grhl2 antibody  (Bio-Techne corporation)

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: A tetracycline-inducible model of high GRHL2 expression in breast cancer cells expresses GRHL2 protein and mRNA in a Dox dose-dependent manner. ( A ) Schematic diagram of tet-inducible models. A Dox-inducible GRHL2-GFP construct was created via rtTA with a control or GRHL2-GFP plasmid in a pUHD10-3 backbone along with a tetracycline response element (TRE). Parental (P) cells lack an exogenous GRHL2-GFP gene. Cells with inducible overexpression of GRHL2-GFP are referred to as (OE). ( B ) Representative Western blot of engineered OE cells treated with Dox at the indicated doses. Endogenous GRHL2 (GRHL2) and overexpressed GRHL2 (GRHL2-GFP) are shown. β-actin is shown as a loading control. Uncropped Western blots are included in the . ( C ) Quantification of total GRHL2 protein (endogenous GRHL2 and GRHL2-GFP) in OE cells. Increase in total GRHL2 levels was relative to the amount of endogenous GRHL2 protein present in cells grown in the absence of Dox, set at 1.0. n = 3. *, p < 0.05. ( D ) Representative Western blot as in ( B ) in OE cells treated for the indicated length of time with Dox. Uncropped Western blots are included in the . ( E ) Quantification of GRHL2 protein in OE cells treated with 1 μg/mL Dox for the indicated length of time. Increase in total GRHL2 levels was relative to the amount of endogenous GRHL2 protein present in cells grown in the absence of Dox, set at 1.0. n = 3. *, p < 0.05; **, p < 0.01 relative to vehicle. ( F ) RT-qPCR analysis of GRHL2 in dose response studies. n = 3. *, p < 0.05 relative to vehicle. ( G ) RT-qPCR analysis of GRHL2 in time course studies. n = 3. *, p < 0.05; **, p < 0.01 relative to vehicle.

Article Snippet: Slides were blocked for 45 min in 5% goat serum (Vector Laboratories, Newark, CA, USA, #PK-4001) in TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) prior to an overnight incubation at 4 °C with a primary antibody for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα cl.

Techniques: Expressing, Construct, Control, Plasmid Preparation, Over Expression, Western Blot, Quantitative RT-PCR

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: High GRHL2 expression increases epithelial cell phenotypes. ( A ) Quantification of the % cell gap closure of P and OE cells subjected to a migration assay. n = 3. *, p -value < 0.05 relative to vehicle. ( B ) Quantification of the % cell gap closure over 24 h of MCF7 and CAMA-1 cells transiently transfected with GRHL2 DNA. n = 3. *, p -value < 0.05 relative to vehicle; **, p -value < 0.01 relative to vector control. ( C ) RT-qPCR analyses of representative epithelial genes CDH1 and CLDN4 in OE cells. n = 3. *, p -value < 0.05 relative to no Dox. supports increase in CDH1 in T47D cells transfected with 1 μg/uL of GRHL2-GFP plasmid. ( D ) Absolute quantification of total GRHL2 mRNA in OE cells and MCF7, T47D, and CAMA-1 cells transfected with GRHL2 DNA. n = 3. *, p < 0.05; ** p < 0.01 relative to no Dox treatment or vector control. provides fluorescence microscopy confirmation of elevated GRHL2 levels in these cells.

Article Snippet: Slides were blocked for 45 min in 5% goat serum (Vector Laboratories, Newark, CA, USA, #PK-4001) in TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) prior to an overnight incubation at 4 °C with a primary antibody for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα cl.

Techniques: Expressing, Migration, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Quantitative Proteomics, Fluorescence, Microscopy

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression alters its endogenous transcriptional activity and gene expression. ( A ) Flow cytometry FACS gating to isolate GFPpositive, GRHL2-high cells (green) from GFP-negative, GRHL2-low (red) cells. FACS-sorted GFP-negative and -positive samples were used for RNA sequencing along with a negative untreated control. n = 5. ( B ) Venn diagram of RNA-seq data displaying the differentially expressed (DE) genes between the GFP-negative, GFP-positive, and negative control gene sets. Select genes are referenced, and bolded genes refer to an association with the epithelial to mesenchymal transition (EMT) gene ontology pathway. specifies the 105 DE genes in the Venn diagram. ( C ) Volcano plot of RNA-seq data depicts fold change of downregulated (black) and upregulated (red) DE genes. Fold change represents the GFP-negative vs. GFP-positive gene set comparison. Outlier genes include ENDOD1 and FAM41C . ( D ) RT-qPCR validation of representative genes from the GFP-negative vs. GFP-positive gene set. n = 3. *, p < 0.05 relative to GFP-negative.

Article Snippet: Slides were blocked for 45 min in 5% goat serum (Vector Laboratories, Newark, CA, USA, #PK-4001) in TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) prior to an overnight incubation at 4 °C with a primary antibody for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα cl.

Techniques: Over Expression, Activity Assay, Gene Expression, Flow Cytometry, RNA Sequencing, Control, Negative Control, Comparison, Quantitative RT-PCR, Biomarker Discovery

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression regulates development and growth. ( A ) Gene ontology analysis with MSigDB biological processes performed with clusterProfiler on the unique 105 gene cluster in the GFP-negative versus GFP-positive gene set. Terms related to EMT and development are highlighted in red. ( B ) RT-qPCR analysis of PEA15 from the unique 105 gene cluster in the GFP-negative versus GFP-positive gene set. n = 3. *, p < 0.05 versus GFP-negative. ( C ) Flow cytometry cell cycle analysis shows % of cells in S, G2, and G1 cell cycles in P and OE cells. n = 3. ( D ) RT-qPCR analyses of representative tumor dormancy genes NR2F1 and CDKN1B in OE cells. n = 3. *, p -value < 0.05; **, p < 0.01 versus no Dox.

Article Snippet: Slides were blocked for 45 min in 5% goat serum (Vector Laboratories, Newark, CA, USA, #PK-4001) in TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) prior to an overnight incubation at 4 °C with a primary antibody for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα cl.

Techniques: Over Expression, Quantitative RT-PCR, Flow Cytometry, Cell Cycle Assay

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression inhibits proliferation in vivo. ( A ) Quantification of soft agar colony formation in P and OE cells. n = 3. *, p < 0.05 versus no Dox. ( B ) Quantification of tumor growth in mice injected with P or OE cells. An arrow marks the introduction of Dox treatment. n = 10. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus parental. ( C ) Weight of tumors derived from P or OE tumors. n = 10. **, p < 0.01 versus P. ( D ) RT-qPCR analysis of GFP gene expression in murine tumors. n = 8. ***, p < 0.001 versus P tumors. ( E ) Representative immunohistochemistry (IHC) staining on proliferation and dormancy-associated proteins on excised P and OE tumors. IHC staining portrays: GRHL2, Ki67, p27, and hematoxylin and eosin (H&E) in tissue sections derived from mammary fat pad tumors. All images are shown at 10× magnification.

Article Snippet: Slides were blocked for 45 min in 5% goat serum (Vector Laboratories, Newark, CA, USA, #PK-4001) in TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) prior to an overnight incubation at 4 °C with a primary antibody for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα cl.

