grhl2 antibody  (Bio-Techne corporation)

Journal: Pathobiology

Article Title: Grainyhead-Like 2 Expression Is Associated with Disease Progression, Chemoresistance and Poor Survival in Tubo-Ovarian High-Grade Serous Carcinoma

doi: 10.1159/000548826

Figure Lengend Snippet: IHC. a–d GRHL2 protein expression in high-grade serous carcinoma (HGSC) effusions. a , b Strong intensity. c , d Weak intensity. e–h GRHL2 protein expression in solid HGSC lesions. e , f Strong intensity. g , h Weak intensity.

Article Snippet: The GRHL2 antibody was a rabbit polyclonal antibody purchased from Atlas Antibodies AB (cat #HPA004820; Stockholm, Sweden), applied at a 1:800 dilution following antigen retrieval in LpH buffer (pH 6.0).

Techniques: Expressing

Journal: Pathobiology

Article Title: Grainyhead-Like 2 Expression Is Associated with Disease Progression, Chemoresistance and Poor Survival in Tubo-Ovarian High-Grade Serous Carcinoma

doi: 10.1159/000548826

Figure Lengend Snippet: Survival. a KM survival curve showing the association between GRHL2 protein expression in 198 HGSC effusions and overall survival (OS). Patients with effusions with high (score >4) expression score ( n = 95; red line) had mean OS of 30 months compared to 38 months for patients with effusions showing low (score ≤4) expression ( n = 103, blue line; p = 0.088). b KM survival curve showing the association between GRHL2 protein expression in 105 HGSC surgical specimens and OS. Patients with effusions with high (score >4) expression score ( n = 19; red line) had mean OS of 34 months compared to 54 months for patients with effusions showing low (score ≤4) expression ( n = 86, blue line; p = 0.038). c KM survival curve showing the association between GRHL2 protein expression in 105 HGSC surgical specimens and progression-free survival (PFS). Patients with effusions with high (score >4) expression score ( n = 19; red line) had mean PFS of 9 months compared to 21 months for patients with effusions showing low (score ≤4) expression ( n = 86, blue line; p = 0.024). d KM survival curve showing the association between FIGO stage and OS for 102 HGSC patients with surgical specimens diagnosed with advanced-stage disease. Patients diagnosed with stage IV disease ( n = 32; red line) had mean OS of 36 months compared to 55 months for patients with stage III disease ( n = 70, blue line; p = 0.022). e KM survival curve showing the association between residual disease (RD) volume and OS for 80 patients with surgical specimens who had debulking data. Patients debulked to no macroscopic disease ( n = 17; blue line) had mean OS of 86 months compared to 48 and 38 months for patients debulked to 1 cm ( n = 31, red line) and ≥2 cm ( n = 32, green line), respectively ( p = 0.001). f KM survival curve showing the association between FIGO stage and PFS for 102 HGSC patients with surgical specimens diagnosed with advanced-stage disease. Patients diagnosed with stage IV disease ( n = 32; red line) had mean PFS of 13 months compared to 19 months for patients with stage III disease ( n = 70, blue line; p = 0.016). g KM survival curve showing the association between residual disease (RD) volume and PFS for 80 patients with surgical specimens who had debulking data. Patients debulked to no macroscopic disease ( n = 17; blue line) had mean PFS of 38 months compared to 13 and 11 months for patients debulked to 1 cm ( n = 31, red line) and ≥2 cm ( n = 32, green line), respectively ( p < 0.001).

Article Snippet: The GRHL2 antibody was a rabbit polyclonal antibody purchased from Atlas Antibodies AB (cat #HPA004820; Stockholm, Sweden), applied at a 1:800 dilution following antigen retrieval in LpH buffer (pH 6.0).

Techniques: Expressing

Journal: Nature Genetics

Article Title: KDM4C inhibition blocks tumor growth in basal breast cancer by promoting cathepsin L-mediated histone H3 cleavage

