western blot analysis and quantification of the levels of the γ-H2A.X, BRCA1 and p21 (n = 3; *p < 0.05 vs. NC group; ***p < 0.001 vs. NC group; # p <0.05 vs. s iNEAT1 group; & p <0.05 vs. H 2 O 2 +NC group). (F) Representative images of γ-H2A.X (scale bar = 50 μm) and β -gal staining (scale = 100 µm) of hMSCs treated with siNEAT1 upon H 2 O 2 stimulation. (G) Quantification of γ-H2A.X and β -gal levels in hMSCs (n = 3; ***p < 0.001 vs. NC group; &&& p <0.001 vs. H 2 O 2 +NC group). (H) Flow cytometry analysis of MSC apoptotic rates (n = 3; *p < 0.05 vs. NC group; # p <0.05 vs. siNEAT1 group).

Article Snippet: The membranes were blocked with 5% nonfat milk and incubated with primary antibodies against rabbit anti-TFAM (A1962, ABclonal), rabbit anti-TFAM (22586-1-AP, Proteintech), rabbit anti-p53 (10442-1-AP, Proteintech), rabbit anti-p21 (A19094, ABclonal), rabbit anti-BRCA1 (A11034, ABclonal), rabbit anti-COXIV (11242-1-AP, Proteintech), rabbit anti-NONO (A5282, ABclonal), rabbit anti-PSPC1 (A9209, ABclonal), rabbit anti-sirt1 (13161-1-AP, Proteintech), mouse anti-PSF (sc-271796, Santa Cruz), rabbit anti-phospho-histone H2A.X (#9718, 20E3, Cell Signaling Technology), rabbit anti-ATF2 (A2155, ABclonal), rabbit anti-phospho-BRCA1 (#9009, ser 1524, Cell Signaling Technology), rabbit anti-RPA32 (A2189, ABclonal) and rabbit anti-4HNE (ab46545, Abcam) and mouse anti-GAPDH (AC002, ABclonal) at 4 °C overnight.

Techniques: Western Blot, Staining, Flow Cytometry

Journal: Oncology Reports

Article Title: Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

doi: 10.3892/or.2018.6334

Figure Lengend Snippet: Increased expression of NONO in ESCC tissue samples. (A) Three independent microarray data from the Oncomine database showed that NONO mRNA levels were increased in ESCC patient tissue samples. Comparison of NONO mRNA levels in normal tissue (N), esophageal squamous cell carcinoma (ESCC), and Barrett's esophagus (BE), esophageal adenocarcinoma (EAC) are presented together with P-values. (B) Immunohistochemistry signal of NONO from paired normal and ESCC tissue samples were recorded by quickscore method. Comparison of quickscore distribution between adjacent normal and ESCC samples was performed by χ 2 test. (C) Representative immunohistochemistry (IHC) images of NONO in paired normal and ESCC tissue samples. NONO levels were lower in adjacent normal esophageal epithelium, but were higher in ESCC. (D) NONO quickscores were further divided into three groups: strong, moderate and weak. The percentage of each group in normal and ESCC tissue samples were plotted.

Article Snippet: The cells were permeabilized with 0.3% Triton X-100 for 20 min, blocked with 5% bovine serum albumin for 30 min, and then incubated with the mouse monoclonal anti-NONO antibody (Becton Dickinson) at 4°C overnight, followed by an Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 45 min at 37°C.

Techniques: Expressing, Microarray, Comparison, Immunohistochemistry

Journal: Oncology Reports

Article Title: Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

doi: 10.3892/or.2018.6334

Figure Lengend Snippet: Pathological information of ESCC patients and NONO expression.