Techniques: Over Expression, In Vivo, Injection, Derivative Assay, Quantitative RT-PCR, Gene Expression, Immunohistochemistry

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression enriches stem cell-like characteristics. ( A ) Quantification of primary of P, OE, and OE pool cells. GFP immunofluorescence microscopy confirmed GRHL2 induction after initial Dox treatment. Error bars represent the mean fold change in mammosphere formation efficiency (MFE%) relative to vehicle. n = 4. *, p < 0.05; ****, p < 0.0001. ( B ) Quantification of secondary P, OE, and OE pool cells. GFP immunofluorescence microscopy confirmed GRHL2 induction after initial Dox treatment. Error bars represent the mean fold change in mammosphere formation efficiency (MFE%) relative to vehicle. n = 4. *, p < 0.05; **, p < 0.01. ( C ) Representative flow cytometry profiles of CD24 and CD44 expression in OE cells. Numbers refer to % of cells in the population. n = 3. ( D ) Quantification of flow cytometry analyses on % of cells co-expressing CD24 and CD44 in OE cells. Error bar represents the mean CD24+/CD44+ % ± SEM. n = 3. *, p < 0.05. ( E ) Representative flow cytometry profiles of ALDH1 activity in OE cells using the Aldefluor assay. SSC refers to the side scatter optical detector. Gating represents the % of ALDH1-positive cells in the OE population. Diethylaminobenzaldehyde (DEAB) was used as a control for the background signal. ( F ) Quantification of the % of OE cells expressing ALDH1. Error bar represents the mean ALDH1+ % ± SEM. n = 3 for DEAB-negative cells. **, p < 0.01. ns = not significant. ( G ) Quantification of fold change ALDH1+ % in OE cells as compared to the DEAB control. Error bars represent the mean ALDH % ± SEM relative to DEAB control. n = 3. *, p < 0.05.

Article Snippet: Slides were blocked for 45 min in 5% goat serum (Vector Laboratories, Newark, CA, USA, #PK-4001) in TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) prior to an overnight incubation at 4 °C with a primary antibody for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα cl.

Techniques: Over Expression, Immunofluorescence, Microscopy, Flow Cytometry, Expressing, Activity Assay, Control

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression leads to a complex epithelial–mesenchymal hybrid phenotype. ( A ) Fluorescence microscopy immunocytochemistry of E-cadherin and vimentin in P and OE cells treated with 1 μg/mL Dox for 72 h. Fluorescence imaging attained by 600× oil microscopy with 0.33 μm/pixels, scale bar of 20 μm. ( B ) RT-qPCR analysis of VIM in OE cells treated with 1 μg/mL Dox and harvested at the indicated times. Error bars represent the mean mRNA fold change ± SEM relative to the vehicle. n = 3. *, p < 0.05. ( C ) RT-qPCR analyses of VIM mRNA in MCF7 cells transiently transfected with GRHL2-GFP DNA. Error bars represent the mean mRNA fold change ± SEM relative to the vector control. n = 3. *, p < 0.05. ( D ) RT-qPCR analyses of VIM mRNA in T47D cells transiently transfected with GRHL2-GFP DNA. Error bars represent the mean mRNA fold change ± SEM relative to the vector control. n = 3. *, p < 0.05. ( E ) RT-qPCR analyses of VIM mRNA in CAMA-1 cells transiently transfected with GRHL2-GFP DNA. Error bars represent the mean mRNA fold change ± SEM relative to the vector control. n = 3. *, p < 0.05.

Article Snippet: Slides were blocked for 45 min in 5% goat serum (Vector Laboratories, Newark, CA, USA, #PK-4001) in TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) prior to an overnight incubation at 4 °C with a primary antibody for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα cl.

Techniques: Over Expression, Fluorescence, Microscopy, Immunocytochemistry, Imaging, Quantitative RT-PCR, Transfection, Plasmid Preparation, Control

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression alters GRHL2 genome binding in a dynamic manner. ( A ) Venn diagram displaying differentially bound sites between +Dox and −Dox datasets in GRHL2-overexpressing OE cells. −Dox and +Dox datasets represent the overlap of 24, 48, and 72 h datasets under −Dox and +Dox conditions, respectively. Three separate binding groups were established: −Dox only (485 sites), Dox independent (3481 sites), and +Dox only (512 sites). ( B ) Representative genome track of a GRHL2 binding site. Internal numbers represent ChIP signal intensity. ( C ) RT-qPCR analysis of SP6 and NR2F1 , a representative +Dox binding group and dormancy gene, respectively, under −Dox (red) or +Dox (blue) treatment at the indicated times. Error bars represent the mean ± SEM, n = 3. *, p < 0.05.

Article Snippet: Slides were blocked for 45 min in 5% goat serum (Vector Laboratories, Newark, CA, USA, #PK-4001) in TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) prior to an overnight incubation at 4 °C with a primary antibody for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα cl.

Techniques: Over Expression, Binding Assay, Quantitative RT-PCR

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression changes motifs found near GRHL2 binding sites. ( A ) Consensus sequence logos representing the top motifs in the −Dox and +Dox peak sets, acquired from HOMER de novo motif analysis. ( B ) Motif analyses using top motifs from −Dox (red) and +Dox (blue) peak sets relative to background (gray). Data are shown as % of binding sites that contain the specific motif. p -values are derived from chi-square test and HOMER de novo motif analysis.

Article Snippet: Slides were blocked for 45 min in 5% goat serum (Vector Laboratories, Newark, CA, USA, #PK-4001) in TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) prior to an overnight incubation at 4 °C with a primary antibody for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα cl.

Techniques: Over Expression, Binding Assay, Sequencing, Derivative Assay

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: A tetracycline-inducible model of high GRHL2 expression in breast cancer cells expresses GRHL2 protein and mRNA in a Dox dose-dependent manner. ( A ) Schematic diagram of tet-inducible models. A Dox-inducible GRHL2-GFP construct was created via rtTA with a control or GRHL2-GFP plasmid in a pUHD10-3 backbone along with a tetracycline response element (TRE). Parental (P) cells lack an exogenous GRHL2-GFP gene. Cells with inducible overexpression of GRHL2-GFP are referred to as (OE). ( B ) Representative Western blot of engineered OE cells treated with Dox at the indicated doses. Endogenous GRHL2 (GRHL2) and overexpressed GRHL2 (GRHL2-GFP) are shown. β-actin is shown as a loading control. Uncropped Western blots are included in the . ( C ) Quantification of total GRHL2 protein (endogenous GRHL2 and GRHL2-GFP) in OE cells. Increase in total GRHL2 levels was relative to the amount of endogenous GRHL2 protein present in cells grown in the absence of Dox, set at 1.0. n = 3. *, p < 0.05. ( D ) Representative Western blot as in ( B ) in OE cells treated for the indicated length of time with Dox. Uncropped Western blots are included in the . ( E ) Quantification of GRHL2 protein in OE cells treated with 1 μg/mL Dox for the indicated length of time. Increase in total GRHL2 levels was relative to the amount of endogenous GRHL2 protein present in cells grown in the absence of Dox, set at 1.0. n = 3. *, p < 0.05; **, p < 0.01 relative to vehicle. ( F ) RT-qPCR analysis of GRHL2 in dose response studies. n = 3. *, p < 0.05 relative to vehicle. ( G ) RT-qPCR analysis of GRHL2 in time course studies. n = 3. *, p < 0.05; **, p < 0.01 relative to vehicle.

Article Snippet: Protein preparation and Western blotting were performed as previously described using antibodies for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα clone sc-7207; Santa Cruz Biotechnology, Santa Cruz, CA, USA, #H-184), and β-actin (clone AC-15; Millipore Sigma, #A5441) [ ].

Techniques: Expressing, Construct, Control, Plasmid Preparation, Over Expression, Western Blot, Quantitative RT-PCR

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: High GRHL2 expression increases epithelial cell phenotypes. ( A ) Quantification of the % cell gap closure of P and OE cells subjected to a migration assay. n = 3. *, p -value < 0.05 relative to vehicle. ( B ) Quantification of the % cell gap closure over 24 h of MCF7 and CAMA-1 cells transiently transfected with GRHL2 DNA. n = 3. *, p -value < 0.05 relative to vehicle; **, p -value < 0.01 relative to vector control. ( C ) RT-qPCR analyses of representative epithelial genes CDH1 and CLDN4 in OE cells. n = 3. *, p -value < 0.05 relative to no Dox. supports increase in CDH1 in T47D cells transfected with 1 μg/uL of GRHL2-GFP plasmid. ( D ) Absolute quantification of total GRHL2 mRNA in OE cells and MCF7, T47D, and CAMA-1 cells transfected with GRHL2 DNA. n = 3. *, p < 0.05; ** p < 0.01 relative to no Dox treatment or vector control. provides fluorescence microscopy confirmation of elevated GRHL2 levels in these cells.