doi: 10.1038/s41588-025-02197-z

Figure Lengend Snippet: a , Heatmap showing histone peptide abundance by MS in the indicated cell lines expressing Dox-inducible sh KDM4C following control (no Dox, dimethyl sulfoxide (DMSO)), sh KDM4C induction (1 μg ml −1 Dox, DMSO) or 10 μm ML324 (no Dox) treatment. Peptide abundances were normalized to the mean values of vehicle group within each cell line and ranked from N to C terminus. b , Schematic illustration of proteolytic cleavage sites in histones H3 and H4 after KDM4C blockade. c , Bar plot showing the clipped H3 peptide (TKAAR) total ion chromatogram signal intensity in the indicated groups. Mean ± s.d. are shown. Two-sided Dunnett’s test was used within each cell line for groups with biological triplicates, except T47D sh KDM4C group. d , Immunoblot for histone H3 using C (C′-H3) and N (N′-H3) terminal antibodies in 5 cell lines with inducible sh KDM4C infection, following control (DMSO, no Dox), 1 μg ml −1 Dox (sh KDM4C ), 10 μm ML324 (no Dox) or 1 μm QC6352 (no Dox) for 5 days. Tubulin was used as a loading control. Clipped H3 bands are marked with red arrow. Experiments were repeated independently three times (HCC1954, SUM149 and HCC38) or twice (HDQP1 and HCC1806) with similar results. e , Heatmap showing histone peptide abundance by MS in SUM149 cell line following DMSO (vehicle) or 10 μm ML324 treatment in the presence or absence of the indicated protease inhibitors (100 μm AEBSF HCl, 100 μm pepstatin A, 10 μm SID2668150, 10 μm E64d and 5 μm CTSLi-III) for 24 h. Peptide abundances were normalized to the mean values of vehicle group within each cell line and ranked from N to C terminus. f , Box plots depicting differences in N-terminal histone H3 (amino acid positions 0–26) peptide abundances between vehicle and KDM4C-inhibited samples following the indicated protease treatment in the SUM149 cell line. Box plots span the upper quartile (upper limit), median (center) and lower quartile (lower limit). Whiskers extend a maximum of 1.5× IQR. Statistical significance of differences was determined by two-sided Kruskal–Wallis test. g , Bar plot showing the ML324-induced FC of clipped H3 peptide (TKAAR) total ion chromatogram signal intensity in the indicated groups. Mean ± s.d. are shown for each group with n = 3 (CTSLi-III group) and n = 2 (all the rest) biological replicates. h , Representative flow cytometry plots depicting the shift of CTSL magic red signal after 1 μm QC6352 treatment for 5 days. i , Bar plot summarizing the QC6352-induced FCs in CTSL activity merging from n = 5 (SUM149), n = 4 (HCC1954) and n = 3 (other cell lines) independent experiments in each cell line (mean ± s.d.). Two-sided Mann–Whitney U test was used to compare average FCs between four KDM4C -amplified and four non-amplified cell lines. SID, SID2668150; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride.

Article Snippet: Inducible SUM149 sh KDM4C -infected cells were treated with control (no Dox, DMSO), sh KDM4C induction (plus 1 μg ml −1 Dox), 10 μm ML324 (no Dox), 1 μm QC6352 (no Dox) for 5 days or 1 μm Leu–Leu methyl ester hydrobromide for 24 h. Cells were then washed, fixed in 4% paraformaldehyde and blocked in 5% BSA/0.3% Triton X-100/PBS solution for 1 h. Primary antibodies against CTSL (Novus Biologicals, AF952; 1:100), GRHL2 (Sigma-Aldrich, HPA004820; 1:100) and KDM4C (Novus Biologicals, NBP1-49600; 1:100) were applied for 1 h at room temperature.

Techniques: Expressing, Control, Western Blot, Infection, Flow Cytometry, Activity Assay, MANN-WHITNEY, Amplification

Journal: Nature Genetics

Article Title: KDM4C inhibition blocks tumor growth in basal breast cancer by promoting cathepsin L-mediated histone H3 cleavage

doi: 10.1038/s41588-025-02197-z

Figure Lengend Snippet: a – c , Immunoblot for C′-H3 in three cell lines following DMSO, 10 μm ML324 or 1 μm QC6352 treatment for 5 days ( a ), SUM149 doxycycline-inducible models with or without 1 μg/ml doxycycline for 5 days ( b ) and SUM149 cells overexpressing KDM4C WT and KDM4C S198M with siRNA against KDM4C 5′UTR for 5 days ( c ; two independent repeats for all with similar results). d , Heatmap showing normalized histone peptide abundance in parental and MLR models with or without ML324 treatment. e , i , Box plots showing average N-terminal peptides abundances of histone H3 (n = 27 peptides) or H4 (n = 16 peptides) in the indicated groups (two-sided Kruskal–Wallis test). Box plots span the upper quartile (upper limit), median (center) and lower quartile (lower limit). Whiskers extend a maximum of 1.5× IQR. f , Immunoblot for total and phospho-histone H3 (Ser10) in SUM149 parental and MLR cells (two independent repeats with similar results). g , Cell viability curves normalized to vehicle group in response to CDK8 or AURKA/AURKB inhibitors in SUM149 parental and MLR cells (mean ± s.d. of n = 6 from one experiment). h , Immunoblot for C′-H3 in SUM149-inducible-sh KDM4C cells with either 1 μg/ml doxycycline or 10 μm ML324 with different protease inhibitors (5 μm CTSL-inhibitor III, 10 μm SID2668150 or 100 μm AEBSF), with tubulin as loading control (three independent repeats for CTSLi-III experiment with similar results and one experiment for the rest). j , Representative plots depicting the CTSL activity signal in the indicated sh KDM4C cell models with 1 μg/ml doxycycline treatment for 5 days. k , Immunoblot analysis for CTSL in the indicated cell lines from one experiment. l , m Representative plot ( l ) and quantification of mean ± s.d. from three independent experiments ( m ) of indicated SUM149 models with or without 1 μg/ml doxycycline for 5 days (two-sided ordinary one-way ANOVA). n , o , Representative images of CTSL activity signal in SUM149 cells overexpressing KDM4C WT and KDM4C S198M with siRNA interference against KDM4C 5′UTR for 5 days ( n ) and quantification of signals of 120 cells from three to four representative regions in mean ± s.d. (two-sided ordinary one-way ANOVA; o ). Scale bar, 100 μm. Tubulin was used as loading control for all the immunoblots.

