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: Oncogene

Article Title: PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer

doi: 10.1038/s41388-020-01617-0

Figure Lengend Snippet: a NONO protein is highly expressed in CRC tissue. NONO protein level was analyzed in 28 paired colorectal tumor (T) and adjacent normal (N) tissue samples by western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. b NONO mRNA level is higher in T than in N. NONO mRNA level was examined by qPCR in 29 paired tissue samples. U6 was used as an internal control (right panel). NONO mRNA expression level in TCGA N ( n = 41) and T ( n = 286) CRC samples (left panel). c , d Higher NONO protein level is correlated with shorter overall survival in CRC patients. The 93 patients were divided into NONO low ( n = 48) and high ( n = 45) subgroups ( c ) and subjected to Kaplan–Meier survival analysis ( d ). Scale bar, 1200 μm. e NONO overexpression promotes CRC cell growth. KM12 (1 × 10 4 ) and HCT8 (2 × 10 4 ) CRC cells were seeded on day 0 and counted on days 2 and 4. f NONO KO leads to a decrease in tumor size. KM12 WT or NONO KO cells (1 × 10 6 ) were injected into nude mice ( n = 5) and tumors were weighed on day 21. g–j NONO overexpression enhances CRC cell migration and invasion. The wound-healing assay was performed using KM12 (8 × 10 4 ) or HCT8 (1 × 10 5 ) cells ( g , i ). The transwell assay was performed using KM12 (4 × 10 4 ) or HCT8 (8 × 10 4 ) cells for 24 h ( h , j ). Scale bar, 400 μm. EV empty vector (pCDH-CMV)-transfected cells, NONO-OE NONO-overexpressing cells (transfected with pCDH-CMV-Flag-NONO). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies were used: Flag (cat. no. ab49763; abcam, Cambridge, MA, USA), HA (cat. no. ab1265; abcam), Myc (cat. no. ab9106; abcam), GAPDH (cat. no. 60004–1-Ig; Proteintech, Chicago, IL, USA), NONO (cat. no. 611279; BD Bioscience, Franklin lakes, NJ, USA), PRMT1 (cat. no. 2449; Cell Signaling Technology, CST, Beverly, MA, USA), PRMT5 (cat. no. D160716; Sangon Biotech, Shanghai, China), pan-ADMA (cat. no. 13522; CST), pan-MMA (cat. no. 8015; CST).

Techniques: Western Blot, Expressing, Over Expression, Injection, Migration, Wound Healing Assay, Transwell Assay, Plasmid Preparation, Transfection

Journal: Oncogene

Article Title: PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer

doi: 10.1038/s41388-020-01617-0

Figure Lengend Snippet: a Compared to adjacent normal (N) tissue, PRMT1 mRNA level was higher in CRC tissue (T), as determined by qPCR ( n = 29 paired tissues). There were no differences in PRMT3, PRMT4, and PRMT5 mRNA levels between groups. b PRMT1 protein is highly expressed in CRC tissue. PRMT5 protein expression was similar between T and N tissues, as determined by western blotting ( n = 28 paired tissues; left panel). Protein band intensity was quantified using ImageJ software (right panel). c NONO and the aDMA modification colocalized in the nucleus. Immunofluorescence analysis was performed using anti-NONO and -ADMA antibodies (right panel) in KM12 and HCT8 cells, and colocalization was analyzed using ZEN v3.0 software (Zeiss, Oberkochen, Germany) (left panel). Scale bar, 10 μm. d , e PRMT1 silencing reduces NONO aDMA level. Endogenous NONO was immunoprecipitated (IP) from control and PRMT1 -deficient KM12 and HCT8 cells and analyzed by western blotting ( d ). NONO methylation was detected using an anti-ADMA antibody. For Duolink PLA ( e ), NONO aDMA level was detected using anti-NONO and -ADMA antibodies (left panel). At least 100 nuclei were counted in each group (right panel). ** P < 0.01, *** P < 0.001; ns no significance.

Article Snippet: The following antibodies were used: Flag (cat. no. ab49763; abcam, Cambridge, MA, USA), HA (cat. no. ab1265; abcam), Myc (cat. no. ab9106; abcam), GAPDH (cat. no. 60004–1-Ig; Proteintech, Chicago, IL, USA), NONO (cat. no. 611279; BD Bioscience, Franklin lakes, NJ, USA), PRMT1 (cat. no. 2449; Cell Signaling Technology, CST, Beverly, MA, USA), PRMT5 (cat. no. D160716; Sangon Biotech, Shanghai, China), pan-ADMA (cat. no. 13522; CST), pan-MMA (cat. no. 8015; CST).