Article Snippet: Protein preparation and Western blotting were performed as previously described using antibodies for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα clone sc-7207; Santa Cruz Biotechnology, Santa Cruz, CA, USA, #H-184), and β-actin (clone AC-15; Millipore Sigma, #A5441) [ ].

Techniques: Expressing, Migration, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Quantitative Proteomics, Fluorescence, Microscopy

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression alters its endogenous transcriptional activity and gene expression. ( A ) Flow cytometry FACS gating to isolate GFPpositive, GRHL2-high cells (green) from GFP-negative, GRHL2-low (red) cells. FACS-sorted GFP-negative and -positive samples were used for RNA sequencing along with a negative untreated control. n = 5. ( B ) Venn diagram of RNA-seq data displaying the differentially expressed (DE) genes between the GFP-negative, GFP-positive, and negative control gene sets. Select genes are referenced, and bolded genes refer to an association with the epithelial to mesenchymal transition (EMT) gene ontology pathway. specifies the 105 DE genes in the Venn diagram. ( C ) Volcano plot of RNA-seq data depicts fold change of downregulated (black) and upregulated (red) DE genes. Fold change represents the GFP-negative vs. GFP-positive gene set comparison. Outlier genes include ENDOD1 and FAM41C . ( D ) RT-qPCR validation of representative genes from the GFP-negative vs. GFP-positive gene set. n = 3. *, p < 0.05 relative to GFP-negative.

Article Snippet: Protein preparation and Western blotting were performed as previously described using antibodies for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα clone sc-7207; Santa Cruz Biotechnology, Santa Cruz, CA, USA, #H-184), and β-actin (clone AC-15; Millipore Sigma, #A5441) [ ].

Techniques: Over Expression, Activity Assay, Gene Expression, Flow Cytometry, RNA Sequencing, Control, Negative Control, Comparison, Quantitative RT-PCR, Biomarker Discovery

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression regulates development and growth. ( A ) Gene ontology analysis with MSigDB biological processes performed with clusterProfiler on the unique 105 gene cluster in the GFP-negative versus GFP-positive gene set. Terms related to EMT and development are highlighted in red. ( B ) RT-qPCR analysis of PEA15 from the unique 105 gene cluster in the GFP-negative versus GFP-positive gene set. n = 3. *, p < 0.05 versus GFP-negative. ( C ) Flow cytometry cell cycle analysis shows % of cells in S, G2, and G1 cell cycles in P and OE cells. n = 3. ( D ) RT-qPCR analyses of representative tumor dormancy genes NR2F1 and CDKN1B in OE cells. n = 3. *, p -value < 0.05; **, p < 0.01 versus no Dox.

Article Snippet: Protein preparation and Western blotting were performed as previously described using antibodies for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα clone sc-7207; Santa Cruz Biotechnology, Santa Cruz, CA, USA, #H-184), and β-actin (clone AC-15; Millipore Sigma, #A5441) [ ].

Techniques: Over Expression, Quantitative RT-PCR, Flow Cytometry, Cell Cycle Assay

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression inhibits proliferation in vivo. ( A ) Quantification of soft agar colony formation in P and OE cells. n = 3. *, p < 0.05 versus no Dox. ( B ) Quantification of tumor growth in mice injected with P or OE cells. An arrow marks the introduction of Dox treatment. n = 10. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus parental. ( C ) Weight of tumors derived from P or OE tumors. n = 10. **, p < 0.01 versus P. ( D ) RT-qPCR analysis of GFP gene expression in murine tumors. n = 8. ***, p < 0.001 versus P tumors. ( E ) Representative immunohistochemistry (IHC) staining on proliferation and dormancy-associated proteins on excised P and OE tumors. IHC staining portrays: GRHL2, Ki67, p27, and hematoxylin and eosin (H&E) in tissue sections derived from mammary fat pad tumors. All images are shown at 10× magnification.

Article Snippet: Protein preparation and Western blotting were performed as previously described using antibodies for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα clone sc-7207; Santa Cruz Biotechnology, Santa Cruz, CA, USA, #H-184), and β-actin (clone AC-15; Millipore Sigma, #A5441) [ ].

Techniques: Over Expression, In Vivo, Injection, Derivative Assay, Quantitative RT-PCR, Gene Expression, Immunohistochemistry

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression enriches stem cell-like characteristics. ( A ) Quantification of primary of P, OE, and OE pool cells. GFP immunofluorescence microscopy confirmed GRHL2 induction after initial Dox treatment. Error bars represent the mean fold change in mammosphere formation efficiency (MFE%) relative to vehicle. n = 4. *, p < 0.05; ****, p < 0.0001. ( B ) Quantification of secondary P, OE, and OE pool cells. GFP immunofluorescence microscopy confirmed GRHL2 induction after initial Dox treatment. Error bars represent the mean fold change in mammosphere formation efficiency (MFE%) relative to vehicle. n = 4. *, p < 0.05; **, p < 0.01. ( C ) Representative flow cytometry profiles of CD24 and CD44 expression in OE cells. Numbers refer to % of cells in the population. n = 3. ( D ) Quantification of flow cytometry analyses on % of cells co-expressing CD24 and CD44 in OE cells. Error bar represents the mean CD24+/CD44+ % ± SEM. n = 3. *, p < 0.05. ( E ) Representative flow cytometry profiles of ALDH1 activity in OE cells using the Aldefluor assay. SSC refers to the side scatter optical detector. Gating represents the % of ALDH1-positive cells in the OE population. Diethylaminobenzaldehyde (DEAB) was used as a control for the background signal. ( F ) Quantification of the % of OE cells expressing ALDH1. Error bar represents the mean ALDH1+ % ± SEM. n = 3 for DEAB-negative cells. **, p < 0.01. ns = not significant. ( G ) Quantification of fold change ALDH1+ % in OE cells as compared to the DEAB control. Error bars represent the mean ALDH % ± SEM relative to DEAB control. n = 3. *, p < 0.05.

Article Snippet: Protein preparation and Western blotting were performed as previously described using antibodies for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα clone sc-7207; Santa Cruz Biotechnology, Santa Cruz, CA, USA, #H-184), and β-actin (clone AC-15; Millipore Sigma, #A5441) [ ].

Techniques: Over Expression, Immunofluorescence, Microscopy, Flow Cytometry, Expressing, Activity Assay, Control

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression leads to a complex epithelial–mesenchymal hybrid phenotype. ( A ) Fluorescence microscopy immunocytochemistry of E-cadherin and vimentin in P and OE cells treated with 1 μg/mL Dox for 72 h. Fluorescence imaging attained by 600× oil microscopy with 0.33 μm/pixels, scale bar of 20 μm. ( B ) RT-qPCR analysis of VIM in OE cells treated with 1 μg/mL Dox and harvested at the indicated times. Error bars represent the mean mRNA fold change ± SEM relative to the vehicle. n = 3. *, p < 0.05. ( C ) RT-qPCR analyses of VIM mRNA in MCF7 cells transiently transfected with GRHL2-GFP DNA. Error bars represent the mean mRNA fold change ± SEM relative to the vector control. n = 3. *, p < 0.05. ( D ) RT-qPCR analyses of VIM mRNA in T47D cells transiently transfected with GRHL2-GFP DNA. Error bars represent the mean mRNA fold change ± SEM relative to the vector control. n = 3. *, p < 0.05. ( E ) RT-qPCR analyses of VIM mRNA in CAMA-1 cells transiently transfected with GRHL2-GFP DNA. Error bars represent the mean mRNA fold change ± SEM relative to the vector control. n = 3. *, p < 0.05.

