Journal: Scientific Reports

Article Title: Designing and fabrication of electrochemical nano-biosensor for the fast detection of SARS-CoV-2-RNA

doi: 10.1038/s41598-023-32168-5

Figure Lengend Snippet: ( A ) Using different crosslinking agents including 4-ATP, EDC/NHS, and DPA/EDC/NHS. The voltammetric and the impedimetric signals were investigated in FCN/TE. Modified surfaces with the 4 - ATP showed the highest significant response. ( B ) The 4-ATP -based electrode showed a remarkably decrease in the voltammetric signals after the hybridization with the matched oligonucleotides (I). Significant impedimetric signals were obtained for the hybridized attached probe (II).

Article Snippet: Signature probes, matched oligonucleotides, Mismatched in one nucleotide, and completely mismatched oligonucleotides were then designed and sent for synthesis (Introgen co., USA).

Techniques: Modification, Hybridization

Journal: Scientific Reports

Article Title: Designing and fabrication of electrochemical nano-biosensor for the fast detection of SARS-CoV-2-RNA

doi: 10.1038/s41598-023-32168-5

Figure Lengend Snippet: Standard calibration curve of the SARS-CoV-2 RNA biosensor using different concentrations of the virus signature nucleotide sequence ranging from 10 fM to 2 µM. In FCN mediating Tris buffer, the impedimetric signals were investigated before and after the hybridization with the different concentrations of the matched oligos for three individual readings. ΔR ct values determined after fitting the resulted signals using the Randles cell circuit where R1: FCN/TE buffer resistance, C1: capacitance of sensor surface, R2: resistance of immobilized probe, CPE1: constant phase elements of polymerized amino thiophenol monolayer, R3: the resistance of the hybridized matched oligo, and W1: Warburg impedance for the FCN diffusion on the electrode surface. The LOD and LOQ were 298.1 and 993.7 fM, respectively. The obtained R2 from the linear regression was 0.99 and P -value was < 0.0001.

Article Snippet: Signature probes, matched oligonucleotides, Mismatched in one nucleotide, and completely mismatched oligonucleotides were then designed and sent for synthesis (Introgen co., USA).

Techniques: Virus, Sequencing, Hybridization, Diffusion-based Assay

Journal: Scientific Reports

Article Title: Designing and fabrication of electrochemical nano-biosensor for the fast detection of SARS-CoV-2-RNA

doi: 10.1038/s41598-023-32168-5

Figure Lengend Snippet: The impedimetric response of the fabricated SARS-CoV-2-RNA biosensor chips towards the mismatched designed Oligos (one nucleotide, completely mismatched), the chips were used in triplicates. The ΔRct was estimated and the percentage of the interference was obtained. The electrodes showed a response of 79% and 40.6%, respectively. The error bars expressed the standard deviations (SDs) between the three electrode readings.

Article Snippet: Signature probes, matched oligonucleotides, Mismatched in one nucleotide, and completely mismatched oligonucleotides were then designed and sent for synthesis (Introgen co., USA).

Techniques:

Journal: Cancer Medicine

Article Title: High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma

doi: 10.1002/cam4.3041

Figure Lengend Snippet: SOX8 is recognized to be a protein binding to GOLPH3 promote within the tongue squamous cell carcinoma (TSCC) cell lines. Numerous transcriptional factor‐binding sites of SOX8 are identified in the core promotor region of GOLPH3 (A). The 5'‐biotin conjugated DNA probe (585‐bp) corresponding to GOLPH3 promoter region comprising the nucleotides −585 to +1 is synthesized (B). The GOLPH3 promoter‐binding proteins band levels (about 30 kDa) are markedly increased within the TSCC cell lines (SCC25, SCC9, HSC3, and HSC6), compared with the normal oral epithelium (NOK) cells (C). More SOX8 is bound onto GOLPH3 promoter probe within the TSCC cell lines (SCC25, SCC9, HSC3, and HSC6), compared with NOK cells (D). ChIP assay finds that SOX8 strongly binds onto GOLPH3 promoter region among the four TSCC cell types (SCC25, SCC9, HSC3, and HSC6), in comparison with NOK cells (E). SOX8 binding onto GOLPH3 promoter probe is elevated within SCC9 cells with SOX8 over‐expression (F), nonetheless, it is reduced within SCC25 cells with SOX8 knockdown (G). ChIP assay confirms that SOX8 is weakly bound onto GOLPH3 promoter within SCC25 cells with SOX8 knockdown (H), but it is strongly bound onto GOLPH3 promoter within SCC9 cells with SOX8 over‐expression (I)

