Journal: Clinical chemistry

Article Title: Integrative bioinformatics analysis reveals new prognostic biomarkers of clear cell renal cell carcinoma.

doi: 10.1373/clinchem.2014.225854

Figure Lengend Snippet: Fig. 4. AHR, GRHL2, and KIAA0101 expression. (A, F, K), AHR, GHRL2,and KIAA0101 expression in ccRCC vs normal tissue. (B, G, L), ROC analysis for AHR, GRHL2, and KIAA0101. (C, H), Survival analysis of AHR and GRHL2. (D, I, N), AHR, GRHL2, and KIAA0101 immunostaining. (E), AHR protein expression was higher in ccRCC (black boxes) compared to normal tissue (open boxes). (J, O) GRHL2 and KIAA0101 staining is associated with worse prognosis. (M), KIAA0101 Western blot of ccRCC vs normal tissue. *, Statistical significance; N, normal; C, cancer; DFS, disease-free survival; OS, overall survival.

Article Snippet: Immunohistochemistry was performed using primary antibodies against arylhydrocarbon receptor (AHR) (Novus Biologicals), grainyhead-like-2 (GRHL2) (Novus Biologicals), and KIAA0101 (Abnova).

Techniques: Expressing, Immunostaining, Staining, Western Blot

Journal: Journal of Dental Research

Article Title: Human Papillomavirus 16 E6 Induces FoxM1B in Oral Keratinocytes through GRHL2

doi: 10.1177/0022034518756071

Figure Lengend Snippet: Human papillomavirus type 16 (HPV-16) E6 induces GRHL2 and FoxM1B expression. (A) Western blotting was performed for GRHL2 and FoxM1B using whole-cell extracts (WCEs) from normal human oral keratinocytes (NHOKs) with different population doubling (PD15 and PD30), HOK-16B cultured in keratinocyte growth medium (KGM) or Dulbecco’s modified Eagle’s medium, and oral squamous cell carcinoma (OSCC) cell lines (HOK-16B/BaP-T, SCC4, and SCC15). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) GRHL2, FoxM1B, and hTERT messenger RNA (mRNA) levels were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in NHOKs infected by HPV-16 E6 or HPV-16 E7. Bars indicate standard deviation and * indicates statistical significance (P < 0.05), compared with the mean values of the control group (LXSN). (C) Western blotting was performed for GRHL2, FoxM1B, and Cyclin A in 2 strains of NHOKs expressing HPV-16 E6 or HPV-16 E7. (D) GRHL2, FoxM1B, and Cyclin A protein levels in tonsillar epithelial cells (TECs) and FaDu cells infected by HPV-16 E6, HPV-16 E7, or 16E6/E7. (E, F) NHOK cells were infected with HPV-16 virus, and then qRT-PCR was performed to detect gene expression of GRHL2 and FoxM1B and Western blotting was executed for determining the protein levels of GRHL2, FoxM1B, and its target proteins, Cyclin A and Cyclin B1. P53 and P21WAF1 were used as markers of HPV-16 virus infection. (G) HOK-16B/BaP-T cells were infected with HPV-16 E6 small interfering RNA to knockdown HPV-16 E6, and then Western blot was performed to determine protein levels of GRHL2, FoxM1B, and GRHL2 target proteins K14 and FN. GAPDH was used as loading control. Each experiment was repeated 3 times.

Article Snippet: Antibodies The following primary antibodies were used in this study: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), FoxM1B, Cyclin A, Cyclin B1, HELLS, p53, p21 WAF1 , and p16 INK4A from Santa Cruz Biotech; p-Rb Ser807/811 , p-AKT Ser473 , and p-p38 Thr180/182 from Cell Signaling Technology; and GRHL2 antibody from Abnova.