Article Snippet: Protein preparation and Western blotting were performed as previously described using antibodies for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα clone sc-7207; Santa Cruz Biotechnology, Santa Cruz, CA, USA, #H-184), and β-actin (clone AC-15; Millipore Sigma, #A5441) [ ].

Techniques: Over Expression, Fluorescence, Microscopy, Immunocytochemistry, Imaging, Quantitative RT-PCR, Transfection, Plasmid Preparation, Control

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression alters GRHL2 genome binding in a dynamic manner. ( A ) Venn diagram displaying differentially bound sites between +Dox and −Dox datasets in GRHL2-overexpressing OE cells. −Dox and +Dox datasets represent the overlap of 24, 48, and 72 h datasets under −Dox and +Dox conditions, respectively. Three separate binding groups were established: −Dox only (485 sites), Dox independent (3481 sites), and +Dox only (512 sites). ( B ) Representative genome track of a GRHL2 binding site. Internal numbers represent ChIP signal intensity. ( C ) RT-qPCR analysis of SP6 and NR2F1 , a representative +Dox binding group and dormancy gene, respectively, under −Dox (red) or +Dox (blue) treatment at the indicated times. Error bars represent the mean ± SEM, n = 3. *, p < 0.05.

Article Snippet: Protein preparation and Western blotting were performed as previously described using antibodies for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα clone sc-7207; Santa Cruz Biotechnology, Santa Cruz, CA, USA, #H-184), and β-actin (clone AC-15; Millipore Sigma, #A5441) [ ].

Techniques: Over Expression, Binding Assay, Quantitative RT-PCR

Journal: Cancers

Article Title: Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells

doi: 10.3390/cancers16162906

Figure Lengend Snippet: GRHL2 overexpression changes motifs found near GRHL2 binding sites. ( A ) Consensus sequence logos representing the top motifs in the −Dox and +Dox peak sets, acquired from HOMER de novo motif analysis. ( B ) Motif analyses using top motifs from −Dox (red) and +Dox (blue) peak sets relative to background (gray). Data are shown as % of binding sites that contain the specific motif. p -values are derived from chi-square test and HOMER de novo motif analysis.

Article Snippet: Protein preparation and Western blotting were performed as previously described using antibodies for GRHL2 (Millipore Sigma, #HPA004820), estrogen receptor alpha (ERα clone sc-7207; Santa Cruz Biotechnology, Santa Cruz, CA, USA, #H-184), and β-actin (clone AC-15; Millipore Sigma, #A5441) [ ].

Techniques: Over Expression, Binding Assay, Sequencing, Derivative Assay

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet: Loss of GRHL2 upregulates CD73 expression in MCF-7 cells (A) Bru-seq reads of nascent NT5E mRNA in an MCF-7 conditional GRHL2 KO model. Graphs are shown for a control sgRNA (Ctrl) and 2 GRHL2 sgRNA models (KO-1 and 2), and colors represent the indicated time points after doxycycline-induced GRHL2 deletion. The reference sequence annotation is shown above with exons in green blocks. (B) Immunofluorescence images of HOECHST (blue), Cas9 Ab (red), or GRHL2 Ab (green) for Ctrl or GRHL2 KO MCF-7 cells after 48 h 1 μg/ml doxycycline treatment. (C) Graph showing log2 fold changes of nascent NT5E mRNA for the indicated time points after doxycycline exposure in Ctrl or GRHL2 KO MCF-7 cells. (D) RT-qPCR analysis showing changes in total NT5E mRNA expression at the indicated time points after doxycycline-induced GRHL2 deletion. Values were normalized to the untreated samples for each time point. Data analyzed using 2–ΔΔCt method. Mean and SD of three biological replicates is shown. (two-way ANOVA test; n.s., non-significant; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (E) Western blot analysis of Cas9, GRHL2, and CD73 in MCF-7 control sgRNA (Ctrl) and 2 GRHL2 sgRNA models (KO-1 and KO-2) at the indicated time points after doxycycline induced GRHL2 deletion. Tubulin serves as a loading control. One out of three biological replicates shown. MDA-MB-231 basal B cells serve as positive control for CD73 expression.

Article Snippet: Cells were fixed and permeabilized by incubation with 4% formaldehyde and 0.1% Triton X-100 in Phosphate-buffered saline (PBS) for 15 min, followed by a blocking step with 0.5% w/v bovine serum albumin (BSA, Sigma Aldrich) in (PBS) for 30 min. Then, cells were incubated with the GRHL2 primary antibody (1:500, Atlas-Antibodies, hpa004820) in 0.5% w/v BSA in PBS overnight at 4°C.

Techniques: Expressing, Control, Sequencing, Immunofluorescence, Quantitative RT-PCR, Western Blot, Positive Control

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet: NT5E/CD73 levels are inversely correlated with GRHL2 in breast cancer subtypes and breast cancer patient tumor samples (A) Violin plots showing gene expression levels of GRHL2 and NT5E based on RNA-seq data for 52 human breast cancer cell lines grouped according to the indicated subtypes. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. p values calculated using one-way ANOVA as described in a previous study. (B) RT-qPCR analysis showing CD73 (left panel) and GRHL2 mRNA expression for the indicated cell lines. Data analyzed using 2–ΔCt method for the gene of interest corrected for GAPDH control. Mean and SD of two experiments performed in triplicate is shown. (two-way ANOVA test). (C) Western blot analysis of CD73 and GAPDH loading control in the indicated cell lines. One experiment of 2 is shown. (D) Flow cytometry analysis of CD73 surface expression in the indicated cell lines. Mean and SD of two experiments performed in duplicate is shown. (E) Correlation between gene expression levels of NT5E and GRHL2 in breast cancer tumors using METABRIC dataset. Correlation scores and p values shown as determined in cBioPortal. (F) CD73 score derived from IHC, on whole slides of a series of metaplastic (TNBC) and high and low GRHL2 mRNA tumors, separated for GRHL2 IHC positive and GRHL2 IHC negative cases. Mean and SEM is shown. p value calculated using non-parametric t test with unequal variance. (G) Representative IHC images for GRHL2 and CD73 in breast cancer tissues. Tumor 1, 2, and 4 are metaplastic tumors. Tumor 1 containing ∗area with non-invasive tumor cells staining positive for GRHL2 and negative for CD73. ∗∗area with stroma containing invasive tumor cells staining negative for GRHL2 and positive for CD73. Tumor 2 Arrow indicates milk duct with GRHL2 positive/CD73 negative epithelial cells surrounded by GRHL2 negative/CD73 positive stroma. ∗∗area with invasive tumor cells staining positive for GRHL2 and negative for CD73. Tumor 3 mRNA high tumor containing ∗∗area with stroma containing invasive tumor cells staining positive for GRHL2 and negative for CD73. Tumor 4 ∗∗area containing invasive tumor cells staining negative for GRHL2 and positive for CD73. Bars, 50 μm; note that magnification is higher for tumor 1, 3, and 4 versus tumor 2.

Article Snippet: Cells were fixed and permeabilized by incubation with 4% formaldehyde and 0.1% Triton X-100 in Phosphate-buffered saline (PBS) for 15 min, followed by a blocking step with 0.5% w/v bovine serum albumin (BSA, Sigma Aldrich) in (PBS) for 30 min. Then, cells were incubated with the GRHL2 primary antibody (1:500, Atlas-Antibodies, hpa004820) in 0.5% w/v BSA in PBS overnight at 4°C.