Article Snippet: Inducible SUM149 sh KDM4C -infected cells were treated with control (no Dox, DMSO), sh KDM4C induction (plus 1 μg ml −1 Dox), 10 μm ML324 (no Dox), 1 μm QC6352 (no Dox) for 5 days or 1 μm Leu–Leu methyl ester hydrobromide for 24 h. Cells were then washed, fixed in 4% paraformaldehyde and blocked in 5% BSA/0.3% Triton X-100/PBS solution for 1 h. Primary antibodies against CTSL (Novus Biologicals, AF952; 1:100), GRHL2 (Sigma-Aldrich, HPA004820; 1:100) and KDM4C (Novus Biologicals, NBP1-49600; 1:100) were applied for 1 h at room temperature.

Techniques: Western Blot, Control, Activity Assay

Journal: Nature Genetics

Article Title: KDM4C inhibition blocks tumor growth in basal breast cancer by promoting cathepsin L-mediated histone H3 cleavage

doi: 10.1038/s41588-025-02197-z

Figure Lengend Snippet: a , Immunoblot depicting CTSL in sgScramble and CTSL KO models in the indicated cell lines with vinculin as a loading control (two independent repeats with similar results). b , Immunofluorescence staining of CTSL and nuclei in the corresponding sgScramble and CTSL KO derivatives from one experiment. Scale bar, 10 μm. c , Immunoblot depicting CTSL in different fractions in SUM149 sgScramble and CTSL KO derivatives with tubulin, AIF and histone H3 as controls for subcellular fractionation (two independent repeats with similar results). d , Immunofluorescence for CTSL and nuclei in SUM149-inducible-sh KDM4C cells treated with DMSO, 1 μg/ml doxycycline (sh KDM4C ), 10 μm ML324 or 1 μm QC6352 for 3 days from one experiment. Treatment of 1 μm LLoMe for 24 h was a positive control. Scale bar, 10 μm. e , Immunoblot for CTSL in the indicated fractions of SUM149-inducible-sh KDM4C cells under the same treatment as d for 5 days (three independent repeats with similar results). f , Immunoblot showing C′-H3 and CTSL in HCC1806 and HDQP1 sgScramble and CTSL KO derivatives with 10 μm ML324 or 1 μm QC6352 treatment for 5 days with tubulin as loading control (two independent repeats with similar results). g , Binding and Expression Target Analysis showing association between ML324-induced gained CTSL sites and differentially expressed genes in SUM149 cells (one-sided Kolmogorov–Smirnov test). h , Intensity plots depicting CTSL ChIP–seq signal in vehicle and ML324-treated SUM149 parental and MLR cells at lost CTSL sites at ±2 kb range of the peak centers. The 95% confidence interval is presented. i , Immunoblot showing H3K4me3, total H3 levels in SUM149 cells ectopically expressing GFP- (left) and V5-tagged (right) histone H3 from one experiment. Ectopic proteins were differentiated by molecular weight and indicated by an asterisk. j , Heatmap showing intrachromosomal and interchromosomal CTSL interactions in SUM149 cells with vehicle or ML324 treatment. k , Box plot showing ML324-induced differential intrachromosomal CTSL interactions with frequency >10 and adjusted p value < 0.05. Box plots span the upper quartile (upper limit), median (center) and lower quartile (lower limit). Whiskers extend a maximum of 1.5× IQR. l , Venn diagrams showing intersections among ML324-induced gained or lost CTSL binding sites in ChIP–seq and intrachromosomal or interchromosomal interaction sites in Hi-ChIP.

Article Snippet: Inducible SUM149 sh KDM4C -infected cells were treated with control (no Dox, DMSO), sh KDM4C induction (plus 1 μg ml −1 Dox), 10 μm ML324 (no Dox), 1 μm QC6352 (no Dox) for 5 days or 1 μm Leu–Leu methyl ester hydrobromide for 24 h. Cells were then washed, fixed in 4% paraformaldehyde and blocked in 5% BSA/0.3% Triton X-100/PBS solution for 1 h. Primary antibodies against CTSL (Novus Biologicals, AF952; 1:100), GRHL2 (Sigma-Aldrich, HPA004820; 1:100) and KDM4C (Novus Biologicals, NBP1-49600; 1:100) were applied for 1 h at room temperature.

