Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 1. Loss of DYRK1A suppresses self-renewal and tumorigenic potential of OSCC. (A) Deletion of DYRK1A was performed in two OSCC cell lines (SCC4 and FaDu) by CRISPR/Cas9 genome editing technique. Deletion of DYRK1A was confirmed by Western blot. GAPDH was used as a loading control. WT; Wild-type, KO; DYRK1A knockout. Arrowhead indicates the DYRK1A-specific band, and asterisk indicates a nonspecific cross-reacting band. (B) Effect of DYRK1A deletion on self-renewal capacity was determined by tumorsphere formation assay. Representative images of tumorspheres formed by the WT and DYRK1A KO cells were shown on the right of each bar graph. Bar indicates 200 μm. Data are means ± SD of triplicate experiment. *P < 0.01 compared to WT. (C) Effect of DYRK1A deletion on anchorage- independent growth ability was determined by soft agar assay. *P < 0.01 compared to WT. (D) Effect of DYRK1A deletion on in vivo tumorigenicity was determined by xenograft tumor assay. FaDu WT and FaDu DYRK1A KO were injected subcutaneously into 5 nude mice. Tumor volumes were measured for 3 weeks. (E) Tumor images and tumor incidence of FaDu WT and FaDu DYRK1A KO were obtained 3 weeks after the injections.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: CRISPR, Western Blot, Control, Knock-Out, Tube Formation Assay, Soft Agar Assay, In Vivo, Injection

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 2. Loss of DYRK1A diminishes CSC-enriched ALDH1HIGH population and CSC properties in OSCC. (A) Effect of DYRK1A deletion on ALDH1HIGH cell population in SCC4 and FaDu was determined by Aldefluor assay. Cells were labeled with Aldefluor with and without the ALDH inhibitor DEAB and analyzed by flow cytometry. The gate for ALDH1HIGH cells is determined in relation to the DEAB control (+DEAB) and shows the brightly fluorescent ALDH population versus the side scatter, a population that is absent/decreased in the presence of DEAB. The number shown in each panel reflects the percentage of ALDH1HIGH cells in each cell type. A similar result was obtained from an independent experiment (Supplemental Fig. 2). (B) Effect of DYRK1A deletion on migration ability was determined by transwell migration assay. Migration ability was described as number of migrated cells per field with data as mean ± SD for three randomly selected fields. Representative images of migrated cells from each group were shown on the right of each bar graph. Bar indicates 200 μm *P < 0.01 (C) Effect of DYRK1A deletion on chemo resistance was determined by MTT assay. SCC4 WT and SCC4 DYRK1A KO cells were treated with the indicated concentrations of cisplatin for 2 days, and their viability was determined by MTT assay. **P < 0.05.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Labeling, Flow Cytometry, Control, Migration, Transwell Migration Assay, MTT Assay

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 3. Ectopic DYRK1A expression promotes CSC properties in OSCC. DYRK1A was ectopically overexpressed in SCC9 by transfecting with vector expressing DYRK1A and empty vector (EV) as a control. (A) Ectopic expression of DYRK1A in SCC9 was validated by qPCR (left) and Western blot (right). (B) Effect of DYRK1A overexpression on self-renewal capacity was determined by tumorsphere formation assay. Representative images of tumorspheres formed from each group were shown on the right of the bar graph. *P < 0.01 compared to EV. (C) Effect of DYRK1A overexpression on migration ability was measured by a transwell migration assay. Representative images of migrated cells from each group were shown on the right of the bar graph. *P < 0.05 (D) Effect of DYRK1A overexpression on chemoresistance was determined by MTT assay. The cells were treated with indicated concentrations of cisplatin for 2 days, and their viability was determined. ns, not significant.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Over Expression, Tube Formation Assay, Migration, Transwell Migration Assay, MTT Assay

