Journal: Cancers

Article Title: NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression

doi: 10.3390/cancers11030388

Figure Lengend Snippet: NAB2 expression in head and neck squamous cell carcinoma (HNSCC) patient tissues. NAB2 protein expression was compared in normal epithelial cells ( A ) and primary tumors ( B , C ), and normal lymph nodes (Scale bar in C: 100 μM) ( E ) and paired metastatic lymph nodes ( F , G ) by immunohistochemistry. ( C ) and ( G ) are two-fold magnifications of the black rectangle images in ( B ) and ( F ), respectively. (Scale bar in G: 100 μM) Nuclei were counterstained with hematoxylin. Red arrows indicate some representative NAB2-expressing interstitial cancer-associated fibroblasts (CAFs) of primary and metastatic lymph node tissues. Black arrows indicate NAB2-negative cancer cells. NAB2-positive tumor cells and CAFs were counted in primary tumor tissues ( D ) and metastatic lymph node tissues ( H ). The boxes show the upper 75% and lower 25% quartiles of the measurements with respect to the median value (horizontal line in each box). Each dot represents an individual patient’s data, while a small open rectangle represents the mean value. Each bar represents the standard deviation.

Article Snippet: Fibroblasts were transiently transfected with a siRNA mixture targeting the NAB2 transcript (siNAB2; Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemistry, Standard Deviation

Journal: Cancers

Article Title: NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression

doi: 10.3390/cancers11030388

Figure Lengend Snippet: Effect of NAB2 on CAF marker and MMP expression in patient fibroblasts. ( A ) NAB2 mRNA and protein expression in CAFs and paired NTFs from tissue specimens of three HNSCC patients was analyzed by qPCR and Western blotting, respectively. ( B ) NAB2 mRNA level is compared in cultured CAFs and paired HNSCC tissues. ( C ) CAFs and NTFs form P3 were transfected with NAB2 overexpression (NAB2-over) or control vector for 48 h. ( D , E ) mRNA and protein levels of CAF markers were analyzed in NTFs transfected with NAB2-over or control vector. ( F ) mRNA and protein levels of MMPs were analyzed under the same conditions. ( G ) CAFs were transfected with control siRNA or siNAB2 mixture for 48 h, and mRNA and protein levels of NAB2, CAF markers, and MMPs were analyzed. Results represent the mean ± standard deviation of two or three experiments.

Article Snippet: Fibroblasts were transiently transfected with a siRNA mixture targeting the NAB2 transcript (siNAB2; Santa Cruz Biotechnology).

Techniques: Marker, Expressing, Western Blot, Cell Culture, Transfection, Over Expression, Control, Plasmid Preparation, Standard Deviation

Journal: Cancers

Article Title: NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression

doi: 10.3390/cancers11030388

Figure Lengend Snippet: Effect of NAB2 expressed by CAFs on FaDu cell or spheroid invasion. ( A ) CAFs were transfected with NAB2 overexpression or control vector and seeded in a bottom well of the plate. FaDu cells were added to the Matrigel-coated transwell. ( B ) After culturing for 48 h, cells in the transwell were stained with crystal violet and those that had migrated to the lower surface of the transwell insert were counted (5× magnification). Cell invasion index was calculated as the difference in the number invaded cells between the NAB2 overexpression and control groups. The effects of siNAB2 or control siRNA transfection were compared under the same conditions. To evaluate the effect of conditioned medium (CM) from CAF cultures on FaDu spheroid invasion, short-term primary culture of CAFs and paired NTFs were transfected with NAB2 overexpression or control vector, or with siNAB2 or control siRNA. After 48 h, the culture supernatant was collected from each plate and passed through a 0.45 μM filter and used as CM. ( C ) FaDu spheroid (>500 μm in diameter) was formed by culturing in a 96-well U-bottom ultra-low attachment plate (5000 cells/well) for 3 days. Matrigel (50 μL) was added to each well containing 100 μL CM, and spheroid invasion was monitored for 14 days by phase-contrast microscopy (5× magnification). ( D ) Cell invasion was quantified by measuring the average length or number of tube-like structures extending from the surface of each spheroid. ( E ) MMP mRNA expression in FaDu spheroids was analyzed by qPCR on day 14. Results represent the mean ± standard deviation of two or three experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Fibroblasts were transiently transfected with a siRNA mixture targeting the NAB2 transcript (siNAB2; Santa Cruz Biotechnology).