Techniques: Western Blot, Control, Immunofluorescence, Staining, Fractionation, Positive Control, Binding Assay, Expressing, ChIP-sequencing, Molecular Weight, HiChIP

Journal: Nature Genetics

Article Title: KDM4C inhibition blocks tumor growth in basal breast cancer by promoting cathepsin L-mediated histone H3 cleavage

doi: 10.1038/s41588-025-02197-z

Figure Lengend Snippet: a , Immunoblot showing H3 protein detected with C-terminal antibodies and CTSL in three KDM4C -amplified cell lines after 5 days of treatments with DMSO, 10 μm ML324 or 1 μm QC6352 in sgScramble and CTSL KO models. Tubulin was used as a loading control. All experiments were repeated independently at least twice with similar results. b , Genomic track view of KDM4C and CTSL binding signals in SUM149 cell lines with or without ML324 treatment at the NFATC4 gene locus. Chr14, chromosome 14. c , Heatmap showing differential and unchanged CTSL peaks after ML324 treatment in SUM149 cell line. Signal intensity is illustrated in a 4 kb window. Venn diagram on the right side illustrating the intersection of unchanged and lost CTSL peaks with KDM4C binding sites. Fisher’s exact test (two-sided) was used. d , Line plot showing Binding and Expression Target Analysis (BETA) to assess the association between lost CTSL sites and DEGs in SUM149 cells following ML324 treatment. Statistical comparison to the background genes was performed using one-sided Kolmogorov–Smirnov test. e – g , Intensity plots representing ATAC–seq signal at CTSL peaks lost following ML324 treatment in SUM149 cell line ( e ), histone H3 signal using the indicated antibodies for ChIP in vehicle and ML324-treated SUM149 cells expressing N-terminal GFP- ( f ) or V5-tagged ( g ) histone H3 at CTSL binding sites at the range of ±2 kb of the PC. The 95% confidence interval of each curve is presented. h , Box plots showing ML324-induced H3 signal changes in each indicated ChIP–seq sample at CTSL peaks or at the same number of random peaks ( n = 16,141 peaks). Box plots span the upper quartile (upper limit), median (center) and lower quartile (lower limit). Whiskers extend a maximum of 1.5× IQR. Two-sided Mann–Whitney U test was used. PC, peak center.

Article Snippet: Inducible SUM149 sh KDM4C -infected cells were treated with control (no Dox, DMSO), sh KDM4C induction (plus 1 μg ml −1 Dox), 10 μm ML324 (no Dox), 1 μm QC6352 (no Dox) for 5 days or 1 μm Leu–Leu methyl ester hydrobromide for 24 h. Cells were then washed, fixed in 4% paraformaldehyde and blocked in 5% BSA/0.3% Triton X-100/PBS solution for 1 h. Primary antibodies against CTSL (Novus Biologicals, AF952; 1:100), GRHL2 (Sigma-Aldrich, HPA004820; 1:100) and KDM4C (Novus Biologicals, NBP1-49600; 1:100) were applied for 1 h at room temperature.

Techniques: Western Blot, Amplification, Control, Binding Assay, Expressing, Comparison, ChIP-sequencing, MANN-WHITNEY

Journal: Nature Genetics

Article Title: KDM4C inhibition blocks tumor growth in basal breast cancer by promoting cathepsin L-mediated histone H3 cleavage

doi: 10.1038/s41588-025-02197-z

Figure Lengend Snippet: a , Scatter plot showing the correlation of log 2 (FC) (normalized to IgG control) MS signal of proteins detected in CTSL immunoprecipitants in SUM149 cell models sh KDM4C #17 and sh KDM4C #20 at baseline without Dox treatment. The linear regression line with 95% confidence interval is shown. P values were derived from two-sided Pearson correlation. b , Heatmap depicting rank order of transcription factor binding site motifs enriched in CTSL binding sites of vehicle-treated cells in SUM149 and HCC1954 cell lines. log 10 ( E values) were used to define the significance of enrichment. c , Immunoblot analysis of KDM4C, GRHL2 and CTSL immunoprecipitants and 10% of input detected with the indicated antibodies in SUM149 cells. CTCF was used as negative control. Signal intensity normalized to input is indicated for each protein. All experiments were repeated at least twice independently with similar results. d , Venn diagrams showing intersections of CTSL and GRHL2 binding sites in HCC1954 and SUM149 cells. e , Heatmap showing overall intensities of CTSL chromatin binding in scramble control and GRHL2 KO SUM149 cell line. Signal intensity is illustrated in a 4 kb window (PC). f , Heatmaps illustrating triple (CTSL + GRHL2 + KDM4C + ) and double (CTSL + GRHL2 + ) overlapping peaks in SUM149 cell lines. Signal intensity is depicted in a 4 kb window. g , Line plot showing BETA for assessing the association of triple and double overlapping peaks with differentially expressed genes in SUM149 following ML324 treatment. One-sided Kolmogorov–Smirnov test was applied to calculate the P values. h , Immunoblot for GRHL2 and CTSL in 10% input and immunoprecipitants of pan-lysine methylation and IgG antibody from cells with the indicated treatments. GRHL2 and CTSL signal normalized to input is indicated for each condition. The experiment was repeated three times independently with similar results. i , Schematic view of GRHL2 protein structure indicating the location of the lysine methylation sites. j , Immunoblot for GHRL2 and C-terminal histone H3 following 3 days of treatment with vehicle or 1 μm QC6352 of SUM149 cells expressing WT or the indicated mutant GRHL2. This experiment was repeated twice independently with similar results. k , Representative flow cytometry plots depicting the shift of CTSL activity signal in SUM149 cells with the indicated conditions. l , Bar plot summarizing the QC6352-induced CTSL magic red FCs in SUM149 cell models merging three independent experiments (mean ± s.d.). Two-sided ordinary one-way analysis of variance (ANOVA) test was used.

