Journal: Oncogene

Article Title: CRTC1-MAML2 fusion-induced lncRNA LINC00473 expression maintains the growth and survival of human mucoepidermoid carcinoma cells

doi: 10.1038/s41388-017-0104-0

Figure Lengend Snippet: (a,b) Heatmap and volcano plot show differentially expressed genes in shLnc473 knockdown in compared with shRNA control in fusion-positive H3118 cells. The cutoff criteria were absolute fold-change of ≥2 and p<0.05. This analysis led to the identification of a total of 1320 LINC00473-regulated candidate coding genes, with 675 up-regulated and 645 down-regulated genes. (c) Functional classification of LINC00473 down-regulated genes was performed using Ingenuity Pathway Analysis (IPA). The top 6 molecular and cell functions were ranked based on p-value and activation z-score. Negative z-score indicates inhibition. (d) The qRT-PCR analysis showed that LncRNA LINC00473 was significantly enriched in the NONO immunoprecipitates relative to the IgG control in H3118 MEC cells. ASNS was used as a negative control (n=3, ***p<0.0001). (e) Overexpression of LINC00473 in HEK293T cells significantly increased the interaction of CRTC1-MAML2 and NONO by a Gal4-NONO reporter assay (n=3, *p<0.05). (f) Knockdown of NONO or LINC00473 expression significantly reduced the pCRE-luc reporter activities in HEK293T cells with CRTC1-MAML2 overexpression (n=3, *p<0.05 and **p<0.001). (g) A model for the molecular basis of LINC00473 induction by CRTC1-MAML2 and function in fusion-positive MEC cells.

Article Snippet: Cell lysates were collected for immunoprecipitation overnight at 4°C using anti-NONO antibodies (A300-587A, Bethyl Laboratories) control and IgG control (SC-2027, Santa Cruz) and protein A/G beads.

Techniques: Knockdown, shRNA, Control, Functional Assay, Activation Assay, Inhibition, Quantitative RT-PCR, Negative Control, Over Expression, Reporter Assay, Expressing

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