Techniques: Gene Expression, RNA Sequencing, Quantitative RT-PCR, Expressing, Control, Western Blot, Flow Cytometry, Derivative Assay, Staining

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet: Conserved GRHL2 binding site in the NT5E gene across luminal and basal A breast cancer cell lines (A) GRHL2 ChIP tracks showing its interactions along the NT5E DNA in the indicated luminal (labeled red) and basal A cell lines (labeled green). Note that track heights use different scales. Annotation of the reference sequence is displayed below with exons in blue bars. (B) GRHL2 binding motif identified in GRHL2 ChIP-seq peaks on NT5E DNA using MEME ChIP on data retrieved from the JASPAR database. Y axis shows frequency matrix of each base occurrence. (C) Table showing locations and frequency of the GRHL2 motif in the NT5E gene for the indicated breast cancer cell lines as determined using FIMO. Motif occurrence measured with log-odds scores and converted into p values; q values calculated using Benjamini and Hochberg method.

Article Snippet: Cells were fixed and permeabilized by incubation with 4% formaldehyde and 0.1% Triton X-100 in Phosphate-buffered saline (PBS) for 15 min, followed by a blocking step with 0.5% w/v bovine serum albumin (BSA, Sigma Aldrich) in (PBS) for 30 min. Then, cells were incubated with the GRHL2 primary antibody (1:500, Atlas-Antibodies, hpa004820) in 0.5% w/v BSA in PBS overnight at 4°C.

Techniques: Binding Assay, Labeling, Sequencing, ChIP-sequencing

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet: GRHL2 regulates CD73-mediated extracellular adenosine production (A) Immunofluorescence staining showing GRHL2 loss at 7 days after doxycycline-mediated Cas9 induction in sgGRHL2 cells (KO-1 and KO-2) but not sgCtrl cells for the experiment shown in C. Green, GRHL2 Ab; Blue, Hoechst. (B) Cartoon displaying the enzymatic conversion of AMP into adenosine by CD73 resulting in the production inorganic phosphate. Image created using BioRender. (C) Inorganic phosphate concentration measured by malachite green assay in culture supernatants from the MCF-7 conditional KO model taken in parallel to images shown in A and basal B positive control cell lines MDA-MB-231 and Hs578T. Cells were incubated in absence or presence of 100 μM AMP (substrate) with or without 25 μM CD73 inhibitor, APCP for 125 min. Mean ± SD of four biological replicates is shown (two-way ANOVA test; n.s., non-significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: Cells were fixed and permeabilized by incubation with 4% formaldehyde and 0.1% Triton X-100 in Phosphate-buffered saline (PBS) for 15 min, followed by a blocking step with 0.5% w/v bovine serum albumin (BSA, Sigma Aldrich) in (PBS) for 30 min. Then, cells were incubated with the GRHL2 primary antibody (1:500, Atlas-Antibodies, hpa004820) in 0.5% w/v BSA in PBS overnight at 4°C.

Techniques: Immunofluorescence, Staining, Concentration Assay, Malachite Green Assay, Positive Control, Incubation

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet: Enhanced CD8 + T cell recruitment in response to GRHL2 loss (A) TIMER 2.0 scatterplots showing the correlation of NT5E mRNA expression with tumor purity (percentage of malignant cells in a tumor tissue; left) and the predicted presence of CD8 + T cells (right) in all breast cancer (BRCA) lesions tested. (B) Table showing TIMER 2.0 Spearman correlation scores for association between NT5E mRNA and predicted presence of CD8 + T cells across breast cancer patient tumors separated for different molecular subtypes. (C) CD8 score (% CD8 positive T cells) derived from IHC, on whole slides of a series of metaplastic (TNBC) and high and low GRHL2 mRNA tumors, separated for IHC determined CD73 membrane staining positive and negative cases. Mean and SEM is shown. p value calculated using non-parametric t test with unequal variance. (D) Representative IHC images for GRHL2, CD73, and CD8 in breast cancer tissues. Arrow indicates area of infiltrated CD8 T cells in tumor 2 that is enlarged in the lower right corner. Bar, 50 μm. (E) Quantification of the number of CD8 + T cells recruited toward the lower compartment of transwells at increasing concentrations of CXCL12 in presence or absence of 100 μM stable adenosine analog, NECA. Average and SEM of 3 biological replicates is shown (two-way ANOVA test; ∗∗∗ p < 0.001. (F) Immunofluorescence staining showing GRHL2 loss at 8 days after doxycycline-mediated Cas9 induction in sgGRHL2 cells but not sgCtrl cells for the experiment shown in G. Green, GRHL2 Ab; Blue, Hoechst. (G) Quantification of CD8 + T cells recruited toward the lower compartment of transwells seeded with control or GRHL2 KO-1 MCF-7 cells (8 days 1 μg/ml doxycycline treatment) in absence or presence of 100 ng/mL CXCL12 and 200 μM APCP CD73 inhibitor. Data normalized to the T cells only condition. Mean and SEM of 3 biological replicates is shown (two-way ANOVA test; ∗∗∗ p < 0.001; n.s., non-significant).

Article Snippet: Cells were fixed and permeabilized by incubation with 4% formaldehyde and 0.1% Triton X-100 in Phosphate-buffered saline (PBS) for 15 min, followed by a blocking step with 0.5% w/v bovine serum albumin (BSA, Sigma Aldrich) in (PBS) for 30 min. Then, cells were incubated with the GRHL2 primary antibody (1:500, Atlas-Antibodies, hpa004820) in 0.5% w/v BSA in PBS overnight at 4°C.

Techniques: Expressing, Derivative Assay, Membrane, Staining, Immunofluorescence, Control

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet:

Article Snippet: Cells were fixed and permeabilized by incubation with 4% formaldehyde and 0.1% Triton X-100 in Phosphate-buffered saline (PBS) for 15 min, followed by a blocking step with 0.5% w/v bovine serum albumin (BSA, Sigma Aldrich) in (PBS) for 30 min. Then, cells were incubated with the GRHL2 primary antibody (1:500, Atlas-Antibodies, hpa004820) in 0.5% w/v BSA in PBS overnight at 4°C.

Techniques: Recombinant, Blocking Assay, Western Blot, cDNA Synthesis, Cell Isolation, RNA Sequencing, CRISPR, Plasmid Preparation, Software

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet: Loss of GRHL2 upregulates CD73 expression in MCF-7 cells (A) Bru-seq reads of nascent NT5E mRNA in an MCF-7 conditional GRHL2 KO model. Graphs are shown for a control sgRNA (Ctrl) and 2 GRHL2 sgRNA models (KO-1 and 2), and colors represent the indicated time points after doxycycline-induced GRHL2 deletion. The reference sequence annotation is shown above with exons in green blocks. (B) Immunofluorescence images of HOECHST (blue), Cas9 Ab (red), or GRHL2 Ab (green) for Ctrl or GRHL2 KO MCF-7 cells after 48 h 1 μg/ml doxycycline treatment. (C) Graph showing log2 fold changes of nascent NT5E mRNA for the indicated time points after doxycycline exposure in Ctrl or GRHL2 KO MCF-7 cells. (D) RT-qPCR analysis showing changes in total NT5E mRNA expression at the indicated time points after doxycycline-induced GRHL2 deletion. Values were normalized to the untreated samples for each time point. Data analyzed using 2–ΔΔCt method. Mean and SD of three biological replicates is shown. (two-way ANOVA test; n.s., non-significant; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (E) Western blot analysis of Cas9, GRHL2, and CD73 in MCF-7 control sgRNA (Ctrl) and 2 GRHL2 sgRNA models (KO-1 and KO-2) at the indicated time points after doxycycline induced GRHL2 deletion. Tubulin serves as a loading control. One out of three biological replicates shown. MDA-MB-231 basal B cells serve as positive control for CD73 expression.

Article Snippet: To block non-specific binding sites, a Protein Block, Serum-free solution (Dako Agilent, X090930-02) was used for 30 min. Then, slides were incubated for 1 h with, 1:1250 mouse- anti -human GRHL2 antibody (Clone CL3760; Cat.nr AMAb91226; Atlas/Sigma), 1:400 mouse- anti -human 5′-Nucleotidase/CD73 (Clone 4G6E3; Cat.nr.