Article Snippet: Inducible SUM149 sh KDM4C -infected cells were treated with control (no Dox, DMSO), sh KDM4C induction (plus 1 μg ml −1 Dox), 10 μm ML324 (no Dox), 1 μm QC6352 (no Dox) for 5 days or 1 μm Leu–Leu methyl ester hydrobromide for 24 h. Cells were then washed, fixed in 4% paraformaldehyde and blocked in 5% BSA/0.3% Triton X-100/PBS solution for 1 h. Primary antibodies against CTSL (Novus Biologicals, AF952; 1:100), GRHL2 (Sigma-Aldrich, HPA004820; 1:100) and KDM4C (Novus Biologicals, NBP1-49600; 1:100) were applied for 1 h at room temperature.

Techniques: Control, Derivative Assay, Binding Assay, Western Blot, Negative Control, Methylation, Expressing, Mutagenesis, Flow Cytometry, Activity Assay

Journal: Nature Genetics

Article Title: KDM4C inhibition blocks tumor growth in basal breast cancer by promoting cathepsin L-mediated histone H3 cleavage

doi: 10.1038/s41588-025-02197-z

Figure Lengend Snippet: a , Volcano plots showing CTSL-interacting proteins in SUM149 sh KDM4C models (17 and 20 without doxycycline and 17 with doxycycline treatment) identified by mass spectrometry of CTSL immunoprecipitants. Red and blue indicate targets with FDR < 0.05 compared to IgG control. b , Immunofluorescence staining of CTSL (red), nuclei (gray) and GRHL2 or KDM4C (green) in SUM149 cell line from one experiment. Scale bar, 10 μm. c , Immunoblot depicting the expression of GRHL2 in HCC1954 and SUM149 sgScramble and GRHL2 KO derivatives with tubulin as a loading control (two independent repeats with similar results). d , e , Immunoblot for KDM4C, GRHL2 and CTSL in the indicated immunoprecipitants of KDM4C in SUM149 GRHL2 KO ( d ) and CTSL in CTSL KO ( e ) derivates for one experiment. f , Genomic track view of GRHL2 (in parental cells) and CTSL ChIP–seq signal in SUM149 sgScramble and GRHL2 KO models at the ASAP3 and NFATC4 loci. g , Immunoblot for C′-H3 and GRHL2 of SUM149 sgScramble and GRHL2 KO models following 1 µM of QC6352 treatment for 5 days with tubulin as a loading control (two independent repeats with similar results). h , Intensity plot depicting GRHL2 ChIP–seq signal in vehicle and ML324-treated SUM149 cells on lost CTSL binding sites. i , Venn diagram showing the intersection of upregulated or downregulated differentially expressed genes associated with triple (KDM4C + GRHL2 + CTSL) and double (GRHL2 + CTSL) overlap peaks. j , Dot plot depicting Hallmark signature enrichment predicted from top 300 triple (KDM4C + GRHL2 + CTSL) and double (GRHL2 + CTSL) overlap peaks-associated genes. (Fisher’s exact test using Enrichr). k , l , Immunoblot for GRHL2 in 10% input and immunoprecipitants of pan-lysine methylation and IgG antibody in KDM4C -amplified HCC1954 ( k ) and KDM4C non-amplified HCC1806 and HDQP1 cells ( l ) grown in the indicated conditions from one experiment. m , Annotated mass spectra for methylated lysine 94 and 453. Lowercase amino acids indicate these are modified ( c is alkylated cysteine, and k is monomethylated lysine). n , Immunoblot of GRHL2 in SUM149 cells following 3 days of siRNA transfection targeting the 3′UTR region of GRHL2, following 3 days of WT or mutant GRHL2 lentiviral infection (two independent repeats with similar results). Target proteins from IP experiments were quantified by normalizing to the corresponding input and labeled below each band.

Article Snippet: Inducible SUM149 sh KDM4C -infected cells were treated with control (no Dox, DMSO), sh KDM4C induction (plus 1 μg ml −1 Dox), 10 μm ML324 (no Dox), 1 μm QC6352 (no Dox) for 5 days or 1 μm Leu–Leu methyl ester hydrobromide for 24 h. Cells were then washed, fixed in 4% paraformaldehyde and blocked in 5% BSA/0.3% Triton X-100/PBS solution for 1 h. Primary antibodies against CTSL (Novus Biologicals, AF952; 1:100), GRHL2 (Sigma-Aldrich, HPA004820; 1:100) and KDM4C (Novus Biologicals, NBP1-49600; 1:100) were applied for 1 h at room temperature.