Article Snippet: The cells were permeabilized with 0.3% Triton X-100 for 20 min, blocked with 5% bovine serum albumin for 30 min, and then incubated with the mouse monoclonal anti-NONO antibody (Becton Dickinson) at 4°C overnight, followed by an Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 45 min at 37°C.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

doi: 10.3892/or.2018.6334

Figure Lengend Snippet: Real-time PCR and western blot analysis of NONO expression in esophageal squamous cell carcinoma (ESCC) cells. (A) Real-time PCR measured relative NONO mRNA levels to GAPDH in 9 ESCC cell lines. (B) Western blot analysis of endogenous expression of NONO in all 9 ESCC cell lines. β-actin served as a loading control. (C and D) Transfection efficiencies of NONO siRNA knockdown in TE-1, KYSE70 cells were measured by real-time PCR and western blotting. (E) Immunofluorescence staining detected expression and nuclear localization of NONO protein in human ESCC cell lines. NONO expression is reduced after NONO siRNA knockdown in TE-1, KYSE70 cells.

Article Snippet: The cells were permeabilized with 0.3% Triton X-100 for 20 min, blocked with 5% bovine serum albumin for 30 min, and then incubated with the mouse monoclonal anti-NONO antibody (Becton Dickinson) at 4°C overnight, followed by an Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 45 min at 37°C.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control, Transfection, Knockdown, Immunofluorescence, Staining

Journal: Oncology Reports

Article Title: Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

doi: 10.3892/or.2018.6334

Figure Lengend Snippet: Examination of NONO targets in esophageal squamous cell carcinoma (ESCC) cells. (A) The cleavages of caspase-3, and PARP-1 were compared between siRNA control and siRNA NONO-transfected cells by western blotting at the indicated time-points. β-actin served as a loading control. NONO depletion upregulated caspase-3 and PARP-1 cleavage in TE-1 and KYSE70 ESCC cell lines.

Article Snippet: The cells were permeabilized with 0.3% Triton X-100 for 20 min, blocked with 5% bovine serum albumin for 30 min, and then incubated with the mouse monoclonal anti-NONO antibody (Becton Dickinson) at 4°C overnight, followed by an Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 45 min at 37°C.

Techniques: Control, Transfection, Western Blot

Journal: Oncology Reports

Article Title: Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

doi: 10.3892/or.2018.6334

Figure Lengend Snippet: NONO modulates cell proliferation and apoptosis. (A and B) MTS assay showed that NONO knockdown inhibited proliferation in TE-1 and KYSE70 ESCC cell lines. (C and D) Apoptotic cell death was determined by flow cytometric analysis with Annexin V and PI staining. NONO knockdown increased the level of apoptosis in TE-1 and KYSE70 cell lines. Results shown are representative of three independent experiments with Student's t-test. Statistical significance *P<0.05 and **P<0.01.

Article Snippet: The cells were permeabilized with 0.3% Triton X-100 for 20 min, blocked with 5% bovine serum albumin for 30 min, and then incubated with the mouse monoclonal anti-NONO antibody (Becton Dickinson) at 4°C overnight, followed by an Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 45 min at 37°C.

Techniques: MTS Assay, Knockdown, Staining

Journal: Oncology Reports

Article Title: Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

doi: 10.3892/or.2018.6334

Figure Lengend Snippet: NONO knockdown decreases esophageal squamous cell carcinoma (ESCC) cell migration and Matrigel invasion. TE-1 and KYSE70 cells were transfected with NONO or control siRNA for 24 h. (A and B) Wound healing assay showed that cell motility was inhibited by NONO knockdown. Representative images at time 0 and 24 h after scratching (left). Cells penetrated through the Matrigel were captured by microscope and representative images are shown (right). The numbers of cells invaded through Matrigel in each condition were counted and plotted. (C) Results shown are representative of three independent experiments with Student's t-test, the statistical significance was *P<0.05, **P<0.01 and ***P<0.001.

Article Snippet: The cells were permeabilized with 0.3% Triton X-100 for 20 min, blocked with 5% bovine serum albumin for 30 min, and then incubated with the mouse monoclonal anti-NONO antibody (Becton Dickinson) at 4°C overnight, followed by an Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 45 min at 37°C.

Techniques: Knockdown, Migration, Transfection, Control, Wound Healing Assay, Microscopy

Journal: Oncology Reports

Article Title: Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

doi: 10.3892/or.2018.6334

Figure Lengend Snippet: Akt and Erk1/2 are likely the downstream targets of NONO-mediated signaling. p-Akt, Akt, p-Erk1/2 and Erk1/2 were compared between TE-1 and KYSE70 cell lines treated with control siRNA and NONO siRNA by western blot analysis. β-actin served as a loading control.