Techniques: Expressing, Western Blot, Software, Modification, Immunofluorescence, Immunoprecipitation, Methylation

Journal: Oncogene

Article Title: PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer

doi: 10.1038/s41388-020-01617-0

Figure Lengend Snippet: a Myc-NONO and Flag-PRMT1 interact in HEK 293T cells. pCDH-CMV-Myc-NONO and pCDH-CMV-Flag-PRMT1 were cotransfected into HEK 293T cells for 24 h before coIP analysis. b NONO interacts with PRMT1 in situ. CoIP was performed in KM12 and HCT8 cells using anti-NONO and -PRMT1 antibodies. Arrows indicate NONO or PRMT1, and circles indicate the heavy chain. c NONO and PRMT1 colocalize in the nucleus. Immunofluorescence analysis was performed using anti-NONO and -PRMT1 antibodies in KM12 and HCT8 cells (right panel). NONO and PRMT1 colocalization was analyzed using ZEN v3.0 software (left panel). Scale bar, 10 μm. d NONO interacts with PRMT1. Duolink PLA was performed with indicated antibodies. Blank, no primary antibody added in Duolink PLA. Scale bar, 10 μm. e NONO binds to the catalytic domain of PRMT1. NONO truncations are illustrated schematically at the top. HEK 293T cells were cotransfected with Myc-NONO and Flag-PRMT1 truncations for 24 h before CoIP analysis. aa amino acid, CD catalytic domain, POD post domain, PRD pre domain.

Article Snippet: The following antibodies were used: Flag (cat. no. ab49763; abcam, Cambridge, MA, USA), HA (cat. no. ab1265; abcam), Myc (cat. no. ab9106; abcam), GAPDH (cat. no. 60004–1-Ig; Proteintech, Chicago, IL, USA), NONO (cat. no. 611279; BD Bioscience, Franklin lakes, NJ, USA), PRMT1 (cat. no. 2449; Cell Signaling Technology, CST, Beverly, MA, USA), PRMT5 (cat. no. D160716; Sangon Biotech, Shanghai, China), pan-ADMA (cat. no. 13522; CST), pan-MMA (cat. no. 8015; CST).

Techniques: In Situ, Immunofluorescence, Software

Journal: Oncogene

Article Title: PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer

doi: 10.1038/s41388-020-01617-0

Figure Lengend Snippet: a Flag-NONO protein were immunoprecipitated from control or PRMT1 -silenced KM12 cells, separated by SDS–PAGE, and subjected to silver staining. b LC–MS/MS analysis of the NONO R251 methylation site. The fragmentation pattern of the typical NONO peptide EREQPPR is shown. Fragment ions are shown as b and y ions; ++ represents the loss of doubly charged ions. c R251K mutation abolishes PRMT1-mediated aDMA modification of NONO. NONO R-to-K mutants are illustrated schematically at the top. WT or R-to-K mutant NONO protein was immunoprecipitated with Flag beads and then immunoblotted with anti-ADMA antibody. d NONO is methylated by PRMT1 at R251, as determined with the in vitro methylation assay. GST-tagged GAR, NONO, and R251K were incubated with purified GST-PRMT1 in the presence or absence of 0.6 μM SAM. Proteins were separated on SDS–PAGE and subjected to western blotting analysis and Coomassie blue staining. GAR, glycine- and arginine-rich N-terminal region of fibrillarin. e NONO R251K mutation reduces cell proliferation. KM12 (1 × 10 4 ) and HCT8 (2 × 10 4 ) cells were seeded on day 0 and counted on days 2 and 4. NONO R251K mutation inhibited KM12 cell migration in the wound-healing assay ( f ) and invasion in the transwell assay ( g ). For experiments shown in e – g , KM12 cells were transfected with pCDH-CMV-Flag-NONO (WT) or pCDH-CMV-Flag-NONO-R251K (R251K mutant) for 24 h before the indicated assay. Scale bar, 400 μm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies were used: Flag (cat. no. ab49763; abcam, Cambridge, MA, USA), HA (cat. no. ab1265; abcam), Myc (cat. no. ab9106; abcam), GAPDH (cat. no. 60004–1-Ig; Proteintech, Chicago, IL, USA), NONO (cat. no. 611279; BD Bioscience, Franklin lakes, NJ, USA), PRMT1 (cat. no. 2449; Cell Signaling Technology, CST, Beverly, MA, USA), PRMT5 (cat. no. D160716; Sangon Biotech, Shanghai, China), pan-ADMA (cat. no. 13522; CST), pan-MMA (cat. no. 8015; CST).