Techniques: Mass Spectrometry, Control, Immunofluorescence, Staining, Western Blot, Expressing, ChIP-sequencing, Binding Assay, Methylation, Amplification, Modification, Transfection, Mutagenesis, Infection, Labeling

Journal: Nature Genetics

Article Title: KDM4C inhibition blocks tumor growth in basal breast cancer by promoting cathepsin L-mediated histone H3 cleavage

doi: 10.1038/s41588-025-02197-z

Figure Lengend Snippet: a , Heatmap showing clustering of 248 polar metabolites in Dox-inducible sh KDM4C- infected HCC1954, SUM149 and T47D cells following control (no Dox, DMSO), sh KDM4C induction (1 μg ml −1 Dox, DMSO) or 10 μm ML324 (no Dox) treatment and in HCC70 parental cells with or without 10 μm ML324 treatment. Metabolite abundances in each condition were normalized to the mean value of vehicle group of each cell line. b , Venn diagrams showing intersections of upregulated or downregulated metabolites in sh KDM4C -expressing HCC1954 and SUM149 cells with either Dox (sh KDM4C ) or ML324 treatment. c , Bar plot representing the top ten consistently decreased metabolites in sh KDM4C -expressing HCC1954 and SUM149 cells with either Dox or ML324 treatments. d , Dot plots depicting normalized reduced (GSH) and oxidized (GSSG) GSH levels and their ratios in HCC1954 and SUM149 cell lines with sh KDM4C or ML324 treatment. Mean ± s.d. from n = 3 is shown. Dunnett’s test (two-sided) was used. m / z , mass-to-charge ratio of ions. e , Line plot depicting oxygen consumption rate (OCR) changes recorded by seahorse mito-stress assay in SUM149 cell lines treated with DMSO, 10 μm ML324 or 1 μm QC6352 for 3 days. Three time points were recorded for each state. This experiment was repeated three times independently with similar results. f , Representative flow cytometry plots depicting the shift of CellROX green signal in 8 cell lines after 1 μm QC6352 for 5 days. g , Bar plot showing QC6352-induced FCs in CellROX green signal merging five (SUM149), four (HCC1954) and three (all the other cell lines) independent experiments of each cell line (mean ± s.d.). Mann–Whitney U test was used to compare average FCs between four KDM4C -amplified and four non-amplified cell lines. h , Dot plot depicting quantification of CTSL activity signal quantified from 120 individual cells from 3 representative fluorescence images of inducible sh KDM4C -expressing SUM149 cells treated with DMSO (vehicle), 1 μg ml −1 Dox (sh KDM4C ), 10 μm ML324 or 1 μm QC6352 or combined with 2 mM GSH-EE for 5 days. Mean and s.d. are shown. Statistical significance of differences was determined by two-sided ordinary one-way ANOVA. i , Immunoblot analysis of histone H3 using antibodies for the C terminus in SUM149 cells treated with 1 µM of QC6352 in the presence or absence of 2 mM GSH-EE for 3 days. Tubulin was used as a loading control. Experiment was repeated three times independently with similar results. j , Line plot illustrating the QC6352-induced log 2 (FCs) of CTSL magic red and CellROX green signals at the indicated time points in SUM149 cells. Data represent mean ± s.d. merged from three independent experiments. Two-sided two-way ANOVA at each time point was used for statistical comparison.

Article Snippet: Inducible SUM149 sh KDM4C -infected cells were treated with control (no Dox, DMSO), sh KDM4C induction (plus 1 μg ml −1 Dox), 10 μm ML324 (no Dox), 1 μm QC6352 (no Dox) for 5 days or 1 μm Leu–Leu methyl ester hydrobromide for 24 h. Cells were then washed, fixed in 4% paraformaldehyde and blocked in 5% BSA/0.3% Triton X-100/PBS solution for 1 h. Primary antibodies against CTSL (Novus Biologicals, AF952; 1:100), GRHL2 (Sigma-Aldrich, HPA004820; 1:100) and KDM4C (Novus Biologicals, NBP1-49600; 1:100) were applied for 1 h at room temperature.

Techniques: Infection, Control, Expressing, Flow Cytometry, MANN-WHITNEY, Amplification, Activity Assay, Fluorescence, Western Blot, Comparison