Techniques: Transfection, Over Expression, Control, Plasmid Preparation, Staining, Microscopy, Expressing, Standard Deviation

Journal: Cancers

Article Title: NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression

doi: 10.3390/cancers11030388

Figure Lengend Snippet: Effect of NAB2-overexpressing CAFs on the growth of FaDu spheroid-derived tumors in nude mice. FaDu spheroids (>400 μm in diameter) were prepared in 96-well U-bottom ultra-low attachment plates. CAFs from P3 transfected with NAB2 overexpression or control vector; 50 FaDu spheroids and 5 × 10 5 CAFs transfected with NAB2 overexpression or control vector were co-injected into the oral mucosa of the left and right cheeks, respectively, of BALB/c mice. ( A ) Mice were sacrificed after 6 weeks and tumor size was measured. ( B ) Hematoxylin and eosin (H&E) staining was performed using tumor tissues from nude mice. The red and black arrows indicate some representative lobulated margins and increased invasion into the surrounding connective tissue in tumor, respectively. ( C ) mRNA expression of MMPs and invasion-related genes in tumor tissues was investigated at the same time. * p < 0.05, ** p < 0.01.

Article Snippet: Fibroblasts were transiently transfected with a siRNA mixture targeting the NAB2 transcript (siNAB2; Santa Cruz Biotechnology).

Techniques: Derivative Assay, Transfection, Over Expression, Control, Plasmid Preparation, Injection, Staining, Expressing

Journal: Cancers

Article Title: NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression

doi: 10.3390/cancers11030388

Figure Lengend Snippet: Putative schematic of HNSCC progression by NAB2 derived from CAF. Results represent the mean ± standard deviation of two experiments.

Article Snippet: Fibroblasts were transiently transfected with a siRNA mixture targeting the NAB2 transcript (siNAB2; Santa Cruz Biotechnology).

Techniques: Derivative Assay, Standard Deviation

Journal: Cancers

Article Title: NGFI-A Binding Protein 2 Promotes EGF-Dependent HNSCC Cell Invasion

doi: 10.3390/cancers11030315

Figure Lengend Snippet: Effect of NAB2 on EGF-dependent head and neck squamous cell carcinoma (HNSCC) cell invasion. ( A ) NAB2 mRNA and protein expression following EGF treatment (50 ng/mL) for 24 h was analyzed in FaDU cells by qPCR and Western blot analysis. ( B ) FaDU cells were transfected with an siNAB2 mixture (40 nm/mL) for 48 h, after which the mRNA and protein expression of NAB2 were analyzed. ( C ) Two-dimensional Transwell Matrigel invasion assays were performed to analyze the effect of NAB2 knockdown, followed by EGF treatment, on FaDU cell invasion. Control siRNA- or siNAB2-transfected FaDU cells were added to each Matrigel-coated Transwell. After culturing for 48 h, the cells were washed twice with phosphate-bufferd saline and stained with 0.2% crystal violet in 10% ethanol (5× magnification). The cell invasion index was calculated as the difference between the number of invading cells in the siNAB2-transfected group compared to that in the control siRNA-transfected group (* p < 0.01). ( D ) Matrigel invasion assays using YD-10B cells were performed with the same conditions (5× magnification) (** p < 0.005). The results shown are from two or three independent experiments, with each bar representing the standard deviation.