Techniques: Immunoprecipitation, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Methylation, Mutagenesis, Modification, In Vitro, Incubation, Purification, Western Blot, Staining, Migration, Wound Healing Assay, Transwell Assay, Transfection

Journal: Oncogene

Article Title: PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer

doi: 10.1038/s41388-020-01617-0

Figure Lengend Snippet: a PRMT1 promotes CRC cell proliferation. KM12 (1 × 10 4 ) and HCT8 (1.8 × 10 4 or 2 × 10 4 ) cells were seeded on day 0 and counted every 2 days. PRMT1 knockdown inhibited and PRMT1 overexpression promoted, KM12 cell migration in the wound-healing assay ( b , d ) and invasion in the transwell assay ( c , e ). KM12 cells were transfected with pCDH-CMV-Flag-PRMT1 (PRMT1-OE) or empty vector (EV) for 24 h before the indicated assay. EV empty vector (pCDH-CMV) transfected cells, NONO-OE NONO-overexpressing cells (transfected with pCDH-CMV-Flag-NONO). Scale bar, 400 μm. f Elevated PRMT1 expression is correlated with shorter overall survival in CRC patients. The 97 patients were divided into PRMT1 low ( n = 61) and high ( n = 36) expression subgroups. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies were used: Flag (cat. no. ab49763; abcam, Cambridge, MA, USA), HA (cat. no. ab1265; abcam), Myc (cat. no. ab9106; abcam), GAPDH (cat. no. 60004–1-Ig; Proteintech, Chicago, IL, USA), NONO (cat. no. 611279; BD Bioscience, Franklin lakes, NJ, USA), PRMT1 (cat. no. 2449; Cell Signaling Technology, CST, Beverly, MA, USA), PRMT5 (cat. no. D160716; Sangon Biotech, Shanghai, China), pan-ADMA (cat. no. 13522; CST), pan-MMA (cat. no. 8015; CST).

Techniques: Over Expression, Migration, Wound Healing Assay, Transwell Assay, Transfection, Plasmid Preparation, Expressing

Journal: Oncogene

Article Title: PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer

doi: 10.1038/s41388-020-01617-0

Figure Lengend Snippet: a Treatment with the PRMT1 inhibitor AMI-1 reduced NONO aDMA level. KM12 and HCT8 cells treated with AMI-1 (1.2 and 0.6 mM) or DMSO for 48 h were subjected to Duolink PLA with anti-ADMA antibody. b AMI-1 treatment reduced tumor xenograft weight ( n = 6 mice per group). c Treatment with the PRMT1 inhibitors MS023 and C7280948 decrease aDMA NONO level. KM12 and HCT8 cells were treated with 10 μM MS023 and 40 μM C7280948, respectively, for 48 h, and subjected to NONO IP and western blotting analysis with anti-ADMA antibody. d Treatment with MS023 or C7280948 inhibits CRC cell proliferation. KM12 WT/NONO-KO (2 × 10 4 ) and HCT8 WT/NONO-KO (4 × 10 4 ) cells were seeded on day 0 and counted on days 2 and 4 after treatment with the indicated inhibitor. KM12 and HCT8 WT/NONO-KO cells treated with MS023 and C7280948 were subjected to the wound-healing assay ( e ) and transwell assay ( f ), respectively. For ( d – f ), MS023, C7280948, or DMSO was added at the time the cells were seeded. Scale bar, 400 μm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies were used: Flag (cat. no. ab49763; abcam, Cambridge, MA, USA), HA (cat. no. ab1265; abcam), Myc (cat. no. ab9106; abcam), GAPDH (cat. no. 60004–1-Ig; Proteintech, Chicago, IL, USA), NONO (cat. no. 611279; BD Bioscience, Franklin lakes, NJ, USA), PRMT1 (cat. no. 2449; Cell Signaling Technology, CST, Beverly, MA, USA), PRMT5 (cat. no. D160716; Sangon Biotech, Shanghai, China), pan-ADMA (cat. no. 13522; CST), pan-MMA (cat. no. 8015; CST).

Techniques: Western Blot, Wound Healing Assay, Transwell Assay

Journal: Oncogene

Article Title: PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer

doi: 10.1038/s41388-020-01617-0

Figure Lengend Snippet: In CRC cells, PRMT1-mediated methylation of NONO at R251 promotes tumor cell proliferation and metastasis. Malignant progression induced by NONO arginine methylation may be abrogated by treatment with small-molecule inhibitors of PRMT1 (AMI-1, MS023, and C7280948).

Article Snippet: The following antibodies were used: Flag (cat. no. ab49763; abcam, Cambridge, MA, USA), HA (cat. no. ab1265; abcam), Myc (cat. no. ab9106; abcam), GAPDH (cat. no. 60004–1-Ig; Proteintech, Chicago, IL, USA), NONO (cat. no. 611279; BD Bioscience, Franklin lakes, NJ, USA), PRMT1 (cat. no. 2449; Cell Signaling Technology, CST, Beverly, MA, USA), PRMT5 (cat. no. D160716; Sangon Biotech, Shanghai, China), pan-ADMA (cat. no. 13522; CST), pan-MMA (cat. no. 8015; CST).

Techniques: Methylation

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