Techniques: Expressing, Sequencing, Immunofluorescence, Quantitative RT-PCR, Western Blot, Positive Control

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet: NT5E/CD73 levels are inversely correlated with GRHL2 in breast cancer subtypes and breast cancer patient tumor samples (A) Violin plots showing gene expression levels of GRHL2 and NT5E based on RNA-seq data for 52 human breast cancer cell lines grouped according to the indicated subtypes. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. p values calculated using one-way ANOVA as described in a previous study. (B) RT-qPCR analysis showing CD73 (left panel) and GRHL2 mRNA expression for the indicated cell lines. Data analyzed using 2–ΔCt method for the gene of interest corrected for GAPDH control. Mean and SD of two experiments performed in triplicate is shown. (two-way ANOVA test). (C) Western blot analysis of CD73 and GAPDH loading control in the indicated cell lines. One experiment of 2 is shown. (D) Flow cytometry analysis of CD73 surface expression in the indicated cell lines. Mean and SD of two experiments performed in duplicate is shown. (E) Correlation between gene expression levels of NT5E and GRHL2 in breast cancer tumors using METABRIC dataset. Correlation scores and p values shown as determined in cBioPortal. (F) CD73 score derived from IHC, on whole slides of a series of metaplastic (TNBC) and high and low GRHL2 mRNA tumors, separated for GRHL2 IHC positive and GRHL2 IHC negative cases. Mean and SEM is shown. p value calculated using non-parametric t test with unequal variance. (G) Representative IHC images for GRHL2 and CD73 in breast cancer tissues. Tumor 1, 2, and 4 are metaplastic tumors. Tumor 1 containing ∗area with non-invasive tumor cells staining positive for GRHL2 and negative for CD73. ∗∗area with stroma containing invasive tumor cells staining negative for GRHL2 and positive for CD73. Tumor 2 Arrow indicates milk duct with GRHL2 positive/CD73 negative epithelial cells surrounded by GRHL2 negative/CD73 positive stroma. ∗∗area with invasive tumor cells staining positive for GRHL2 and negative for CD73. Tumor 3 mRNA high tumor containing ∗∗area with stroma containing invasive tumor cells staining positive for GRHL2 and negative for CD73. Tumor 4 ∗∗area containing invasive tumor cells staining negative for GRHL2 and positive for CD73. Bars, 50 μm; note that magnification is higher for tumor 1, 3, and 4 versus tumor 2.

Article Snippet: To block non-specific binding sites, a Protein Block, Serum-free solution (Dako Agilent, X090930-02) was used for 30 min. Then, slides were incubated for 1 h with, 1:1250 mouse- anti -human GRHL2 antibody (Clone CL3760; Cat.nr AMAb91226; Atlas/Sigma), 1:400 mouse- anti -human 5′-Nucleotidase/CD73 (Clone 4G6E3; Cat.nr.

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Flow Cytometry, Derivative Assay, Staining

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet: Conserved GRHL2 binding site in the NT5E gene across luminal and basal A breast cancer cell lines (A) GRHL2 ChIP tracks showing its interactions along the NT5E DNA in the indicated luminal (labeled red) and basal A cell lines (labeled green). Note that track heights use different scales. Annotation of the reference sequence is displayed below with exons in blue bars. (B) GRHL2 binding motif identified in GRHL2 ChIP-seq peaks on NT5E DNA using MEME ChIP on data retrieved from the JASPAR database. Y axis shows frequency matrix of each base occurrence. (C) Table showing locations and frequency of the GRHL2 motif in the NT5E gene for the indicated breast cancer cell lines as determined using FIMO. Motif occurrence measured with log-odds scores and converted into p values; q values calculated using Benjamini and Hochberg method.

Article Snippet: To block non-specific binding sites, a Protein Block, Serum-free solution (Dako Agilent, X090930-02) was used for 30 min. Then, slides were incubated for 1 h with, 1:1250 mouse- anti -human GRHL2 antibody (Clone CL3760; Cat.nr AMAb91226; Atlas/Sigma), 1:400 mouse- anti -human 5′-Nucleotidase/CD73 (Clone 4G6E3; Cat.nr.

Techniques: Binding Assay, Labeling, Sequencing, ChIP-sequencing

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet: GRHL2 regulates CD73-mediated extracellular adenosine production (A) Immunofluorescence staining showing GRHL2 loss at 7 days after doxycycline-mediated Cas9 induction in sgGRHL2 cells (KO-1 and KO-2) but not sgCtrl cells for the experiment shown in C. Green, GRHL2 Ab; Blue, Hoechst. (B) Cartoon displaying the enzymatic conversion of AMP into adenosine by CD73 resulting in the production inorganic phosphate. Image created using BioRender. (C) Inorganic phosphate concentration measured by malachite green assay in culture supernatants from the MCF-7 conditional KO model taken in parallel to images shown in A and basal B positive control cell lines MDA-MB-231 and Hs578T. Cells were incubated in absence or presence of 100 μM AMP (substrate) with or without 25 μM CD73 inhibitor, APCP for 125 min. Mean ± SD of four biological replicates is shown (two-way ANOVA test; n.s., non-significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: To block non-specific binding sites, a Protein Block, Serum-free solution (Dako Agilent, X090930-02) was used for 30 min. Then, slides were incubated for 1 h with, 1:1250 mouse- anti -human GRHL2 antibody (Clone CL3760; Cat.nr AMAb91226; Atlas/Sigma), 1:400 mouse- anti -human 5′-Nucleotidase/CD73 (Clone 4G6E3; Cat.nr.

Techniques: Immunofluorescence, Staining, Concentration Assay, Malachite Green Assay, Positive Control, Incubation

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet: Enhanced CD8 + T cell recruitment in response to GRHL2 loss (A) TIMER 2.0 scatterplots showing the correlation of NT5E mRNA expression with tumor purity (percentage of malignant cells in a tumor tissue; left) and the predicted presence of CD8 + T cells (right) in all breast cancer (BRCA) lesions tested. (B) Table showing TIMER 2.0 Spearman correlation scores for association between NT5E mRNA and predicted presence of CD8 + T cells across breast cancer patient tumors separated for different molecular subtypes. (C) CD8 score (% CD8 positive T cells) derived from IHC, on whole slides of a series of metaplastic (TNBC) and high and low GRHL2 mRNA tumors, separated for IHC determined CD73 membrane staining positive and negative cases. Mean and SEM is shown. p value calculated using non-parametric t test with unequal variance. (D) Representative IHC images for GRHL2, CD73, and CD8 in breast cancer tissues. Arrow indicates area of infiltrated CD8 T cells in tumor 2 that is enlarged in the lower right corner. Bar, 50 μm. (E) Quantification of the number of CD8 + T cells recruited toward the lower compartment of transwells at increasing concentrations of CXCL12 in presence or absence of 100 μM stable adenosine analog, NECA. Average and SEM of 3 biological replicates is shown (two-way ANOVA test; ∗∗∗ p < 0.001. (F) Immunofluorescence staining showing GRHL2 loss at 8 days after doxycycline-mediated Cas9 induction in sgGRHL2 cells but not sgCtrl cells for the experiment shown in G. Green, GRHL2 Ab; Blue, Hoechst. (G) Quantification of CD8 + T cells recruited toward the lower compartment of transwells seeded with control or GRHL2 KO-1 MCF-7 cells (8 days 1 μg/ml doxycycline treatment) in absence or presence of 100 ng/mL CXCL12 and 200 μM APCP CD73 inhibitor. Data normalized to the T cells only condition. Mean and SEM of 3 biological replicates is shown (two-way ANOVA test; ∗∗∗ p < 0.001; n.s., non-significant).

Article Snippet: To block non-specific binding sites, a Protein Block, Serum-free solution (Dako Agilent, X090930-02) was used for 30 min. Then, slides were incubated for 1 h with, 1:1250 mouse- anti -human GRHL2 antibody (Clone CL3760; Cat.nr AMAb91226; Atlas/Sigma), 1:400 mouse- anti -human 5′-Nucleotidase/CD73 (Clone 4G6E3; Cat.nr.