Article Snippet: The cells were permeabilized with 0.3% Triton X-100 for 20 min, blocked with 5% bovine serum albumin for 30 min, and then incubated with the mouse monoclonal anti-NONO antibody (Becton Dickinson) at 4°C overnight, followed by an Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 45 min at 37°C.

Techniques: Control, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells

doi: 10.1111/jcmm.16243

Figure Lengend Snippet: USP11 interacts with NONO. A, Flag‐USP11 plasmid and Myc‐NONO plasmid were introduced into HEK293. Antibodies against Flag or Myc, an isotype‐matched normal IgG immuno‐precipitated total cell lysates, then the indicated bands were checked in the precipitations. B, SK‐Mel‐28 cell, (C) A375 cell lysates were immuno‐precipitated with control IgG, anti‐USP11/anti‐NONO antibody. The indicated proteins were checked in the precipitations. To detect the IPed protein, the prey proteins’ bands were visualized in long exposure. D, Myc‐NONO was expressed in HEK293 cell, GST and GST‐USP11 purified from E.coli were incubated with equal Myc‐NONO, and then loaded onto GST‐tag Purification Resin, Myc‐NONO in the elution was analysed. E, The subcellular localization of USP11 (green) and NONO (red) in A375 was visualized. DNA was counterstained with DAPI, and the views of USP11 and NONO were merged

Article Snippet: The arrays were incubated in Anti‐USP11(Santa Cruz, 1:100) or anti‐NONO (Sangon Biotech, 1:100) at room temperature overnight, rinsed twice with DPBS (Gibco), and incubated with a secondary antibody.

Techniques: Plasmid Preparation, Purification, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells

doi: 10.1111/jcmm.16243

Figure Lengend Snippet: USP11 affects NONO levels. A, SK‐Mel28 and A375 were transfected with Flag‐USP11 or control vector, and the indicated proteins were assessed. B, C, A375 cells were transfected with increasing amounts of USP11 WT (B) or USP11 Mut (C275S/C283S) vector (C), total proteins were extracted and the indicated proteins were checked. D, SK‐Mel‐28 and A375 were infected with USP11‐specific shRNA #1 or #2, and the indicated proteins were analysed. E, SK‐Mel28 and A375 were introduced with either scrambled or USP11 siRNA, the indicated proteins were probed

Article Snippet: The arrays were incubated in Anti‐USP11(Santa Cruz, 1:100) or anti‐NONO (Sangon Biotech, 1:100) at room temperature overnight, rinsed twice with DPBS (Gibco), and incubated with a secondary antibody.

Techniques: Transfection, Plasmid Preparation, Infection, shRNA

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells

doi: 10.1111/jcmm.16243

Figure Lengend Snippet: USP11 regulates NONO protein levels in a proteasome‐dependent manner. A, B, C, D, SK‐Mel‐28 cells (A and B), A375 cells (C and D) were introduced with Flag‐USP11/ control vector (A and C) or control scrambled siRNA/USP11 siRNA (B and D) , total RNA was subjected to RT‐PCR. E, F, SK‐Mel28 or A375 cells transfected with Flag‐USP11/control vector(E) or infected with indicated lentivirus shRNA (F) were treated with DMSO or MG132 (20 µmol/L) for 6 h, and the indicated proteins were probed

Article Snippet: The arrays were incubated in Anti‐USP11(Santa Cruz, 1:100) or anti‐NONO (Sangon Biotech, 1:100) at room temperature overnight, rinsed twice with DPBS (Gibco), and incubated with a secondary antibody.