Techniques: Expressing, Western Blot, Cell Culture, Modification, Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Standard Deviation, Virus, Small Interfering RNA, Knockdown

Journal: Journal of Dental Research

Article Title: Human Papillomavirus 16 E6 Induces FoxM1B in Oral Keratinocytes through GRHL2

doi: 10.1177/0022034518756071

Figure Lengend Snippet: FoxM1B promotes malignant transformation of human oral keratinocytes (HOKs) harboring human papillomavirus type 16 (HPV-16). (A) HOK-16B cells were infected with pBabe empty vector (B0) or pBabe-FoxM1B and then selected with puromycin (1 µg/mL). The puromycin-resistant cells were exposed to medium with physiologic Ca++ level (1.5 mM) and maintained in serial subcultures. Their replication kinetics was plotted against time in culture. (B) Colony formation was performed using HOK-16B cells infected with pBabe empty vector (B0) or pBabe-FoxM1B (200 cells per well). (C) Western blotting was performed for FoxM1B, Cyclin A, Cyclin B1, HELLS, GRHL2, and p-RBser807/811 in HOK-16B cells infected with empty vector (B0) or pBabe-FoxM1B exposed to medium with physiologic Ca++ level for 7 d or 60 d. (D) Anchorage-independent growth (AIG) assay was performed with HOK-16B/B0, HOK-16B/FoxM1B, and HOK-16B/BaP-T in soft agar plates. Representative colony sizes were shown after 14 d postseeding in the upper panel. In the bottom panel, tumor spheroid assay was performed with HOK-16B/B0, HOK-16B/FoxM1B, and HOK-16B/BaP-T cells in suspension culture for 7 d. Growth of colonies in soft agar and tumor spheroids in ultra-low attachment plate was visualized under light microscopy. (E) Quantification of the number of colonies formed in the soft agar and the number and size of tumor spheroids formed by HOK-16B/B0, HOK-16B/FoxM1B, and HOK-16B/BaP-T cells in an ultra-low attachment plate. (F) Multistep oral carcinogenesis involves stepwise conversion of normal human oral keratinocytes (NHOKs), immortalized and nontumorigenic cells (HOK-16B), and tumorigenic cells (HOK-16B/BaP-T). HPV infection leads to FoxM1 induction, in part, through GRHL2. During tumorigenic conversion, additional unknown factors may lead to further elevation of FoxM1B, which is required to maintain the transformed phenotype.

Article Snippet: Antibodies The following primary antibodies were used in this study: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), FoxM1B, Cyclin A, Cyclin B1, HELLS, p53, p21 WAF1 , and p16 INK4A from Santa Cruz Biotech; p-Rb Ser807/811 , p-AKT Ser473 , and p-p38 Thr180/182 from Cell Signaling Technology; and GRHL2 antibody from Abnova.

Techniques: Transformation Assay, Infection, Plasmid Preparation, Western Blot, Suspension, Light Microscopy

Journal: Journal of Dental Research

Article Title: Human Papillomavirus 16 E6 Induces FoxM1B in Oral Keratinocytes through GRHL2

doi: 10.1177/0022034518756071

Figure Lengend Snippet: Loss of GRHL2 suppresses FoxM1B expression in 4-nitroquinolin 1-oxide (4-NQO)–induced tongue tumors. (A) Morphological change of tongues from Grhl2 wild-type (WT) and knockout (KO) mice treated without or with 4-NQO (30 µg/mL) for 16 wk to induce oral cancer formation. (B) Western blotting was performed with tongue tissues from Grhl2 WT or KO mice for FoxM1B, Cyclin A, Cyclin B1, and Cyclin D with or without 4-NQO exposure for 16 wk. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. (C) Western blotting signals were quantitated by densitometric analysis and plotted with the mean values for Grhl2 WT or KO mice with or without 4-NQO exposures. (D) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed with total RNA isolated tongue tissues. Data were derived from 3 independent experiments, and qRT-PCR assays were performed in triplicates. Error bars indicate standard deviation of the mean; *P < 0.05.

Article Snippet: Antibodies The following primary antibodies were used in this study: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), FoxM1B, Cyclin A, Cyclin B1, HELLS, p53, p21 WAF1 , and p16 INK4A from Santa Cruz Biotech; p-Rb Ser807/811 , p-AKT Ser473 , and p-p38 Thr180/182 from Cell Signaling Technology; and GRHL2 antibody from Abnova.

