Journal: Oncology Reports

Article Title: Suprabasin enhances the invasion, migration, and angiogenic ability of oral squamous cell carcinoma cells under hypoxic conditions

doi: 10.3892/or.2023.8520

Figure Lengend Snippet: Effects of knockdown and overexpression of SBSN on growth of SAS cells. In this experiment, siRNA for SBSN or control siRNA, and HaloTag-SBSN (OE) or HaloTag control vector, were transfected into SAS cells and incubated under normoxic conditions for 24 h. Next, the cells were exposed to normoxic or hypoxic conditions. After 0, 1 and 3 days, cells were subjected to the (A) MTT assay and (B) BrdU assay, and the (C and D) cell cycle assay. For the MTT assay, the value at day 0 is designated as ‘1’, and relative values are shown. The value of results was subjected to ANOVA. *P<0.05 vs. control. In panel D, the percentage of cell-cycle phase (G0 and G1, S, and G2/M) in each sample is shown in a table. All experiments were performed four times at least, and a representative result is shown. SBSN, suprabasin; siRNA, small interfering RNA; OE, overexpression; BrdU, 5-bromo-2′-deoxyuridine; OE, overexpression.

Article Snippet: The expression vectors HaloTag-SBSN (pFN21AE2798) and HaloTag control vector (G659) were purchased from Promega Corporation.

Techniques: Over Expression, Plasmid Preparation, Transfection, Incubation, MTT Assay, BrdU Staining, Cell Cycle Assay, Small Interfering RNA

Journal: Oncology Reports

Article Title: Suprabasin enhances the invasion, migration, and angiogenic ability of oral squamous cell carcinoma cells under hypoxic conditions

doi: 10.3892/or.2023.8520

Figure Lengend Snippet: Effects of knockdown and overexpression of SBSN on apoptosis and autophagy of SAS cells. (A) First, siRNA for SBSN or control siRNA, and HaloTag-SBSN (OE) or HaloTag control vector, were transfected into SAS cells and incubated under normoxic conditions for 24 h. Next, the cells were exposed to normoxic or hypoxic conditions. After 0, 1 and 3 days, either (A) the cells were subjected to caspase 3/7 assays or (B) total cellular proteins were subjected to western blot analysis to detect SBSN and HaloTag (overexpressed and endogenous proteins are indicated by arrows along with each molecular weight on the right side of right panel), as well as HIF-1α, HIF-2α, LC3-I, LC3-II, p62 and β-actin. For caspase 3/7 assays, the value at day 0 is designated as ‘1’, and relative values are shown. The value of results was subjected to ANOVA. *P<0.05 vs. control. All experiments were performed four times at least, and a representative result is shown. SBSN, suprabasin; siRNA, small interfering RNA; OE, overexpression; HIF, hypoxia-inducible factor.

Article Snippet: The expression vectors HaloTag-SBSN (pFN21AE2798) and HaloTag control vector (G659) were purchased from Promega Corporation.

Techniques: Over Expression, Plasmid Preparation, Transfection, Incubation, Western Blot, Molecular Weight, Small Interfering RNA

Journal: Oncology Reports

Article Title: Suprabasin enhances the invasion, migration, and angiogenic ability of oral squamous cell carcinoma cells under hypoxic conditions

doi: 10.3892/or.2023.8520

Figure Lengend Snippet: Effects of knockdown and overexpression of SBSN on cell invasion, migration, MMP activities, and EMT. First, siRNA for SBSN or control siRNA, and HaloTag-SBSN (OE) or HaloTag control vector, were transfected into SAS cells. (A and B) Cell invasion assay in which the transfected cells were incubated under normoxic or hypoxic conditions for 48 h. From these, 1-ml aliquots of the condition medium were collected and used as chemoattractants. Freshly suspended SAS cells were subjected to cell invasion assay for 48 h. Then, the invasive cells were stained by crystal violet. (A) Images of the invasive cells were captured and (B) the optical absorbance of destained solution from the membrane was measured. (C and D) Wound healing assay. The transfected cells were reseeded to a 24-well tissue culture plate, exposed to normoxic or hypoxic conditions for 24 h, and subjected to a cell migration assay. (E) Gelatin zymography, in which the transfected cells were exposed to normoxic or hypoxic conditions for 48 h, the condition medium was collected and concentrated, and 10-ml aliquots of each concentrated condition medium were subjected to gelatin-zymography. (F) Western blotting analysis for epithelial-mesenchymal transition, in which the transfected cells were exposed to normoxic or hypoxic conditions for 0, 1 and 3 days. Next, total cellular proteins were subjected to western blot analysis to detect E-cadherin, N-cadherin and β-actin. The values in panels (B) and (D) were subjected to ANOVA. *P<0.05 vs. control. Scale bars: (A) 200 µm (×100) and (C) 1 mm (×40). All experiments were performed four times at least, and a representative result is shown. SBSN, suprabasin; MMP, matrix metalloprotease; siRNA, small interfering RNA; OE, overexpression.