Article Snippet: Cells were suspended in serum-free medium, which was followed by transfection with control siRNA (sc-37007) or an siNAB2 mixture (sc-36014) consisting of three specific targeting oligonucleotides (Santa Cruz Biotechnology) using an electroporation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Techniques: Expressing, Western Blot, Transfection, Knockdown, Control, Saline, Staining, Standard Deviation

Journal: Cancers

Article Title: NGFI-A Binding Protein 2 Promotes EGF-Dependent HNSCC Cell Invasion

doi: 10.3390/cancers11030315

Figure Lengend Snippet: Effect of NAB2 on EGF-dependent FaDU spheroid invasion. ( A ) FaDU cells were seeded in a 96-well ultralow attachment U-bottom plate (6000 cells per well) and cultured for 4 days to form one spheroid per well (>700 μm in diameter). Control siRNA or siNAB2 was transfected into FaDU spheroids using Lipofectamine 2000 for 10 days and NAB2 mRNA expression was evaluated by qPCR. ( B ) The Matrigel invasion of FaDU spheroids transfected with siNAB2 was investigated after EGF treatment (50 ng/mL). FaDU spheroids cultured for 5 days were transfected with control siRNA or siNAB2 using Lipofectamine 2000. After 8 h, the spheroids were centrifuged and 50 μL of Matrigel matrix was added directly to each well (100 μL of medium) to provide a semi-solid matrix into which tumor cells could migrate from the spheroid body. Matrigel invasion was monitored over a period of 10 days by phase-contrast microscopy (5× magnification). ( C ) Matrigel invasion was quantified based on the average length of tube-like structures protruding from the surface of each spheroid (* p < 0.005). The results shown are from two or three independent experiments, with each bar representing the standard deviation.

Article Snippet: Cells were suspended in serum-free medium, which was followed by transfection with control siRNA (sc-37007) or an siNAB2 mixture (sc-36014) consisting of three specific targeting oligonucleotides (Santa Cruz Biotechnology) using an electroporation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Techniques: Cell Culture, Control, Transfection, Expressing, Microscopy, Standard Deviation

Journal: Cancers

Article Title: NGFI-A Binding Protein 2 Promotes EGF-Dependent HNSCC Cell Invasion

doi: 10.3390/cancers11030315

Figure Lengend Snippet: Effect of knockdown or overexpression of EGR1, NAB2, and Sp1 on MMP expression. ( A ) FaDU cells were transfected with control siRNA or siNAB2 for 24 h, and then subjected to EGF treatment (50 ng/mL) for another 24 h. Transfected cells were analyzed for mRNA and protein expression of MMP2 and MMP9. Protein expression was quantified and plotted in three independent experiments. ( B ) A schematic representation of the promoter regions containing the consensus EGR1/Sp1-binding sites (black arrowheads). FaDU cells were transfected with control siRNA or siNAB2 and chromatin was immunoprecipitated with ( C ) anti-Sp1 antibody or ( D ) anti-EGR1 antibody. The resulting immunoprecipitates were analyzed by qPCR to detect the consensus promoter sequences. ( E ) EGR1 / NAB2 genes were overexpressed in FaDU cells, after which chromatin was immunoprecipitated with an anti-Sp1 antibody. The resulting immunoprecipitates were analyzed by qPCR to detect the consensus promoter sequences. ( F ) For luciferase reporter assays using MMP2-1 and MMP9-1, promoter fragments were synthesized and cloned into the pGL4.70 vector. After 24 h of vector transfection, dual luciferase assays were performed to determine the effect of different combinations of EGR1/NBA2 overexpression (over) and/or Sp1 knockdown. The results shown are based on two or three independent experiments, with each bar representing the standard deviation.

Article Snippet: Cells were suspended in serum-free medium, which was followed by transfection with control siRNA (sc-37007) or an siNAB2 mixture (sc-36014) consisting of three specific targeting oligonucleotides (Santa Cruz Biotechnology) using an electroporation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Techniques: Knockdown, Over Expression, Expressing, Transfection, Control, Binding Assay, Immunoprecipitation, Luciferase, Synthesized, Clone Assay, Plasmid Preparation, Standard Deviation