Journal: Nature Genetics

Article Title: KDM4C inhibition blocks tumor growth in basal breast cancer by promoting cathepsin L-mediated histone H3 cleavage

doi: 10.1038/s41588-025-02197-z

Figure Lengend Snippet: a , Plots showing the CellROX green signal of five inducible-sh KDM4C models treated with or without 1 μg/ml doxycycline for 5 days. b , Representative images (left) and quantification (right) of CellROX orange signal in SUM149 cells overexpressing KDM4C WT and KDM4C S198M with siRNA against KDM4C 5′UTR for 5 days. Mean ± s.d. of 120 cells from three to four representative regions are shown (two-sided ordinary one-way ANOVA). Scale bar, 100 μm. c , Immunoblot for C′-H3 from SUM149 cells treated with 0.5 mM H 2 O 2 for 1 day (top) or 1 μm BSO for 3 days (bottom; two (BSO) and three (H 2 O 2 ) independent repeats with similar results). d , f , Representative images of CTSL activity and CellROX green signal in SUM149 cells treated with water, 0.5 mM H 2 O 2 with or without 2 mM GSH-EE for 1 day ( d ) or 1 μm BSO for 3 days ( f ). Scale bar, 100 μm. e , g , Quantification of d and f . Mean ± s.d. are shown from 60 ( e ) or 120 ( g ) cells from three representative regions of each condition (two-sided ordinary one-way ANOVA for e and Student’s t test for g ). h , Magnified images of H 2 O 2 -treated SUM149 cells. Overlapped CTSL and ROS nuclei signals are highlighted. Scale bar, 30 μm. i , Bar plot showing intracellular GSH levels in inducible-sh KDM4C SUM149 cells with DMSO, 1 μg/ml doxycycline (sh KDM4C ), 10 μm ML324 or 1 μm QC6352 with or without 2 mM GSH-EE for 2 days normalized to the corresponding cell numbers. Mean ± s.d. are shown from n = 3 from one experiment (two-sided ordinary one-way ANOVA). j , Representative images of CTSL activity and CellROX green signal under the same conditions as i for 5 days. Scale bar, 100 μm. k , Mean ± s.d. are shown for ROS signal of 120 cells from three representative regions (two-sided ordinary one-way ANOVA). l , Immunoblot of CTSL isoforms in different fractions of SUM149 cells under the same conditions as i for 5 days with tubulin and histone H3 as loading controls (three independent repeats with similar results). m , Plots showing CTSL activity and CellROX green signals in SUM149 cells treated with 1 μm QC6352 for the indicated time. Representative experiment from three independent repeats is shown.

Article Snippet: Inducible SUM149 sh KDM4C -infected cells were treated with control (no Dox, DMSO), sh KDM4C induction (plus 1 μg ml −1 Dox), 10 μm ML324 (no Dox), 1 μm QC6352 (no Dox) for 5 days or 1 μm Leu–Leu methyl ester hydrobromide for 24 h. Cells were then washed, fixed in 4% paraformaldehyde and blocked in 5% BSA/0.3% Triton X-100/PBS solution for 1 h. Primary antibodies against CTSL (Novus Biologicals, AF952; 1:100), GRHL2 (Sigma-Aldrich, HPA004820; 1:100) and KDM4C (Novus Biologicals, NBP1-49600; 1:100) were applied for 1 h at room temperature.

Techniques: Western Blot, Activity Assay

Journal: Nature Genetics

Article Title: KDM4C inhibition blocks tumor growth in basal breast cancer by promoting cathepsin L-mediated histone H3 cleavage

doi: 10.1038/s41588-025-02197-z

Figure Lengend Snippet: a , Schematic view of glutathione synthesis pathway. Enzymes or transporters analyzed in b are indicated with red and blue representing increase and decrease upon KDM4C blockade, respectively. b , Heatmap showing fold change in expression of nine key enzymes or transporters involved in glutathione biosynthesis in HCC1954 and SUM149 Dox-inducible shKDM4C cell lines following treatment with 0.1 μg/ml doxycycline (Dox), 10 μm ML324 or 1 μm QC6352 for 5 days. Gene expression was normalized to the corresponding vehicle controls. c , Left: scatter plots showing the correlation of GCLC expression with GSH abundance from 34 TNBC cell lines. R and p values were derived from two-sided Pearson correlation. Right: dot plot depicting mean ± s.d. of GCLC expression (log 2 (FPKM)) in GSH-high (n = 24) and GSH-low (n = 10) TNBC cell lines (two-sided Mann–Whitney U test). d , Immunoblot for KDM4C and GCLC in SUM149 cells overexpressing KDM4C WT and KDM4C S198M with siRNA against KDM4C 5′UTR for 3 days with tubulin as a loading control (two independent repeats with similar results). e , Immunoblots showing CTSL and GCLC protein levels in SUM149 sgScramble and CTSL KO cell lines treated with or without 10 μm ML324 or 1 μm QC6352 for 5 days, with vinculin as a loading control (three independent repeats with similar results). f , Principal component analysis plot of RNA-seq profiles of SUM149 sgScramble and CTSL KO models treated with DMSO (vehicle) or 1 μm QC6352 for 3 days. g , Volcano plots showing QC6352-induced differentially expressed genes in SUM149 sgScramble and CTSL KO models. DEGs were selected with adjusted p < 0.05 and log 2 (FC) > 2, and specific numbers were labeled on the plots. FDR values were calculated by the Wald test following Benjamini–Hochberg correction using DESeq2. h , Venn diagram illustrating the overlap of upregulated and downregulated DEGs induced by QC6352 in SUM149 sgScramble and CTSL KO models.

