human tongue squamous cell cancer cell line  (ATCC)

Journal: Experimental Hematology & Oncology

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

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

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

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

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

Journal: Experimental Hematology & Oncology

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

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

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

Journal: Experimental Hematology & Oncology

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

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

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: Expressing, Western Blot

Journal: Experimental Hematology & Oncology

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

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

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: Expressing, Transfection, Western Blot, Quantitation Assay

Journal: Experimental Hematology & Oncology

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

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

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