Article Snippet: The streptavidin‐agarose pull‐down experiment was carried out according to previous description to analyze the proteins binding to GOLPH3 promoter., Firstly, extracts of nuclear protein (0.5 mg) isolated based on TSCC cancer cells would be subjected to incubation with the biotin‐conjugated double strand DNA probes (20 µg)matched with the nucleotides from −585 to +1 in the promoter region of GOLPH3 (Sigma‐Aldrich), together with the agarose‐streptavidin beads (200 µL, Sigma‐Aldrich) overnight under 4°C.

Techniques: Protein Binding, Binding Assay, Synthesized, Over Expression

Journal: Cancer Medicine

Article Title: High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma

doi: 10.1002/cam4.3041

Figure Lengend Snippet: SOX8 increased GOLPH3 expression and promoter activity within the tongue squamous cell carcinoma (TSCC) cell lines. Luciferase reporter gene assay finds that, SOX8 over‐expression enhances activity of GOLPH3 promoter within SCC9 and HS3 cells, compared to the vector‐treated group (A), whereas SOX8 knockdown dramatically decreases activity of GOLPH3 promoter within SCC25 as well as HSC6 cells, compared with groups treated with nonspecific shRNA (shNC) (B). SOX8 knockdown is found to inhibit GOLPH3 expression at the translational and transcriptional levels within HSC6 (C), while SOX8 over‐expression enhances the GOLPH3 mRNA and protein levels in HSC3 cells (D). Also, western blotting is carried out, which confirms that SOX8 knockdown inhibits GOLPH3 expression in SCC25 cells, and that SOX8 over‐expression enhances the activity of GOLPH3 promoter in SCC9 cells (E). Besides, western blotting and RT‐qPCR show that neither SOX9 (F) nor SOX10 (G) regulates GOLPH3 expression in TSCC cells at transcriptional and translational levels

Article Snippet: The streptavidin‐agarose pull‐down experiment was carried out according to previous description to analyze the proteins binding to GOLPH3 promoter., Firstly, extracts of nuclear protein (0.5 mg) isolated based on TSCC cancer cells would be subjected to incubation with the biotin‐conjugated double strand DNA probes (20 µg)matched with the nucleotides from −585 to +1 in the promoter region of GOLPH3 (Sigma‐Aldrich), together with the agarose‐streptavidin beads (200 µL, Sigma‐Aldrich) overnight under 4°C.

Techniques: Expressing, Activity Assay, Luciferase, Reporter Gene Assay, Over Expression, Plasmid Preparation, shRNA, Western Blot, Quantitative RT-PCR

Journal: Cancer Medicine

Article Title: High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma

doi: 10.1002/cam4.3041

Figure Lengend Snippet: SOX8 promotes tongue squamous cell carcinoma (TSCC) growth via the GOLPH3 signaling pathway in vitro . Western blotting confirms SOX8 knockdown in SCC25 cells by SOX8‐specific shRNAs (sh#1 and sh#2) (A). SOX8 knockdown decreases the viability (B) and colony‐forming capacity of SCC25 cells (C). Western Blotting confirms the over‐expression of SOX8 in SCC25 cells (D). SOX8 over‐expression promotes the proliferation and viability (E), and the colony‐forming capacity (F) of SCC25 cells. In SOX8‐depleted cells, GOLPH3 over‐expression rescues the GOLPH3 protein expression (G), together with cell viability (H) and colony forming capacity (I). Moreover western blotting suggests that SOX8 over‐expression up‐regulates the activation of p‐PI3K, p‐GSK3β, and p‐FOXO1, but not the total expression of PI3K, GSK3β, and FOXO1 in SCC9 cells (J). Immunoblotting test indicates that GOLPH3 over‐expression rescues the protein expression of p‐AKT, p‐GSK3β, and p‐FOXO1, which is markedly down‐regulated following SOX8 knockdown, respectively, in SCC25 cells (K)

Article Snippet: The streptavidin‐agarose pull‐down experiment was carried out according to previous description to analyze the proteins binding to GOLPH3 promoter., Firstly, extracts of nuclear protein (0.5 mg) isolated based on TSCC cancer cells would be subjected to incubation with the biotin‐conjugated double strand DNA probes (20 µg)matched with the nucleotides from −585 to +1 in the promoter region of GOLPH3 (Sigma‐Aldrich), together with the agarose‐streptavidin beads (200 µL, Sigma‐Aldrich) overnight under 4°C.

Techniques: In Vitro, Western Blot, Over Expression, Expressing, Activation Assay

Journal: Cancer Medicine

Article Title: High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma

doi: 10.1002/cam4.3041

Figure Lengend Snippet: SOX8 regulates the invasion and migration of tongue squamous cell carcinoma (TSCC) cells via GOLPH3. SOX8 knockdown remarkably inhibits the wound healing rate (A and B), as well as migration and invasion rates (C and D) in SCC25 cells. Inversely, SOX8 over‐expression increases the wound healing rate (E and F), together with the migration and invasion rates (G and H) of SCC9 cells. It is also found that GOLPH3 knockdown also evidently inhibited the invasion and migration of SCC25 (I and J) and HSC6 cells (K and L). But, GOLPH3over‐expression rescues the migration and invasion rates in SOX8‐depleted cells. Western blotting finds that, only SOX8 knockdown or GOLPH3 knockdown notably down‐regulates the protein expression of β‐catenin, E‐cadherin, Vimentin, Snail, and c‐Myc in SCC25 and HSC6 cells. However, the over‐expression of GOLPH3 in cells with stable SOX8 knockdown distinctly antagonized β‐catenin, Vimentin, E‐cadherin, c‐Myc, and Snail protein expression (M)

Article Snippet: The streptavidin‐agarose pull‐down experiment was carried out according to previous description to analyze the proteins binding to GOLPH3 promoter., Firstly, extracts of nuclear protein (0.5 mg) isolated based on TSCC cancer cells would be subjected to incubation with the biotin‐conjugated double strand DNA probes (20 µg)matched with the nucleotides from −585 to +1 in the promoter region of GOLPH3 (Sigma‐Aldrich), together with the agarose‐streptavidin beads (200 µL, Sigma‐Aldrich) overnight under 4°C.

Techniques: Migration, Over Expression, Expressing, Western Blot

Journal: Cancer Medicine

Article Title: High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma

doi: 10.1002/cam4.3041

Figure Lengend Snippet: SOX8 promotes tongue squamous cell carcinoma (TSCC) tumor growth and metastasis via GOLPH3. SCC25 xenografts with SOX8 over‐expression grow fastest than those in other groups. In contrast, SCC25 xenografts with GOLPH3 knockdown grow at the slowest rate (A), but SCC25 xenografts with SOX8 over‐expression combined with GOLPH3 knockdown grow lower than those with SOX8 over‐expression alone. The ranks of tumor size and weight are in accordance with tumor growth, which follow the order of SOX8 over‐expression, vector, SOX8 over‐expression combined with GOLPH3 knockdown, and GOLPH3 knockdown in succession (B and C). HE staining and metastatic tumor counting suggests that, SOX8 over‐expression enhances metastasis formation in lungs (D). In contrast, GOLPH3 knockdown markedly reduces metastasis number in lungs (E). Immunochemical staining is carried out to analyze the protein levels of SOX8, GOLPH3, and Ki67 in xenografts (F). The results show that, GOLPH3 expression is markedly up‐regulated in the SOX8 over‐expression group, and that GOLPH3 staining is notably increased in SOX8 over‐expression combined with GOLPH3 knockdown group, compared with that in GOLPH3 knockdown group. Silencing GOLPH3 expression dramatically reduces Ki67 expression relative to control groups. However, SOX8 over‐expression up‐regulates Ki67 expression level. Interestingly, Ki67 expression is up‐regulated in the SOX8 over‐expression combined with GOLPH3 knockdown group, compared with that in GOLPH3 knockdown group