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 4. DYRK1A is overexpressed in OSCC. (A) Level of DYRK1A mRNA was determined in normal human oral keratinocyte (NHOK) and 9 OSCC cell lines (BapT, FaDu, SCC4, SCC9, SCC15, SCC105, SNU1066, UM6 and YD38) by qPCR. The Ct values of DYRK1A were normalized by the Ct value of GAPDH. (B) Level of DYRK1A protein was determined in NHOK and OSCC cell lines by Western blot analysis. (C) Self-renewal capacity in three groups (high, medium, and low based on their level of DYRK1A protein) was determined by tumorsphere formation assay. (D) ALDH1HIGH cell population in three groups was determined by Aldefluor assay. (E) In vivo DYRK1A expression was determined in normal human oral epithelia (NHOE) and OSCC tissues by immunohistochemical (IHC) staining. (F) Representative examples of DYRK1A IHC staining in NHOE and OSCC tissues in vivo. Bar indicates 100 μm.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Western Blot, Tube Formation Assay, In Vivo, Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 5. DYRK1A regulates CSC property via FGF2 expression. (A) Effect of DYRK1A deletion on FGF2 expression was assessed in SCC4 and FaDu by qPCR (upper) and Western blot analysis (lower). (B) Effect of DYRK1A deletion on FGF2 secretion was determined by ELISA. (C) Effect of DYRK1A overexpression on FGF2 expression was assessed in SCC9 by qPCR (upper) and Western blot analysis (lower). (D) Effect of DYRK1A overexpression on FGF2 secretion was determined by determined by ELISA. (E) Effect of FGF2 on self-renewal capacity of SCC4 WT and DYRK1A KO was measured by tumorsphere formation assay. The assays were performed in the absence and presence of FGF2 (50, 100 and 200 ng/ml). *P < 0.05 compared to untreated control cells. (F) Effect of FGF2 on migration ability of SCC4 WT and DYRK1A KO was measured by transwell migration assay. The assays were performed in the absence and presence of FGF2. (G) Effect of FGF2 neutralizing antibody on self-renewal capacity of DYRK1A-overerxpressing SCC9 was determined by tumorsphere assay. The assays were performed in the absence and presence of FGF2 antibody (1, 2.5, and 5 ng/ml). *P < 0.05 and **P < 0.01 compared to untreated control cells. (H) Effect of FGF2 neutralizing antibody on migration ability of DYRK1A-overerxpressing SCC9 was measured by transwell migration assay. The assays were performed in the absence and presence of FGF2 antibody.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression, Tube Formation Assay, Control, Migration, Transwell Migration Assay

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 6. Expression of DYRK1A and FGF2 are elevated in CSC-enriched populations, and their levels are positively correlated in OSCC cells. (A) Cisplatin-resistant SCC4 (CR-SCC4) cells were isolated from SCC4 treated with 25 μM cisplatin for 2 days. The IC50 value for cisplatin in SCC4 and CR-SCC4 was determined by MTT assay. Cells were treated with various concentrations of cisplatin for 48 h, and their viability was determined. (B) Self-renewal capacity of SCC4 and CR-SCC4 was determined by tumorsphere formation assay. *P < 0.05 and **P < 0.01 compared to SCC4. (C) Expression of DYRK1A and FGF2 was assessed in SCC4 and CR- SCC4 by qPCR (left) and Western blot (right). (D) ALDH1HIGH (CSC-enriched population) and ALDH1low (non-CSC population) cell populations were sorted from SCC4 cells by flow cytometry. Expression of DYRK1A and FGF2 was assessed in ALDH1low and ALDH1HIGH cell populations by qPCR. (E) Expression of DYRK1A and FGF2 was assessed in tumorspheres (S) and their corresponding adherent monolayer cells (M) derived from multiple OSCC cell lines by qPCR. (F) Correlation analysis of DYRK1A and FGF2 mRNA was determined based on their expression levels in 14 human SCC cell lines by qPCR.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Expressing, Isolation, MTT Assay, Tube Formation Assay, Western Blot, Flow Cytometry, Derivative Assay