Techniques: Expressing, Derivative Assay, Membrane, Staining, Immunofluorescence

Journal: iScience

Article Title: GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

doi: 10.1016/j.isci.2024.109738

Figure Lengend Snippet:

Article Snippet: To block non-specific binding sites, a Protein Block, Serum-free solution (Dako Agilent, X090930-02) was used for 30 min. Then, slides were incubated for 1 h with, 1:1250 mouse- anti -human GRHL2 antibody (Clone CL3760; Cat.nr AMAb91226; Atlas/Sigma), 1:400 mouse- anti -human 5′-Nucleotidase/CD73 (Clone 4G6E3; Cat.nr.

Techniques: Recombinant, Blocking Assay, Western Blot, Cell Isolation, RNA Sequencing Assay, CRISPR, Plasmid Preparation, Software

Journal: Cell Communication and Signaling : CCS

Article Title: GRHL2-controlled gene expression networks in luminal breast cancer

doi: 10.1186/s12964-022-01029-5

Figure Lengend Snippet: GRHL2 ChIP-seq in luminal breast cancer cells. a Percentage of GRHL2 binding sites found at promoter regions, 5′ untranslated regions (UTRs), 3′ UTRs, exons, introns, intergenic regions, transcription termination sites (TTSs) and unknown regions in the indicated luminal breast cancer cell lines. Promoter regions are defined as − 1000 to + 100 bp from the transcription start sites (TSS). b Read count frequency and density profile of GRHL2 binding sites within − 6000 to + 6000 bp of the TSS. Left panels show GRHL2 ChIP-seq read count frequencies in indicated cell lines (Y axis, read count frequency; X axis, genomic region). Right panels show density of ChIP-seq reads for GRHL2 binding sites in the indicated cell lines. c Venn diagrams showing overlap of GRHL2 binding sites among the three indicated cell lines. Top panel shows overlap for all peaks. Bottom panel shows overlap for peaks within the − 1000 to + 100 promoter region

Article Snippet: GRHL2-bound chromatin fragments were immunoprecipitated with anti-GRHL2 antibody (Sigma; HPA004820).

Techniques: ChIP-sequencing, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: GRHL2-controlled gene expression networks in luminal breast cancer

doi: 10.1186/s12964-022-01029-5

Figure Lengend Snippet: Association of GRHL2 motif with ER transcriptional complex in luminal breast cancer cells. a DNA-binding motif of GRHL2 in luminal breast cancer. From left to right, the first panel shows the identified motifs in the indicated cell lines. The second panel shows distribution of the best matches to the motif in the sequences. The third panel shows the E -value, representing the significance of the motif according to the motif discovery. The last panel shows the number of regions that match the corresponding motif. b Heatmaps showing the coverage of identified GRHL2 peaks shared between MCF7, BT474 and T47D at GRHL2 motifs (red) (n = 20,766), ER⍺ motifs (blue) (n = 76,564), FOXA1 motifs (Green) (n = 88,923) and GATA3 motifs (Orange) (n = 93,403). Note that the read coverage scale differs for the different heatmaps. c Table indicating the occurrence of ER⍺ consensus motif in a region spanning 1000 bp up- and downstream of all GRHL2 peaks either identified in the indicated cell lines (left 3 columns) or shared between the indicated cell lines (right column). d Table indicating the occurrence of published ER⍺ binding events in a region spanning 1000 bp up- and downstream of all GRHL2 peaks shared between MCF7, BT474 and T47D (upper row) or shared between 2 MCF7 datasets (bottom row)

Article Snippet: GRHL2-bound chromatin fragments were immunoprecipitated with anti-GRHL2 antibody (Sigma; HPA004820).

Techniques: Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: GRHL2-controlled gene expression networks in luminal breast cancer

doi: 10.1186/s12964-022-01029-5

Figure Lengend Snippet: Bru-seq analysis of transcriptional changes in response to GRHL2 loss in luminal breast cancer MCF7 cells. a Bru-seq sample preparation . Bromouridine (Bru) labeling of nascent RNA was carried out for 30 min at the indicated time points after doxycycline (dox)-induced GRHL2 deletion. b Western blot analysis of GRHL2 expression levels at the indicated time points in sgCTR and sgGRHL2 transduced MCF7 cells. Cas9 induction is monitored and GAPDH serves as loading control. c Bru-seq data analysis approach. Each circle represents a gene set with differential transcription relative to the condition where no doxycycline was added. d Heatmap for genes whose transcription was altered in response to GRHL2 depletion. e Graphs depicting clusters of genes with distinct patterns of transcriptional changes in response to GRHL2 depletion. Graphs represent log 2 AFC of transcription in sgGRHL2(1) and sgGRHL2(2) cells. “Dynamic”: genes with AFC > 2; p < 0.05 at some and AFC < 0.5; p < 0.05 at other time points. “Sustained induction”: genes with AFC > 2; p < 0.05 at all time points. “Sustained repression”: genes with AFC < 0.5; p < 0.05 at all time points. “Induction reset”: genes with AFC > 2; p < 0.05 at early time points followed by a return to 1 < AFC < 2 at day 16. “Repression reset”: genes with AFC < 0.5; p < 0.05 at early time points followed by a return to 0.5 < AFC < 1 at day 16

Article Snippet: GRHL2-bound chromatin fragments were immunoprecipitated with anti-GRHL2 antibody (Sigma; HPA004820).

Techniques: Sample Prep, Labeling, Western Blot, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: GRHL2-controlled gene expression networks in luminal breast cancer

doi: 10.1186/s12964-022-01029-5

Figure Lengend Snippet: EHF represents a direct GRHL2 regulated gene. a Bru-seq reads for EHF at indicated time points after to GRHL2 deletion. Track colors: green, sgCTR; red, sgGRHL2(1); blue, sgGRHL2(2). Positive y-axis indicates the plus-strand signal of RNA synthesis from left to right and the negative y-axis represents the minus-strand signal of RNA synthesis from right to left. b Line graph depicting the log 2 AFC of EHF transcription in sgGRHL2(1) and sgGRHL2(2) cells. c ChIP-qPCR showing enrichment of GRHL2 binding sites in EHF promoter region but not in the control GAPDH gene. Graph represents the efficiency of indicated genomic DNA co-precipitation with anti-GRHL2 Ab (black bars) or IgG control Ab (grey bars). Signals for IgG control and GRHL2 antibody pulldown samples are normalized to input DNA and are presented as % input with SEM from 3 technical replicates. Data are statistically analyzed by t-test and *indicates p < 0.05. d EHF mRNA expression in a panel of 52 human breast cancer cell lines covering luminal-, basal A-, and basal B subtypes extracted from RNA-seq data. Data is statistically analyzed by t-test and *indicates p < 0.05. e qRT-PCR analysis of expression level of EHF mRNA after 4 days of doxycycline treatment of MCF7 cells transduced with dox-inducible Cas9 and sgCTR or sgGRHL2 constructs, in combination with ectopic expression of EHF or empty vector (EV) plasmids. Data are presented as mean ± SEM from three technical replicates. Data are statistically analyzed by t-test. *Indicates p < 0.05. f Graph showing results from SRB assay after 4 days doxycycline-treatment as in ( e ) and subsequent culture for the indicated time periods

Article Snippet: GRHL2-bound chromatin fragments were immunoprecipitated with anti-GRHL2 antibody (Sigma; HPA004820).