Techniques: Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Transfection, Infection, shRNA

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells

doi: 10.1111/jcmm.16243

Figure Lengend Snippet: USP11 maintains NONO levels by Deubiquitination. A, HEK293 cells were transfected with the indicated vectors and then treated with MG132 (20 µM) for 6 h followed by the harvest. Myc‐NONO fusion protein was immuno‐precipitated with a Myc‐specific antibody, and the precipitations were checked with the antibody against Ub or Myc. (*WT: Wild‐Type Flag‐USP11, *Mu: Mutated Flag‐USP11 (C275S/C283S)) (B) A375 cells infected with USP11 shRNA or scrambled control shRNA, were transfected with Myc‐NONO and ub vectors, after 24 h later, the cells were treated with MG132 (20 µM) for 6 h followed by the harvest. Myc‐NONO fusion protein was immuno‐precipitated with anti‐Myc antibody, and the precipitations were checked with the antibody against Ub or Myc. C, SK‐Mel‐28 cells infected with the USP11 shRNA or scrambled shRNA were treated with MG132 (20µM) for 6 h followed by the harvest. Endogenous NONO protein was immuno‐precipitated with anti‐NONO antibody, and the precipitations were checked with the antibody against Ub or NONO

Article Snippet: The arrays were incubated in Anti‐USP11(Santa Cruz, 1:100) or anti‐NONO (Sangon Biotech, 1:100) at room temperature overnight, rinsed twice with DPBS (Gibco), and incubated with a secondary antibody.

Techniques: Transfection, Infection, shRNA

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells

doi: 10.1111/jcmm.16243

Figure Lengend Snippet: USP11 promotes melanoma cell proliferation via NONO. A, Colony formation assay was performed. A375 cells infected with lentivirus USP11 shRNA and transfected with the indicated vectors, were seeded with density 15 000 cells per well. 24 h later, A375 cells were subcultured and selected using G418 (200 µg/mL), and surviving colonies were counted 2 weeks later. Colonies were visualized and quantified. Relative cell viability was summarized from three independent experiments and was presented on the right. Statistical significance was determined by a two‐tailed, unpaired Student's t test. Data represent the mean (±SD) of three independent experiments (* P ≤ 0.05). B, A375 was infected with USP11‐specific shRNA #1 or #2 and then introduced with Myc‐NONO plasmid. CCK‐8 assays were performed to check relative cell viability at the indicated time points. C, A375 cells used in colony formation assay and CCK‐8 assays were lysed and analysed using Western blotting

Article Snippet: The arrays were incubated in Anti‐USP11(Santa Cruz, 1:100) or anti‐NONO (Sangon Biotech, 1:100) at room temperature overnight, rinsed twice with DPBS (Gibco), and incubated with a secondary antibody.

Techniques: Colony Assay, Infection, shRNA, Transfection, Two Tailed Test, Plasmid Preparation, CCK-8 Assay, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells

doi: 10.1111/jcmm.16243

Figure Lengend Snippet: USP11 promotes melanoma progress in a NONO‐dependent manner in vivo. A, B, C, The 8 × 10 6 indicated shRNA‐transduced SK‐Mel‐28 cells with or without ectopic expression of NONO were subcutaneously injected into mice. Tumour size (A), tumour weight (B) and tumour images (C) were shown. D, SK‐Mel‐28 cells implanted into nude mice were lysed and analysed using Western blotting

Article Snippet: The arrays were incubated in Anti‐USP11(Santa Cruz, 1:100) or anti‐NONO (Sangon Biotech, 1:100) at room temperature overnight, rinsed twice with DPBS (Gibco), and incubated with a secondary antibody.

Techniques: In Vivo, shRNA, Expressing, Injection, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells

doi: 10.1111/jcmm.16243

Figure Lengend Snippet: USP11 is overexpressed and positively correlates with NONO in melanoma. A, Representative images of USP11 and NONO protein in normal skin tissue and melanoma skin tissue. B, C, NONO (B) and USP11 (C) protein levels in normal skin and melanoma skin tissues. D, Correlation study between USP11 and NONO in clinical melanoma skin tissues. The statistical analysis was determined by a χ 2 test

Article Snippet: The arrays were incubated in Anti‐USP11(Santa Cruz, 1:100) or anti‐NONO (Sangon Biotech, 1:100) at room temperature overnight, rinsed twice with DPBS (Gibco), and incubated with a secondary antibody.

Techniques: