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human tongue squamous cell carcinoma scc9 cell line (ATCC)

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ATCC human tongue squamous cell carcinoma scc9 cell line

CD109 promotes IL6Rα stability and IL-6 induced phosphorylation of STAT3 in SCC cells. A , B A431, <t>SCC9</t> wild-type (WT) cells and CD109 knockout (KO) cells were treated with IL-6 (20 ng/mL) for 30 min and the expression of CD109, IL6Rα and phosphorylation of the STAT3 (Y705) proteins were analyzed by Western blotting. Densitometric analysis of the data depicted in A and B are shown in supplementary data. C A431-WT and A431-CD109 KO cells were treated with IL-6 (20 ng/ml) for various time periods (0–4 h), and the levels of phospho-STAT3 (Y705) and total STAT3 were assessed by Western blot. β-Actin was used as a loading control. D Fluorescence microscopy showing the levels of phospho-STAT3 (Y705) (upper panels, green) and total STAT3 (lower panels, green) in A431-WT and A431-CD109 KO cells treated with IL-6 (20 ng/ml). Scale bar: 25 μm. E A431-WT and A431-CD109 KO cells transfected with IL6Rα siRNA or control siRNA and treated with IL-6 (20 ng/ml), and the expression of IL6Rα, CD109 and the levels of phospho-STAT3 (Y705) were analyzed by Western blotting. F A431 and SCC9 cells were treated without or with (20 ug) tocilizumab for 24 h and the expression of IL6Rα, CD109 and phosphorylation of STAT-3 (Y705) were analyzed by Western blotting. All results ( A – F ) are representative of at least 3 independent experiments. Significance was calculated using a student T-test. NS: Not significant, * P < 0.05, ** P < 0.01 and *** P < 0.0010

Human Tongue Squamous Cell Carcinoma Scc9 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human tongue squamous cell carcinoma scc9 cell line - by Bioz Stars, 2026-10

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1) Product Images from "IL-6-mediated tumorigenicity and antioxidant state in squamous cell carcinoma cells are driven by CD109 via stabilization of IL-6 receptor-alpha and activation of STAT3/NRF2 pathway"

Journal: Experimental Hematology & Oncology

doi: 10.1186/s40164-025-00630-x

CD109 promotes IL6Rα stability and IL-6 induced phosphorylation of STAT3 in SCC cells. A , B A431, SCC9 wild-type (WT) cells and CD109 knockout (KO) cells were treated with IL-6 (20 ng/mL) for 30 min and the expression of CD109, IL6Rα and phosphorylation of the STAT3 (Y705) proteins were analyzed by Western blotting. Densitometric analysis of the data depicted in A and B are shown in supplementary data. C A431-WT and A431-CD109 KO cells were treated with IL-6 (20 ng/ml) for various time periods (0–4 h), and the levels of phospho-STAT3 (Y705) and total STAT3 were assessed by Western blot. β-Actin was used as a loading control. D Fluorescence microscopy showing the levels of phospho-STAT3 (Y705) (upper panels, green) and total STAT3 (lower panels, green) in A431-WT and A431-CD109 KO cells treated with IL-6 (20 ng/ml). Scale bar: 25 μm. E A431-WT and A431-CD109 KO cells transfected with IL6Rα siRNA or control siRNA and treated with IL-6 (20 ng/ml), and the expression of IL6Rα, CD109 and the levels of phospho-STAT3 (Y705) were analyzed by Western blotting. F A431 and SCC9 cells were treated without or with (20 ug) tocilizumab for 24 h and the expression of IL6Rα, CD109 and phosphorylation of STAT-3 (Y705) were analyzed by Western blotting. All results ( A – F ) are representative of at least 3 independent experiments. Significance was calculated using a student T-test. NS: Not significant, * P < 0.05, ** P < 0.01 and *** P < 0.0010

Figure Legend Snippet: CD109 promotes IL6Rα stability and IL-6 induced phosphorylation of STAT3 in SCC cells. A , B A431, SCC9 wild-type (WT) cells and CD109 knockout (KO) cells were treated with IL-6 (20 ng/mL) for 30 min and the expression of CD109, IL6Rα and phosphorylation of the STAT3 (Y705) proteins were analyzed by Western blotting. Densitometric analysis of the data depicted in A and B are shown in supplementary data. C A431-WT and A431-CD109 KO cells were treated with IL-6 (20 ng/ml) for various time periods (0–4 h), and the levels of phospho-STAT3 (Y705) and total STAT3 were assessed by Western blot. β-Actin was used as a loading control. D Fluorescence microscopy showing the levels of phospho-STAT3 (Y705) (upper panels, green) and total STAT3 (lower panels, green) in A431-WT and A431-CD109 KO cells treated with IL-6 (20 ng/ml). Scale bar: 25 μm. E A431-WT and A431-CD109 KO cells transfected with IL6Rα siRNA or control siRNA and treated with IL-6 (20 ng/ml), and the expression of IL6Rα, CD109 and the levels of phospho-STAT3 (Y705) were analyzed by Western blotting. F A431 and SCC9 cells were treated without or with (20 ug) tocilizumab for 24 h and the expression of IL6Rα, CD109 and phosphorylation of STAT-3 (Y705) were analyzed by Western blotting. All results ( A – F ) are representative of at least 3 independent experiments. Significance was calculated using a student T-test. NS: Not significant, * P < 0.05, ** P < 0.01 and *** P < 0.0010

Techniques Used: Phospho-proteomics, Knock-Out, Expressing, Western Blot, Control, Fluorescence, Microscopy, Transfection

CD109 interacts and co-localizes with IL6Rα in an IL-6-dependent manner. A Immunoprecipitation of total cell lysates from A431-WT versus A431-CD109 KO, and SCC9-WT versus SCC9-CD109 KO cells was done using an anti-CD109 antibody or a control IgG followed by Western blot analysis using an anti-IL6Rα antibody. B The input data showing the levels of CD109 and IL6Rα in the total cell lysates used are shown below with β-Actin as loading control. C Reverse co-immunoprecipitation was performed by immunoprecipitation of total cell lysates from A431-WT versus A431-CD109 KO cells using an anti-IL6Rα antibody or a control IgG followed by Western blot analysis using an anti-CD109 antibody. The input data are same as for panel A. D Representative fluorescence microscopic images showing co-expression and colocalization of CD109 and IL6Rα in A431 cells and oral SCC patient derived tissue. CD109 (red), IL6Rα (green) and DAPI (blue) are shown. All data shown A – D are representative of three independent experiments. Scale bar ( D ): 25 μm

Figure Legend Snippet: CD109 interacts and co-localizes with IL6Rα in an IL-6-dependent manner. A Immunoprecipitation of total cell lysates from A431-WT versus A431-CD109 KO, and SCC9-WT versus SCC9-CD109 KO cells was done using an anti-CD109 antibody or a control IgG followed by Western blot analysis using an anti-IL6Rα antibody. B The input data showing the levels of CD109 and IL6Rα in the total cell lysates used are shown below with β-Actin as loading control. C Reverse co-immunoprecipitation was performed by immunoprecipitation of total cell lysates from A431-WT versus A431-CD109 KO cells using an anti-IL6Rα antibody or a control IgG followed by Western blot analysis using an anti-CD109 antibody. The input data are same as for panel A. D Representative fluorescence microscopic images showing co-expression and colocalization of CD109 and IL6Rα in A431 cells and oral SCC patient derived tissue. CD109 (red), IL6Rα (green) and DAPI (blue) are shown. All data shown A – D are representative of three independent experiments. Scale bar ( D ): 25 μm

Techniques Used: Immunoprecipitation, Control, Western Blot, Fluorescence, Expressing, Derivative Assay

CD109 promotes IL-6-induced expression of stem cell markers and tumorigenicity in SCC cells. A A431-WT versus A431-CD109 KO and B SCC9-WT versus SCC9-CD109 KO and C EV-A431 versus CD109OE-A431 cells were treated with 20 ng/ml of IL-6 for 48 h and Western blot analysis for the indicated stem cell markers was performed. Densitometric analysis of the data depicted in A, B and C are shown in supplementary data. D , E Tumor spheroids were grown using A431-WT versus A431-CD109 KO ( D ) and SCC9-WT versus SCC9-CD109 KO ( E ) cells to assess in vitro tumorigenicity as described in Methods, and the spheroids formed were counted under the microscope. The percentage of cells capable of forming spheroids was calculated as (number of spheroids formed/number of cells plated) × 100. Quantitation of the results shown in D and E are shown at the bottom panels. Scale bar: 100 μm. Significance between either wt-IL6 and KO-IL6 or wt + IL6 and KO + IL6 or EVA431-IL6 and OECD109A431-IL6 or EVA431 + IL6 and OECD109A431 + IL6 was calculated using a Student’s T-test: NS: Non-Significant, * P < 0.05, ** P < 0.01 and *** P < 0.001

Figure Legend Snippet: CD109 promotes IL-6-induced expression of stem cell markers and tumorigenicity in SCC cells. A A431-WT versus A431-CD109 KO and B SCC9-WT versus SCC9-CD109 KO and C EV-A431 versus CD109OE-A431 cells were treated with 20 ng/ml of IL-6 for 48 h and Western blot analysis for the indicated stem cell markers was performed. Densitometric analysis of the data depicted in A, B and C are shown in supplementary data. D , E Tumor spheroids were grown using A431-WT versus A431-CD109 KO ( D ) and SCC9-WT versus SCC9-CD109 KO ( E ) cells to assess in vitro tumorigenicity as described in Methods, and the spheroids formed were counted under the microscope. The percentage of cells capable of forming spheroids was calculated as (number of spheroids formed/number of cells plated) × 100. Quantitation of the results shown in D and E are shown at the bottom panels. Scale bar: 100 μm. Significance between either wt-IL6 and KO-IL6 or wt + IL6 and KO + IL6 or EVA431-IL6 and OECD109A431-IL6 or EVA431 + IL6 and OECD109A431 + IL6 was calculated using a Student’s T-test: NS: Non-Significant, * P < 0.05, ** P < 0.01 and *** P < 0.001

Techniques Used: Expressing, Western Blot, In Vitro, Microscopy, Quantitation Assay

CD109 promotes basal and IL-6-induced expression of antioxidant protein NRF2 and its target genes. A A431-WT versus A431-CD109 KO cells and B SCC9-WT versus SCC9-CD109 KO cells and C EV-A431 and OE CD109A431 cells were treated without or with 20 ng/ml of IL-6 for 24 h. Expression levels of NRF2, SOD1 and HO-1 were analyzed by Western blot. Densitometric analysis of the data depicted in A, B and C are shown in supplementary data. D , E Measurement of ROS Levels in A431-WT versus A431-CD109 KO and SCC9-WT versus SCC9-CD109 KO cells. Cells were treated without or with 50 ng/ml of IL-6 for 2, 4 and 24 h. ROS levels were measured as described in materials and methods. All the results are expressed as mean ± S.D. of three independent experiments. Significance between either WT-IL6 and KO-IL6 or WT + IL6 and KO + IL6 or EVA431-IL6 and OECD109A431-IL6 or EVA431 + IL6 and OECD109A431 + IL6 was calculated using a student T test: NS: Not-significant * P < 0.05, ** P < 0.01 and *** P < 0.001

Figure Legend Snippet: CD109 promotes basal and IL-6-induced expression of antioxidant protein NRF2 and its target genes. A A431-WT versus A431-CD109 KO cells and B SCC9-WT versus SCC9-CD109 KO cells and C EV-A431 and OE CD109A431 cells were treated without or with 20 ng/ml of IL-6 for 24 h. Expression levels of NRF2, SOD1 and HO-1 were analyzed by Western blot. Densitometric analysis of the data depicted in A, B and C are shown in supplementary data. D , E Measurement of ROS Levels in A431-WT versus A431-CD109 KO and SCC9-WT versus SCC9-CD109 KO cells. Cells were treated without or with 50 ng/ml of IL-6 for 2, 4 and 24 h. ROS levels were measured as described in materials and methods. All the results are expressed as mean ± S.D. of three independent experiments. Significance between either WT-IL6 and KO-IL6 or WT + IL6 and KO + IL6 or EVA431-IL6 and OECD109A431-IL6 or EVA431 + IL6 and OECD109A431 + IL6 was calculated using a student T test: NS: Not-significant * P < 0.05, ** P < 0.01 and *** P < 0.001

Techniques Used: Expressing, Western Blot

STAT3 is critical for NRF2 protein expression. A431 ( A ) and SCC9 ( C ) cells were transfected with STAT3-specific siRNA for 72 h and were treated with IL-6 (20 ng/ml) for 30 min or 1 h. The levels of NRF2, phospho-STAT3, total STAT3 were analyzed by Western blot. B A431 cells were treated with Stattic for 1 h followed by treatment with IL-6 (20 ng/mL) for 30 min or 1 h and the levels of NRF2, phospho-STAT3, total STAT3 were analyzed by Western blot. The quantitation of this data is shown on the left bottom panel. Significance between siCtrl-IL6 and siSTAT3-IL6 or siCtrl + IL6 and siSTAT3 + IL6 0.5 h or siCtrl + IL6 and siCtrl + IL6 1 h was calculated using a student T test: NS: non-significant, * P < 0.05, ** P < 0.01 and *** P < 0.001

Figure Legend Snippet: STAT3 is critical for NRF2 protein expression. A431 ( A ) and SCC9 ( C ) cells were transfected with STAT3-specific siRNA for 72 h and were treated with IL-6 (20 ng/ml) for 30 min or 1 h. The levels of NRF2, phospho-STAT3, total STAT3 were analyzed by Western blot. B A431 cells were treated with Stattic for 1 h followed by treatment with IL-6 (20 ng/mL) for 30 min or 1 h and the levels of NRF2, phospho-STAT3, total STAT3 were analyzed by Western blot. The quantitation of this data is shown on the left bottom panel. Significance between siCtrl-IL6 and siSTAT3-IL6 or siCtrl + IL6 and siSTAT3 + IL6 0.5 h or siCtrl + IL6 and siCtrl + IL6 1 h was calculated using a student T test: NS: non-significant, * P < 0.05, ** P < 0.01 and *** P < 0.001

Techniques Used: Expressing, Transfection, Western Blot, Quantitation Assay

NRF2 associates with STAT3 in an IL-6-dependent manner, mediating the IL-6-CD109 driven SCC stemness. A SCC9 cells were incubated with IL-6 (20 ng/ml) overnight and lysates were immunoprecipitated with an anti-NRF2 antibody or control IgG followed by Western blot with an anti-STAT3 antibody (left panel). Reverse co-immunoprecipitation was performed by immunoprecipitating the lysates with an anti-STAT3 antibody followed by Western blot with anti-NRF2 antibody or control IgG (right panel). The input data showing the levels of STAT3 and NRF2 in the total cell lysates used are shown below with β-Actin as loading control. B – G NRF2 knockdown was performed in A431-WT versus A431-CD109 KO ( B ), or in SCC9-WT versus SCC9-CD109 KO cells ( C ), or in EVA431 versus CD109OE A431 cells ( D ), by transfecting with NRF2-specific siRNA (or control siRNA). Alternatively, NRF2 function was inhibited using ML385, an inhibitor of NRF2, in A431-WT versus A431-CD109 KO ( E ), or in SCC9-WT versus SCC9-CD109 KO cells ( F ) or in EVA431 versus CD109OE A431 cells ( G ). A spheroid formation assay was then performed as described in Methods. The number of tumor spheroids formed was quantified by analyzing five random fields. Scale bar: 100 μm. All the results are expressed as the mean ± S.D. of three independent experiments. Significance was calculated using a Student’s T-test: * P < 0.05, ** P < 0.01 and *** P < 0.001

Figure Legend Snippet: NRF2 associates with STAT3 in an IL-6-dependent manner, mediating the IL-6-CD109 driven SCC stemness. A SCC9 cells were incubated with IL-6 (20 ng/ml) overnight and lysates were immunoprecipitated with an anti-NRF2 antibody or control IgG followed by Western blot with an anti-STAT3 antibody (left panel). Reverse co-immunoprecipitation was performed by immunoprecipitating the lysates with an anti-STAT3 antibody followed by Western blot with anti-NRF2 antibody or control IgG (right panel). The input data showing the levels of STAT3 and NRF2 in the total cell lysates used are shown below with β-Actin as loading control. B – G NRF2 knockdown was performed in A431-WT versus A431-CD109 KO ( B ), or in SCC9-WT versus SCC9-CD109 KO cells ( C ), or in EVA431 versus CD109OE A431 cells ( D ), by transfecting with NRF2-specific siRNA (or control siRNA). Alternatively, NRF2 function was inhibited using ML385, an inhibitor of NRF2, in A431-WT versus A431-CD109 KO ( E ), or in SCC9-WT versus SCC9-CD109 KO cells ( F ) or in EVA431 versus CD109OE A431 cells ( G ). A spheroid formation assay was then performed as described in Methods. The number of tumor spheroids formed was quantified by analyzing five random fields. Scale bar: 100 μm. All the results are expressed as the mean ± S.D. of three independent experiments. Significance was calculated using a Student’s T-test: * P < 0.05, ** P < 0.01 and *** P < 0.001

Techniques Used: Incubation, Immunoprecipitation, Control, Western Blot, Knockdown, Tube Formation Assay

Cell Culture:

Article Title: IL-6-mediated tumorigenicity and antioxidant state in squamous cell carcinoma cells are driven by CD109 via stabilization of IL-6 receptor-alpha and activation of STAT3/NRF2 pathway
Article Snippet: The human vulvar squamous cell carcinoma A431 cell line (RRID: CVCL_0037) was cultured in Dulbecco's Modified Eagle’s Medium (DMEM; Gibco Life Technologies; #11995-065) supplemented with 10% fetal bovine serum (FBS, Gibco Life Technologies; #12483-020). .. Human tongue squamous cell carcinoma SCC9 cell line (ATCC CRL-9) was cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham's F12 Medium containing 1.2 g/l sodium bicarbonate, 2.5 mM l -glutamine, 15 mM HEPES, and 0.5 mM sodium pyruvate supplemented with 400 ng/ml hydrocortisone and 10% FBS. .. For the protein degradation assay, we utilized CD109 overexpressing A431 and empty vector (EV) containing stable cell lines, cultured in DMEM supplemented with 10% FBS, geneticin, and penicillin–streptomycin.

Article Title: IL-6-mediated tumorigenicity and antioxidant state in squamous cell carcinoma cells are driven by CD109 via stabilization of IL-6 receptor-alpha and activation of STAT3/NRF2 pathway.
Article Snippet: The human vulvar squamous cell carcinoma A431 cell line (RRID: CVCL_0037) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco Life Technologies; #11995-065) supplemented with 10% fetal bovine serum (FBS, Gibco Life Technologies; #12483- 020). .. Human tongue squamous cell carcinoma SCC9 cell line (ATCC CRL-9) was cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F12 Medium containing 1.2 g/l sodium bicarbonate, 2.5 mM l-glutamine, 15 mM HEPES, and 0.5 mM sodium pyruvate supplemented with 400 ng/ml hydrocortisone and 10% FBS. .. For the protein degradation assay, we utilized CD109 overexpressing A431 and empty vector (EV) containing stable cell lines, cultured in DMEM supplemented with 10% FBS, geneticin, and penicillin–streptomycin.

Modification:

Article Title: IL-6-mediated tumorigenicity and antioxidant state in squamous cell carcinoma cells are driven by CD109 via stabilization of IL-6 receptor-alpha and activation of STAT3/NRF2 pathway.
Article Snippet: The human vulvar squamous cell carcinoma A431 cell line (RRID: CVCL_0037) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco Life Technologies; #11995-065) supplemented with 10% fetal bovine serum (FBS, Gibco Life Technologies; #12483- 020). .. Human tongue squamous cell carcinoma SCC9 cell line (ATCC CRL-9) was cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F12 Medium containing 1.2 g/l sodium bicarbonate, 2.5 mM l-glutamine, 15 mM HEPES, and 0.5 mM sodium pyruvate supplemented with 400 ng/ml hydrocortisone and 10% FBS. .. For the protein degradation assay, we utilized CD109 overexpressing A431 and empty vector (EV) containing stable cell lines, cultured in DMEM supplemented with 10% FBS, geneticin, and penicillin–streptomycin.

Image Search Results

Journal: Experimental Hematology & Oncology

doi: 10.1186/s40164-025-00630-x

Figure Lengend Snippet: CD109 promotes IL6Rα stability and IL-6 induced phosphorylation of STAT3 in SCC cells. A , B A431, SCC9 wild-type (WT) cells and CD109 knockout (KO) cells were treated with IL-6 (20 ng/mL) for 30 min and the expression of CD109, IL6Rα and phosphorylation of the STAT3 (Y705) proteins were analyzed by Western blotting. Densitometric analysis of the data depicted in A and B are shown in supplementary data. C A431-WT and A431-CD109 KO cells were treated with IL-6 (20 ng/ml) for various time periods (0–4 h), and the levels of phospho-STAT3 (Y705) and total STAT3 were assessed by Western blot. β-Actin was used as a loading control. D Fluorescence microscopy showing the levels of phospho-STAT3 (Y705) (upper panels, green) and total STAT3 (lower panels, green) in A431-WT and A431-CD109 KO cells treated with IL-6 (20 ng/ml). Scale bar: 25 μm. E A431-WT and A431-CD109 KO cells transfected with IL6Rα siRNA or control siRNA and treated with IL-6 (20 ng/ml), and the expression of IL6Rα, CD109 and the levels of phospho-STAT3 (Y705) were analyzed by Western blotting. F A431 and SCC9 cells were treated without or with (20 ug) tocilizumab for 24 h and the expression of IL6Rα, CD109 and phosphorylation of STAT-3 (Y705) were analyzed by Western blotting. All results ( A – F ) are representative of at least 3 independent experiments. Significance was calculated using a student T-test. NS: Not significant, * P < 0.05, ** P < 0.01 and *** P < 0.0010

Article Snippet: Human tongue squamous cell carcinoma SCC9 cell line (ATCC CRL-9) was cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham's F12 Medium containing 1.2 g/l sodium bicarbonate, 2.5 mM l -glutamine, 15 mM HEPES, and 0.5 mM sodium pyruvate supplemented with 400 ng/ml hydrocortisone and 10% FBS.

Techniques: Phospho-proteomics, Knock-Out, Expressing, Western Blot, Control, Fluorescence, Microscopy, Transfection

Journal: Experimental Hematology & Oncology

doi: 10.1186/s40164-025-00630-x

Figure Lengend Snippet: CD109 interacts and co-localizes with IL6Rα in an IL-6-dependent manner. A Immunoprecipitation of total cell lysates from A431-WT versus A431-CD109 KO, and SCC9-WT versus SCC9-CD109 KO cells was done using an anti-CD109 antibody or a control IgG followed by Western blot analysis using an anti-IL6Rα antibody. B The input data showing the levels of CD109 and IL6Rα in the total cell lysates used are shown below with β-Actin as loading control. C Reverse co-immunoprecipitation was performed by immunoprecipitation of total cell lysates from A431-WT versus A431-CD109 KO cells using an anti-IL6Rα antibody or a control IgG followed by Western blot analysis using an anti-CD109 antibody. The input data are same as for panel A. D Representative fluorescence microscopic images showing co-expression and colocalization of CD109 and IL6Rα in A431 cells and oral SCC patient derived tissue. CD109 (red), IL6Rα (green) and DAPI (blue) are shown. All data shown A – D are representative of three independent experiments. Scale bar ( D ): 25 μm

Techniques: Immunoprecipitation, Control, Western Blot, Fluorescence, Expressing, Derivative Assay

Journal: Experimental Hematology & Oncology

doi: 10.1186/s40164-025-00630-x

Figure Lengend Snippet: CD109 promotes IL-6-induced expression of stem cell markers and tumorigenicity in SCC cells. A A431-WT versus A431-CD109 KO and B SCC9-WT versus SCC9-CD109 KO and C EV-A431 versus CD109OE-A431 cells were treated with 20 ng/ml of IL-6 for 48 h and Western blot analysis for the indicated stem cell markers was performed. Densitometric analysis of the data depicted in A, B and C are shown in supplementary data. D , E Tumor spheroids were grown using A431-WT versus A431-CD109 KO ( D ) and SCC9-WT versus SCC9-CD109 KO ( E ) cells to assess in vitro tumorigenicity as described in Methods, and the spheroids formed were counted under the microscope. The percentage of cells capable of forming spheroids was calculated as (number of spheroids formed/number of cells plated) × 100. Quantitation of the results shown in D and E are shown at the bottom panels. Scale bar: 100 μm. Significance between either wt-IL6 and KO-IL6 or wt + IL6 and KO + IL6 or EVA431-IL6 and OECD109A431-IL6 or EVA431 + IL6 and OECD109A431 + IL6 was calculated using a Student’s T-test: NS: Non-Significant, * P < 0.05, ** P < 0.01 and *** P < 0.001

Techniques: Expressing, Western Blot, In Vitro, Microscopy, Quantitation Assay

Journal: Experimental Hematology & Oncology

doi: 10.1186/s40164-025-00630-x

Figure Lengend Snippet: CD109 promotes basal and IL-6-induced expression of antioxidant protein NRF2 and its target genes. A A431-WT versus A431-CD109 KO cells and B SCC9-WT versus SCC9-CD109 KO cells and C EV-A431 and OE CD109A431 cells were treated without or with 20 ng/ml of IL-6 for 24 h. Expression levels of NRF2, SOD1 and HO-1 were analyzed by Western blot. Densitometric analysis of the data depicted in A, B and C are shown in supplementary data. D , E Measurement of ROS Levels in A431-WT versus A431-CD109 KO and SCC9-WT versus SCC9-CD109 KO cells. Cells were treated without or with 50 ng/ml of IL-6 for 2, 4 and 24 h. ROS levels were measured as described in materials and methods. All the results are expressed as mean ± S.D. of three independent experiments. Significance between either WT-IL6 and KO-IL6 or WT + IL6 and KO + IL6 or EVA431-IL6 and OECD109A431-IL6 or EVA431 + IL6 and OECD109A431 + IL6 was calculated using a student T test: NS: Not-significant * P < 0.05, ** P < 0.01 and *** P < 0.001

Techniques: Expressing, Western Blot

Journal: Experimental Hematology & Oncology

doi: 10.1186/s40164-025-00630-x

Figure Lengend Snippet: STAT3 is critical for NRF2 protein expression. A431 ( A ) and SCC9 ( C ) cells were transfected with STAT3-specific siRNA for 72 h and were treated with IL-6 (20 ng/ml) for 30 min or 1 h. The levels of NRF2, phospho-STAT3, total STAT3 were analyzed by Western blot. B A431 cells were treated with Stattic for 1 h followed by treatment with IL-6 (20 ng/mL) for 30 min or 1 h and the levels of NRF2, phospho-STAT3, total STAT3 were analyzed by Western blot. The quantitation of this data is shown on the left bottom panel. Significance between siCtrl-IL6 and siSTAT3-IL6 or siCtrl + IL6 and siSTAT3 + IL6 0.5 h or siCtrl + IL6 and siCtrl + IL6 1 h was calculated using a student T test: NS: non-significant, * P < 0.05, ** P < 0.01 and *** P < 0.001

Techniques: Expressing, Transfection, Western Blot, Quantitation Assay

Journal: Experimental Hematology & Oncology

doi: 10.1186/s40164-025-00630-x

Figure Lengend Snippet: NRF2 associates with STAT3 in an IL-6-dependent manner, mediating the IL-6-CD109 driven SCC stemness. A SCC9 cells were incubated with IL-6 (20 ng/ml) overnight and lysates were immunoprecipitated with an anti-NRF2 antibody or control IgG followed by Western blot with an anti-STAT3 antibody (left panel). Reverse co-immunoprecipitation was performed by immunoprecipitating the lysates with an anti-STAT3 antibody followed by Western blot with anti-NRF2 antibody or control IgG (right panel). The input data showing the levels of STAT3 and NRF2 in the total cell lysates used are shown below with β-Actin as loading control. B – G NRF2 knockdown was performed in A431-WT versus A431-CD109 KO ( B ), or in SCC9-WT versus SCC9-CD109 KO cells ( C ), or in EVA431 versus CD109OE A431 cells ( D ), by transfecting with NRF2-specific siRNA (or control siRNA). Alternatively, NRF2 function was inhibited using ML385, an inhibitor of NRF2, in A431-WT versus A431-CD109 KO ( E ), or in SCC9-WT versus SCC9-CD109 KO cells ( F ) or in EVA431 versus CD109OE A431 cells ( G ). A spheroid formation assay was then performed as described in Methods. The number of tumor spheroids formed was quantified by analyzing five random fields. Scale bar: 100 μm. All the results are expressed as the mean ± S.D. of three independent experiments. Significance was calculated using a Student’s T-test: * P < 0.05, ** P < 0.01 and *** P < 0.001

Techniques: Incubation, Immunoprecipitation, Control, Western Blot, Knockdown, Tube Formation Assay

Knockdown of RASSF-1A gene in SCC9 cells promotes cell growth. a Western blot analysis of RASSF-1A protein expression in cells after siRNA interference. (SCC9: blank control group; non-siRNA: siRNA empty control group; RASSF-1A-siRNA: siRNA interference knockdown group). b Western blot results statistics. c CCK8 detects cell growth curve (at 72 h and 96 h *p < 0.05). d Flow cytometry to detect cell cycle distribution. e Western blot analysis of CyclinD2 protein expression in cells. f Western blot analysis of CyclinD2 protein expression (n = 6 in each group; *p < 0.05)

Journal: Cancer Cell International

Article Title: RASSF-1A modulates proliferation-mediated oral squamous cell carcinoma progression

doi: 10.1186/s12935-019-0932-9

Figure Lengend Snippet: Knockdown of RASSF-1A gene in SCC9 cells promotes cell growth. a Western blot analysis of RASSF-1A protein expression in cells after siRNA interference. (SCC9: blank control group; non-siRNA: siRNA empty control group; RASSF-1A-siRNA: siRNA interference knockdown group). b Western blot results statistics. c CCK8 detects cell growth curve (at 72 h and 96 h *p < 0.05). d Flow cytometry to detect cell cycle distribution. e Western blot analysis of CyclinD2 protein expression in cells. f Western blot analysis of CyclinD2 protein expression (n = 6 in each group; *p < 0.05)

Article Snippet: Human tongue squamous cell carcinoma cell line SCC9 was purchased from ATCC, USA and cells were cultured in Dulbecco’s Phosphate Buffered Saline medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco, USA).

Techniques: Knockdown, Western Blot, Expressing, Control, Flow Cytometry

Increased cell migration ability after knockdown of RASSF-1A gene in SCC9 cells. a Scratch test to detect cell migration ability. b The width of the scratch between the cells of the different groups was counted at 0 and 36 h, and the width of the scratch was divided by the width of 36 h by the width of the scratch for 0 h (n = 6 in each group; *p < 0.05). c Transwell chamber for cell migration. d Crystal Violet staining counts the number of cells migrating through the transwell chamber after 36 h of cell culture (n = 6 in each group; *p < 0.05). e Western blot analysis of MMP2 protein expression in cells. f Western blot analysis of MMP2 protein expression (n = 6 in each group; *p < 0.05)

Journal: Cancer Cell International

Article Title: RASSF-1A modulates proliferation-mediated oral squamous cell carcinoma progression

doi: 10.1186/s12935-019-0932-9

Figure Lengend Snippet: Increased cell migration ability after knockdown of RASSF-1A gene in SCC9 cells. a Scratch test to detect cell migration ability. b The width of the scratch between the cells of the different groups was counted at 0 and 36 h, and the width of the scratch was divided by the width of 36 h by the width of the scratch for 0 h (n = 6 in each group; *p < 0.05). c Transwell chamber for cell migration. d Crystal Violet staining counts the number of cells migrating through the transwell chamber after 36 h of cell culture (n = 6 in each group; *p < 0.05). e Western blot analysis of MMP2 protein expression in cells. f Western blot analysis of MMP2 protein expression (n = 6 in each group; *p < 0.05)

Techniques: Migration, Knockdown, Staining, Cell Culture, Western Blot, Expressing

Decreased apoptosis after knockdown of RASFF-1A gene in SCC9 cells. a Flow cytometry to detect the percentage of positive expression of AV protein in cells. b Percentage of AV positive cells in statistical flow assay results (n = 6 in each group; *p < 0.05). c Western blot analysis of caspase3 protein expression in SCC9 cells. d Statistical analysis of the expression of caspase3 protein in cells by western blot (n = 6 in each group; *p < 0.05)

Journal: Cancer Cell International

Article Title: RASSF-1A modulates proliferation-mediated oral squamous cell carcinoma progression

doi: 10.1186/s12935-019-0932-9

Figure Lengend Snippet: Decreased apoptosis after knockdown of RASFF-1A gene in SCC9 cells. a Flow cytometry to detect the percentage of positive expression of AV protein in cells. b Percentage of AV positive cells in statistical flow assay results (n = 6 in each group; *p < 0.05). c Western blot analysis of caspase3 protein expression in SCC9 cells. d Statistical analysis of the expression of caspase3 protein in cells by western blot (n = 6 in each group; *p < 0.05)

Techniques: Knockdown, Flow Cytometry, Expressing, Western Blot

Knockdown of RASSF-1A gene in SCC9 cells promotes CyclinD1 protein expression. a – c qPCR detection of PCNA, p38 MAPK, CyclinD1 mRNA expression in cells. d Western blot analysis of CyclinD1 protein expression in cells. e Western blot analysis of CyclinD1 protein expression in cells (n = 6 in each group; *p < 0.05). f Co-immunoprecipitation assay for RASSF-1A protein and CyclinD1 protein binding in cells

Journal: Cancer Cell International

Article Title: RASSF-1A modulates proliferation-mediated oral squamous cell carcinoma progression

doi: 10.1186/s12935-019-0932-9

Figure Lengend Snippet: Knockdown of RASSF-1A gene in SCC9 cells promotes CyclinD1 protein expression. a – c qPCR detection of PCNA, p38 MAPK, CyclinD1 mRNA expression in cells. d Western blot analysis of CyclinD1 protein expression in cells. e Western blot analysis of CyclinD1 protein expression in cells (n = 6 in each group; *p < 0.05). f Co-immunoprecipitation assay for RASSF-1A protein and CyclinD1 protein binding in cells

Techniques: Knockdown, Expressing, Western Blot, Co-Immunoprecipitation Assay, Protein Binding

$Figure 1. Analysis of cancer stem cell (CSC) properties in SCC4 and SCC9 cell lines. (A) Representative images of the gates used to sort the CD44high and CD44low fractions. (B) Cell morphology of CD44high and CD44low fractions, demonstrating the presence of holoclones in CD44high cells. (C) Colony forma tion ability of CD44high and CD44low fractions. Graphs representing the (D) number of colonies and (E) number of spheres in CD44high and CD44low cells. (F) RT-qPCR for CD44, NANOG, CD133, POU5F1 (OCT4) and BMI1 transcripts. Fold change values represent the relative gene expression in CD44high cells in relation to CD44low cells. *P<0.05, **P<0.01 and ***P<0.001 (t-tests with the Welch correction). Data for SCC9 cells was gathered as previously described by de Andrade et al (51). Microscope images: Magnification, x100 (scale bars, 40 µm).$

Journal: International journal of oncology

Article Title: GLI3 knockdown decreases stemness, cell proliferation and invasion in oral squamous cell carcinoma.

doi: 10.3892/ijo.2018.4572

Figure Lengend Snippet: Figure 1. Analysis of cancer stem cell (CSC) properties in SCC4 and SCC9 cell lines. (A) Representative images of the gates used to sort the CD44high and CD44low fractions. (B) Cell morphology of CD44high and CD44low fractions, demonstrating the presence of holoclones in CD44high cells. (C) Colony forma tion ability of CD44high and CD44low fractions. Graphs representing the (D) number of colonies and (E) number of spheres in CD44high and CD44low cells. (F) RT-qPCR for CD44, NANOG, CD133, POU5F1 (OCT4) and BMI1 transcripts. Fold change values represent the relative gene expression in CD44high cells in relation to CD44low cells. *P<0.05, **P<0.01 and ***P<0.001 (t-tests with the Welch correction). Data for SCC9 cells was gathered as previously described by de Andrade et al (51). Microscope images: Magnification, x100 (scale bars, 40 µm).

Article Snippet: SCC4 (CRL-1624TM) and SCC9 (CRL-1629TM) human tongue squamous cell carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Gene Expression, Microscopy

Figure 3. (A) PCR array heatmap of the differentially expressed genes in SCC9 CD44high in relation to CD44low cells. (B) Fold change values for each gene evaluated. Only genes with a fold change >2.0 and classified as ‘A’ and ‘OKAY’ by the PCR array software were considered overexpressed. Data represents the fold change values of 3 independent experiments.