Techniques: Binding Assay, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Transduction, Construct, Plasmid Preparation, Sulforhodamine B Assay

Journal: Cell Communication and Signaling : CCS

Article Title: GRHL2-controlled gene expression networks in luminal breast cancer

doi: 10.1186/s12964-022-01029-5

Figure Lengend Snippet: Downregulation of RNA synthesis for genes involved in cell cycle progression after GRHL2 loss. a – d Top: Bru-seq reads for indicated genes at indicated time point after to GRHL2 deletion. Track colors: green, sgCTR; red, sgGRHL2(1); blue, sgGRHL2(2). Bottom: Line graphs depicting the log 2 AFC of transcription in sgGRHL2(1) and sgGRHL2(2) cells for the indicated genes. The positive y-axis indicates the plus-strand signal of RNA synthesis from left to right and the negative y-axis represents the minus-strand signal of RNA synthesis from right to left. e Validation of interaction of GRHL2 binding sites with the promoter regions of indicated genes by ChIP-qPCR . Signals for IgG control and GRHL2 antibody pulldown samples are normalized to input DNA and are presented as % input with SEM from 3 technical replicates. Data are statistically analyzed by t-test and *indicates p < 0.05

Article Snippet: GRHL2-bound chromatin fragments were immunoprecipitated with anti-GRHL2 antibody (Sigma; HPA004820).

Techniques: Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: GRHL2-controlled gene expression networks in luminal breast cancer

doi: 10.1186/s12964-022-01029-5

Figure Lengend Snippet: Gene clusters responding to GRHL2 depletion and their correlation with GRHL2 in breast cancer tissues. a STRING derived protein interaction analysis of genes displaying sustained up- or down regulation in response to GRHL2 depletion in MCF7. GO terms are color marked as indicated. i , entire network with boxes showing zoom-in on indicated regions; ii , zoom in on indicated region showing different GO terms. # Indicates GRHL2 targets identified by promoter binding. *Indicates TGFß signaling axis. b Average expression (log2 scale) in the MA-867 patient dataset of the cluster of genes negatively (left panel) or positively associated with GRHL2 (right panel) in MCF7 KO model. Patients were divided in 4 quartiles according to the level of GRHL2 expression. Q1, lowest GRHL2 expression; Q4, GRHL2 highest expression; All, all patients grouped together; ER + , ER positive patients grouped; ER-, ER negative patients grouped. Boxplots display the median with 25–75th percentile and dots represent lower 5% and upper 95% samples. p values determined by t test (two-sided). c Correlation with GRHL2 in MA-867 and METABRIC datasets for indicated genes negatively or positively correlated with GRHL2 in MCF KO model analyzed by Bru-seq. For MA-867 dataset, R-values for all patients grouped together, ER positive patients, or ER negative patients are shown. For METABRIC dataset, correlation, p -value, and q -values are shown as determined in BioPortal

Article Snippet: GRHL2-bound chromatin fragments were immunoprecipitated with anti-GRHL2 antibody (Sigma; HPA004820).

Techniques: Derivative Assay, Binding Assay, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: GRHL2-controlled gene expression networks in luminal breast cancer

doi: 10.1186/s12964-022-01029-5

Figure Lengend Snippet: Regulation of EMT related genes by GRHL2. a ChIP tracks for the indicated genes in three luminal breast cancer cell lines. The track height is scaled from 0 to the indicated number. The locus with its exon/intron structure is presented above the tracks. *Indicates binding sites validated by ChIP-qPCR in (b). b ChIP-qPCR validation of presence and absence of GRHL2 binding sites identified by ChIP-seq. Graphs represent the efficiency of indicated genomic DNA co-precipitation with anti-GRHL2 Ab (grey bars) or IgG control Ab (black bars). Note enrichment of GRHL2 binding at OVOL2 exon and CDH1 intron, but not at ZEB1 promoter regions. For ZEB1 detection, ChIP-qPCR was performed using primers that have been previously reported to amplify ZEB1 promoter DNA sequences bound by GRHL2 in human mammary epithelial cells and in PEO1 but not OVCA429 human ovarian cancer cells (indicated by ##) [ , ] and another primer set that did not confirm GRHL2 promoter interaction in ovarian cancer cells (indicated by #) . Signals for IgG control and GRHL2 antibody pulldown samples were normalized to input DNA and presented as % input with SEM from 3 technical replicates. Data were statistically analyzed by t-test and * indicates p < 0.05. c , e Bru-seq reads for indicated genes at indicated time point after to GRHL2 deletion. Track colors: green, sgCTR; red, sgGRHL2(1); blue, sgGRHL2(2). d , f Line graphs depicting the log 2 AFC of transcription in sgGRHL2(1) and sgGRHL2(2) cells for the indicated genes. The positive y-axis indicates the plus-strand signal of RNA synthesis from left to right and the negative y-axis represents the minus-strand signal of RNA synthesis from right to left

Article Snippet: GRHL2-bound chromatin fragments were immunoprecipitated with anti-GRHL2 antibody (Sigma; HPA004820).

Techniques: Binding Assay, ChIP-sequencing

Journal: Gastroenterology Research and Practice

Article Title: Peritoneal Metastatic Cancer Stem Cells of Gastric Cancer with Partial Mesenchymal-Epithelial Transition and Enhanced Invasiveness in an Intraperitoneal Transplantation Model

doi: 10.1155/2020/3256538

Figure Lengend Snippet: Immunocytofluorescence of E-cad, α -SMA, Survivin, Vimentin, MMP2, MMP9, GRHL2, ZEB1, and OVOL2 (×400) for the CSC-hGC, pMCSC-tGC [G1] , and pMCSC-tGC [G2] cells.

Article Snippet: The reagents used for ICF included primary mouse anti-human E-cad monoclonal antibodies (BD Biosciences, U.S.), primary rabbit anti-human MMP2 polyclonal antibodies (Abcam, Hong Kong, China), primary rabbit anti-human MMP9 monoclonal antibodies (Abcam, Hong Kong, China), a primary rabbit anti-human α -SMA monoclonal antibody (Abcam, Hong Kong, China), a primary rabbit anti-human Survivin monoclonal antibody (Abcam, Hong Kong, China), a primary rabbit anti-human Vimentin monoclonal antibody (Cell Signaling Technology, U.S.), a primary mouse anti-human OVOL2 monoclonal antibody (Santa Cruz Biotechnology, U.S.), primary rabbit anti-human ZEB1 monoclonal antibodies (Abcam, Hong Kong, China), primary rabbit anti-human anti-GRHL2 polyclonal antibodies (Sigma-Aldrich, U.S.), and secondary goat anti-mouse and rabbit antibodies (Life Technologies, U.S.).

Techniques:

Journal: Gastroenterology Research and Practice

Article Title: Peritoneal Metastatic Cancer Stem Cells of Gastric Cancer with Partial Mesenchymal-Epithelial Transition and Enhanced Invasiveness in an Intraperitoneal Transplantation Model

doi: 10.1155/2020/3256538

Figure Lengend Snippet: Quantitative real-time PCR of E-cad, α -SMA, MMP9, Vimentin, GRHL2, OVOL2, ZEB1, and MMP2 for the CSC-hGC, pMCSC-tGC [G1] , and pMCSC-tGC [G2] cells.

Article Snippet: The reagents used for ICF included primary mouse anti-human E-cad monoclonal antibodies (BD Biosciences, U.S.), primary rabbit anti-human MMP2 polyclonal antibodies (Abcam, Hong Kong, China), primary rabbit anti-human MMP9 monoclonal antibodies (Abcam, Hong Kong, China), a primary rabbit anti-human α -SMA monoclonal antibody (Abcam, Hong Kong, China), a primary rabbit anti-human Survivin monoclonal antibody (Abcam, Hong Kong, China), a primary rabbit anti-human Vimentin monoclonal antibody (Cell Signaling Technology, U.S.), a primary mouse anti-human OVOL2 monoclonal antibody (Santa Cruz Biotechnology, U.S.), primary rabbit anti-human ZEB1 monoclonal antibodies (Abcam, Hong Kong, China), primary rabbit anti-human anti-GRHL2 polyclonal antibodies (Sigma-Aldrich, U.S.), and secondary goat anti-mouse and rabbit antibodies (Life Technologies, U.S.).

Techniques: Real-time Polymerase Chain Reaction