Article Snippet: The expression vectors HaloTag-SBSN (pFN21AE2798) and HaloTag control vector (G659) were purchased from Promega Corporation.

Techniques: Over Expression, Migration, Plasmid Preparation, Transfection, Invasion Assay, Incubation, Staining, Wound Healing Assay, Cell Migration Assay, Zymography, Western Blot, Small Interfering RNA

Journal: Oncology Reports

Article Title: Suprabasin enhances the invasion, migration, and angiogenic ability of oral squamous cell carcinoma cells under hypoxic conditions

doi: 10.3892/or.2023.8520

Figure Lengend Snippet: Effects of knockdown and overexpression of SBSN on in vitro angiogenesis. (A) First, siRNA for SBSN or control siRNA, and HaloTag-SBSN (OE) or HaloTag control vector were transfected into SAS cells and incubated under normoxic conditions for 24 h. Next, the culture medium was changed to serum-free EBM-2 medium, and the cells were cultured for another 48 h under normoxic conditions. The conditioned medium of SAS cells was collected for tube formation assay. The experimental HUVEC cells were suspended by the conditioned medium of SAS cells and were subjected to tube formation assay. After 24 h, (Α) images of the cells were captured, and (B) formed junctions of the endothelial tubes were counted under a microscope. The results were subjected to ANOVA. *P<0.05. Scale bars: 200 µm (×200). All experiments were performed four times at least, and a representative result is shown. SBSN, suprabasin; siRNA, small interfering RNA; OE, overexpression.

Article Snippet: The expression vectors HaloTag-SBSN (pFN21AE2798) and HaloTag control vector (G659) were purchased from Promega Corporation.

Techniques: Over Expression, In Vitro, Plasmid Preparation, Transfection, Incubation, Cell Culture, Tube Formation Assay, Microscopy, Small Interfering RNA

Journal: Scientific Reports

Article Title: TDP-43 self-interaction is modulated by redox-active compounds Auranofin, Chelerythrine and Riluzole

doi: 10.1038/s41598-018-20565-0

Figure Lengend Snippet: TDP-43 self-interaction. NanoBit ® luciferase complementation assay for protein interactions has been used to measure interaction of different N-terminal large (LgBit, pFN33), small (SmBit, pFN35), C-terminal large (LgBit, pFC34) and small (SmBit, pFC36) fusion proteins of TDP-43, positive control vectors SmBiT-PRKACA and LgBiT-PRKAR2A (coding the catalytic and regulatory subunits of PKA) and vector SmBiT-Halotag, which contains haloalkane dehalogenase – smBit fusion protein (Halitag) as a negative control, were obtained from Promega (Madison, WI, USA) and have been used in parallel 24 h after transfection of N2a cells and seeding cells into 384-well plates, Mean ± SD, Mann-Whitney-U-Test **p < 0.01.

Article Snippet: NanoBit ® luciferase complementation assay for protein interactions, N-terminal large (LgBiT, pFN33) and small (SmBiT, pFN35), C-terminal LgBiT (pFC34) and SmBiT (pFC36) vectors, constitutive NanoLuc luciferase pNL1.1.TK[Nluc/TK] vector ( = Nluc vector), positive control vectors SmBiT-PRKACA and LgBiT-PRKAR2A and negative control vector SmBiT-Halotag were obtained from Promega (Madison, WI, USA).

Techniques: Luciferase, Positive Control, Plasmid Preparation, Negative Control, Transfection, MANN-WHITNEY