Journal: International journal of oncology

Article Title: GLI3 knockdown decreases stemness, cell proliferation and invasion in oral squamous cell carcinoma.

doi: 10.3892/ijo.2018.4572

Figure Lengend Snippet: Figure 3. (A) PCR array heatmap of the differentially expressed genes in SCC9 CD44high in relation to CD44low cells. (B) Fold change values for each gene evaluated. Only genes with a fold change >2.0 and classified as ‘A’ and ‘OKAY’ by the PCR array software were considered overexpressed. Data represents the fold change values of 3 independent experiments.

Article Snippet: SCC4 (CRL-1624TM) and SCC9 (CRL-1629TM) human tongue squamous cell carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Software

$Figure 4. Effects of GLI3 knockdown on SCC9 stemness. (A) GLI3 mRNA expression levels and (B) GLI3 protein expression in SCC9 cells transfected with control or shRNA-GLI3. (C) FACS analysis of the expression of CD44 (y-axis) and ESA (x-axis) on control and shRNA GLI3 SCC9 cells. GLI3 knockdown decreases CD44high/ESAlow and CD44high/ESAhigh fractions of SCC9 cells, as well as increased the CD44low fraction. (D) Quantification of data depicted in (C), showing the percentage of CD44high/ESAlow, CD44high/ESAhigh and CD44low fractions. (E) Colony formation ability and quantification of the number of colonies formed in shRNA GLI3 and control cells. (F) Number of spheres formed in shRNA GLI3 and control cells. (G) RT-qPCR indicated reduced CD44, BMI1, POU5F1 (OCT4) and SNAI2 (SLUG) mRNA expression and increased S100A9 (Calgranulin) and Involucrin (IVL) in shRNA-GLI3-transfected cells in relation to the control cells. *P<0.05, **P<0.01 and ***P<0.001 (t-tests with the Welch correction). Microscope images: Magnification, x100 (scale bars, 40 µm).$

Journal: International journal of oncology

Article Title: GLI3 knockdown decreases stemness, cell proliferation and invasion in oral squamous cell carcinoma.

doi: 10.3892/ijo.2018.4572

Figure Lengend Snippet: Figure 4. Effects of GLI3 knockdown on SCC9 stemness. (A) GLI3 mRNA expression levels and (B) GLI3 protein expression in SCC9 cells transfected with control or shRNA-GLI3. (C) FACS analysis of the expression of CD44 (y-axis) and ESA (x-axis) on control and shRNA GLI3 SCC9 cells. GLI3 knockdown decreases CD44high/ESAlow and CD44high/ESAhigh fractions of SCC9 cells, as well as increased the CD44low fraction. (D) Quantification of data depicted in (C), showing the percentage of CD44high/ESAlow, CD44high/ESAhigh and CD44low fractions. (E) Colony formation ability and quantification of the number of colonies formed in shRNA GLI3 and control cells. (F) Number of spheres formed in shRNA GLI3 and control cells. (G) RT-qPCR indicated reduced CD44, BMI1, POU5F1 (OCT4) and SNAI2 (SLUG) mRNA expression and increased S100A9 (Calgranulin) and Involucrin (IVL) in shRNA-GLI3-transfected cells in relation to the control cells. *P<0.05, **P<0.01 and ***P<0.001 (t-tests with the Welch correction). Microscope images: Magnification, x100 (scale bars, 40 µm).

Article Snippet: SCC4 (CRL-1624TM) and SCC9 (CRL-1629TM) human tongue squamous cell carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Expressing, Transfection, Control, shRNA, Quantitative RT-PCR, Microscopy

Figure 5. Effects of GLI3 knockdown on SCC9 cellular proliferation, apoptosis and invasion. (A and B) Cells transfected with shRNA GLI3 cells exhibited a reduced number of BrdU-positive cells and (C) a decrease in the number of cells after 48, 72, 96 and 120 h of culture. (D) Number of Annexin V-positive cells. (E and F) GLI3 knockdown in SCC9 cells was associated with significantly lower numbers of invasive cells in relation to the controls. *P<0.05 and ***P<0.001 (t-test with the Welch correction). Microscope images: (A and B) Magnification, x100 (scale bars, 40 µm); (E) x400 (scale bars, 10 µm).

Journal: International journal of oncology

Article Title: GLI3 knockdown decreases stemness, cell proliferation and invasion in oral squamous cell carcinoma.

doi: 10.3892/ijo.2018.4572

Figure Lengend Snippet: Figure 5. Effects of GLI3 knockdown on SCC9 cellular proliferation, apoptosis and invasion. (A and B) Cells transfected with shRNA GLI3 cells exhibited a reduced number of BrdU-positive cells and (C) a decrease in the number of cells after 48, 72, 96 and 120 h of culture. (D) Number of Annexin V-positive cells. (E and F) GLI3 knockdown in SCC9 cells was associated with significantly lower numbers of invasive cells in relation to the controls. *P<0.05 and ***P<0.001 (t-test with the Welch correction). Microscope images: (A and B) Magnification, x100 (scale bars, 40 µm); (E) x400 (scale bars, 10 µm).

Article Snippet: SCC4 (CRL-1624TM) and SCC9 (CRL-1629TM) human tongue squamous cell carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Transfection, shRNA, Microscopy

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human tongue squamous cell carcinoma scc9 cell line (ATCC)

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1) Product Images from "IL-6-mediated tumorigenicity and antioxidant state in squamous cell carcinoma cells are driven by CD109 via stabilization of IL-6 receptor-alpha and activation of STAT3/NRF2 pathway"

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