Techniques: Expressing, Knock-Out, Western Blot, Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Derivative Assay, Standard Deviation

Journal: Journal of Dental Research

Article Title: Human Papillomavirus 16 E6 Induces FoxM1B in Oral Keratinocytes through GRHL2

doi: 10.1177/0022034518756071

Figure Lengend Snippet: Human papillomavirus type 16 (HPV-16) E6 induces GRHL2 and FoxM1B expression in epithelial tissue in vivo. (A) Western blotting was performed with epidermal tissues isolated from wild-type mice (FvB) and HPV-16 E6 or HPV-16 E7 transgenic mice for GRHL2, FoxM1B, EGFR, and p53. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Western blotting signals were quantitated by densitometric analysis and plotted with the mean values for wild-type mice (FvB). Bar indicates mean (SD), and * indicates P < 0.05. (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed with total RNAs isolated from the epidermis of wild-type mice (FvB) and HPV-16 E6 or HPV-16 E7 mice. Experiments were performed in triplicates. Bars indicate standard deviation. * indicates statistically significant change (P < 0.05) compared with wild-type (WT) mice. (D) Skin tissues from HPV-16 E6 transgenic mice were stained for GRHL2, E-cadherin, FoxM1B, and p63 by immunofluorescence staining (IFS). Elevated GRHL2 and FoxM1B were noted in the epidermis of HPV-16 E6 transgenic mice. (E) Immunofluorescence staining was performed with normal human oral mucosa (NHOM) and HPV+ oral squamous cell carcinoma (OSCC) tissues for GRHL2, E-cadherin, p63, and FoxM1B antibody. Numbers of patients with HPV+ OSCC or healthy donors for NHOM were 10. Representative staining results are shown. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

Article Snippet: Antibodies The following primary antibodies were used in this study: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), FoxM1B, Cyclin A, Cyclin B1, HELLS, p53, p21 WAF1 , and p16 INK4A from Santa Cruz Biotech; p-Rb Ser807/811 , p-AKT Ser473 , and p-p38 Thr180/182 from Cell Signaling Technology; and GRHL2 antibody from Abnova.

Techniques: Expressing, In Vivo, Western Blot, Isolation, Transgenic Assay, Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Staining, Immunofluorescence

Journal: Journal of Dental Research

Article Title: Human Papillomavirus 16 E6 Induces FoxM1B in Oral Keratinocytes through GRHL2

doi: 10.1177/0022034518756071

Figure Lengend Snippet: GRHL2 is an upstream regulator of FoxM1B. (A) Normal human oral keratinocytes (NHOKs) stably transduced with retrovirus plasmid expressing GRHL2 and the empty vector (LXSN) at various population doubling (PD) were assessed for change in protein expression of GRHL2, FoxM1B, and Cyclin A1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was using as the loading control. (B) GRHL2 expression was knocked down using lentiviral vector (LV-ShGRHL2) in SCC4 followed by single-cell colony selection. As control, SCC4 was infected with lentiviral vector expressing EGFP (LV-EGFP). GRHL2 and FoxM1B levels were examined in different GRHL2 knockdown clones. (C) GRHL2 was knocked down in SCC15 and SCC4 and led to loss of FoxM1B. When GRHL2 was reexpressed in SCC4 through retroviral infection with LXSN-GRHL2 vector, FoxM1B expression was restored. (D) GRHL2 was overexpressed in HEp2 and SCC9 cells, both of which express low level of endogenous GRHL2. Ectopic expression of GRHL2 in these cells led to enhanced FoxM1B expression. (E) GRHL2 was knocked down in SCC15 or overexpressed in SCC9, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed for FoxM1B messenger RNA (mRNA) levels. (F) In the left panel, the FoxM1B promoter assay was performed in 293 cells with various concentrations of luciferase reporter plasmids, in which the luciferase reporter gene is driven by FoxM1B promoter (pFoxM1). In the right panel, FoxM1B promoter assay was performed in SCC4 cells with or without GRHL2 knockdown, using the luciferase reporter contsruct. * indicates statistical significance (P < 0.05). Empty plasmid, pGL3B, served as the negative control. Experiments were performed in triplicates.

Article Snippet: Antibodies The following primary antibodies were used in this study: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), FoxM1B, Cyclin A, Cyclin B1, HELLS, p53, p21 WAF1 , and p16 INK4A from Santa Cruz Biotech; p-Rb Ser807/811 , p-AKT Ser473 , and p-p38 Thr180/182 from Cell Signaling Technology; and GRHL2 antibody from Abnova.

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Expressing, Control, Selection, Infection, Knockdown, Clone Assay, Retroviral, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Promoter Assay, Luciferase, Negative Control