Article Snippet: The streptavidin‐agarose pull‐down experiment was carried out according to previous description to analyze the proteins binding to GOLPH3 promoter., Firstly, extracts of nuclear protein (0.5 mg) isolated based on TSCC cancer cells would be subjected to incubation with the biotin‐conjugated double strand DNA probes (20 µg)matched with the nucleotides from −585 to +1 in the promoter region of GOLPH3 (Sigma‐Aldrich), together with the agarose‐streptavidin beads (200 µL, Sigma‐Aldrich) overnight under 4°C.

Techniques: Over Expression, Plasmid Preparation, Staining, Expressing

Journal: Cancer Medicine

Article Title: High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma

doi: 10.1002/cam4.3041

Figure Lengend Snippet: SOX8 interacts with TFAP2A to regulate GOLPH3 expression and tongue squamous cell carcinoma (TSCC) cell growth. Co‐immunoprecipitation assay is carried out to test 4 predicted GOLPH3 promoter‐binding proteins, namely, p65, p50, SP1, and TFAP2A, using specific antibodies against each of these proteins in the flag‐SOX8‐overexpressing SCC25 and HSC6 cells. Surprisingly, SOX8 is knocked down via the anti‐TFAP2A antibodies (A). Besides, the SOX8‐TFAP2A interaction is also validated in the other co‐immunoprecipitation assay that uses the SOX8‐specific antibodies (B). Additionally, dual‐immunofluorescence assay is used for further analyzing TFAP2A and SOX8 expression as well as the sub‐cellular localization within SCC9 as well as SCC25 cells. SOX8 (green) is expressed both within the cytoplasm and nucleus, while TFAP2A (red) expression is mainly detected in cytoplasm. The TFAP2A‐SOX8 co‐localization is detected in the nucleus and cytoplasm (yellow). On the other hand, ChIP assay confirms that TFAP2A binds onto GOLPH3 promoter in SCC9 and SCC25 cells (E). Furthermore, the over‐expression of TFAP2A enhances SOX8 binding onto the GOLPH3 promoter. By contrast, the knockdown of TFAP2A weakens SOX8 binding onto GOLPH3 promoter within the SCC25 cells with SOX8 over‐expression. Knockdown of SOX8 weakens SOX8 binding onto the GOLPH3 promoter within SCC25 cells with TFAP2A over‐expression (D). Moreover, luciferase reporter gene assay is carried out, which suggests that TFAP2A over‐expression enhances activity of GOLPH3 promoter, whereas knockdown of TFAP2A suppresses activity of GOLPH3 promoter. Additionally, only SOX8 knockdown or TFAP2A knockdown reduces the GOLPH3 promoter activity in SCC25 cells with TFAP2A or SOX8 over‐expression, respectively (F). Western Blotting (H) and RT‐PCR (G) suggest that, TFAP2A over‐expression up‐regulates GOLPH3 mRNA and protein expression, whereas the expression of GOLPH3 is down‐regulated following TFAP2A knockdown, even in SOX8‐overexpressing SCC25 cells. Finally, CCK8 assay indicates that TFAP2A knockdown inhibits the increased TSCC cell growth induced by SOX8 over‐expression, while over‐expression of TFAP2A partially rescues the inhibition of cell growth mediated by SOX8 knockdown (I)

Article Snippet: The streptavidin‐agarose pull‐down experiment was carried out according to previous description to analyze the proteins binding to GOLPH3 promoter., Firstly, extracts of nuclear protein (0.5 mg) isolated based on TSCC cancer cells would be subjected to incubation with the biotin‐conjugated double strand DNA probes (20 µg)matched with the nucleotides from −585 to +1 in the promoter region of GOLPH3 (Sigma‐Aldrich), together with the agarose‐streptavidin beads (200 µL, Sigma‐Aldrich) overnight under 4°C.

