nab2 transcript  (Santa Cruz Biotechnology)

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: 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