Techniques: Expressing, Co-Immunoprecipitation Assay, Binding Assay, Immunofluorescence, Over Expression, Luciferase, Reporter Gene Assay, Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, CCK-8 Assay, Inhibition

Journal: Cancer Medicine

Article Title: High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma

doi: 10.1002/cam4.3041

Figure Lengend Snippet: SOX8 shows positive correlation with GOLPH3, and these two are highly expressed in tongue squamous cell carcinoma (TSCC) cell lines and tumor tissues. By immunoblotting analysis, the SOX8 and GOLPH3 protein expression is evidently up‐regulated within TSCC tissues compared with that in matched para‐cancer counterparts (A). Besides, the SOX8 protein expression shows positive correlation the GOLPH3 protein expression (Pearson correlation test; n = 16, r = .497, P = .031) (B). Furthermore, our findings show that, both SOX8 and GOLPH3 mRNA levels are obviously increased within TSCC tumor tissues relative to those within matched non‐cancer counterparts (C). In addition, SOX8 mRNA level shows positive correlation with the GOLPH3 mRNA level (D). Finally, the SOX8 and GOLPH3 protein and mRNA expression is analyzed within TSCC cell lines via western blotting and RT‐PCR, respectively. According to our findings, SOX8 expression is positively correlated with GOLPH3 expression (E and F). Analysis of subcellular localization via immunofluorescence indicates that SOX8 and GOLPH3 are mainly expressed in the cytoplasm. The above findings further demonstrate the regulatory effect of SOX8 on GOLPH3 within TSCC (G)

Article Snippet: The streptavidin‐agarose pull‐down experiment was carried out according to previous description to analyze the proteins binding to GOLPH3 promoter., Firstly, extracts of nuclear protein (0.5 mg) isolated based on TSCC cancer cells would be subjected to incubation with the biotin‐conjugated double strand DNA probes (20 µg)matched with the nucleotides from −585 to +1 in the promoter region of GOLPH3 (Sigma‐Aldrich), together with the agarose‐streptavidin beads (200 µL, Sigma‐Aldrich) overnight under 4°C.

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: Cancer Medicine

Article Title: High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma

doi: 10.1002/cam4.3041

Figure Lengend Snippet: High SOX8 and GOLPH3 expression Levels are related to dismal clinical results for tongue squamous cell carcinoma (TSCC) patients. Tissue microarrays (n = 89) are used to investigate the influence of up‐regulated SOX8 and GOLPH3 expression on the clinical results for TSCC patients. The relationships of SOX8 level with clinicopathological parameters from the 89 TSCC patients are displayed in (A). IHC analysis shows the representative results of SOX8 and GOLPH3 in tumor tissues from three different patients (B). High expression of SOX8 and GOLPH3 is also detected, which is positively correlated within TSCC tissues (C, D). Moreover patients that have up‐regulated SOX8 level are associated with reduced overall survival (OS) compared with patients having low SOX8 level (E), and this is also true regarding the GOLPH3 level (F). In addition, the OS for cases with up‐regulated SOX8 and GOLPH3 expression is dramatically reduced compared with that for patients with low SOX8 and GOLPH3 expression (G)

Article Snippet: The streptavidin‐agarose pull‐down experiment was carried out according to previous description to analyze the proteins binding to GOLPH3 promoter., Firstly, extracts of nuclear protein (0.5 mg) isolated based on TSCC cancer cells would be subjected to incubation with the biotin‐conjugated double strand DNA probes (20 µg)matched with the nucleotides from −585 to +1 in the promoter region of GOLPH3 (Sigma‐Aldrich), together with the agarose‐streptavidin beads (200 µL, Sigma‐Aldrich) overnight under 4°C.

Techniques: Expressing