Article Snippet: Inducible SUM149 sh KDM4C -infected cells were treated with control (no Dox, DMSO), sh KDM4C induction (plus 1 μg ml −1 Dox), 10 μm ML324 (no Dox), 1 μm QC6352 (no Dox) for 5 days or 1 μm Leu–Leu methyl ester hydrobromide for 24 h. Cells were then washed, fixed in 4% paraformaldehyde and blocked in 5% BSA/0.3% Triton X-100/PBS solution for 1 h. Primary antibodies against CTSL (Novus Biologicals, AF952; 1:100), GRHL2 (Sigma-Aldrich, HPA004820; 1:100) and KDM4C (Novus Biologicals, NBP1-49600; 1:100) were applied for 1 h at room temperature.

Techniques: Expressing, Gene Expression, Derivative Assay, MANN-WHITNEY, Western Blot, Control, RNA Sequencing, Labeling

Journal: Nature Genetics

Article Title: KDM4C inhibition blocks tumor growth in basal breast cancer by promoting cathepsin L-mediated histone H3 cleavage

doi: 10.1038/s41588-025-02197-z

Figure Lengend Snippet: a , Immunoblot analysis of KDM4C and GCLC protein levels in HCC1954 and SUM149 Dox-inducible sh KDM4C -expressing cell lines treated with control (no Dox, DMSO), sh KDM4C induction (1 μg ml −1 Dox, DMSO), 10 μm ML324 (no Dox) or 1 μm QC6352 (no Dox) treatment for 5 days. Tubulin was used as loading control. Experiment was repeated three times independently with similar results. b , Representative images of GCLC immunofluorescence staining of xenografts derived from SUM149 cells expressing Dox-inducible sh KDM4C from mice fed with ( n = 4) or without ( n = 5) Dox diet. Signal intensity of each tumor was quantified by calculating the mean of three representative regions and shown as mean ± s.d. Two-sided Student’s t test was used. c , Scatter plot depicting correlation between KDM4C and GCLC mRNA levels in 190 basal breast tumors from the TCGA cohort. Two-sided Pearson correlation was used to calculate the P value. The linear regression line with 95% confidence interval is shown. TPM, transcripts per million. d , Genomic track view of CTSL, GRHL2 and KDM4C binding in HCC1954 and SUM149 cells at GCLC genomic locus. ATAC peaks from Dox-inducible sh KDM4C -expressing SUM149 cells treated with vehicle, Dox, ML324 and QC6352 are also displayed using the same scaling. Chr6, chromosome 6. e , Bar plot showing the cell percentage normalized to sgScramble cell models treated with DMSO in the indicated groups. Results are shown as mean ± s.d. from n = 3 as representative experiments from at least 2 independent trials. Two-sided ordinary one-way ANOVA was used within each cell line. f , g , Plots depicting the tumor volumes of xenografts derived from SUM149 ( f ) and HCC1806 ( g ) sgScramble and CTSL KO cells in mice treated with vehicle or QC6352 at the indicated time points. Data are presented as mean ± s.d. with n = 5 (SUM149) and n = 10 (HCC1806) tumors. Two-sided repeated-measure two-way ANOVA was used to compare the tumor growth kinetics. h , Heatmap illustrating unsupervised clustering of samples based on the GSVA enrichment scores of the 50 hallmark gene signatures. QC6352 upregulated and downregulated pathways that were rescued by CTSL depletion are highlighted by magenta and cyan rectangles, respectively. i , Representative flow cytometry plots depicting the shift of CellROX green signal in SUM149 and HCC38 sgScramble and CTSL KO models after 1 μm QC6352 for 5 days. j , Bar plot depicting QC6352-induced CellROX green FCs merging three independent experiments (mean ± s.d.). Two-sided Student’s t test was used. k , Dot plot depicting GSH levels normalized to tumor weight in SUM149 and HCC1806 xenografts collected at endpoint. Data are presented as mean ± s.d. with n = 5 (SUM149) or n = 10 (HCC1806) tumors. Two-sided Kruskal–Wallis test was used for each comparison. RLU, relative light units. l , Schematic illustration of major findings. HDM KDM4C blocks GRHL2-mediated CTSL activation and histone H3 tail clipping, which have a pivotal role in redox balance via maintaining GSH production and promoting basal breast tumor growth. KDM4C blockade activates CTSL either directly or indirectly and induces redox imbalance, which elevates oxidative stress and impairs basal breast tumor growth. Panel l created with BioRender.com .

Article Snippet: Inducible SUM149 sh KDM4C -infected cells were treated with control (no Dox, DMSO), sh KDM4C induction (plus 1 μg ml −1 Dox), 10 μm ML324 (no Dox), 1 μm QC6352 (no Dox) for 5 days or 1 μm Leu–Leu methyl ester hydrobromide for 24 h. Cells were then washed, fixed in 4% paraformaldehyde and blocked in 5% BSA/0.3% Triton X-100/PBS solution for 1 h. Primary antibodies against CTSL (Novus Biologicals, AF952; 1:100), GRHL2 (Sigma-Aldrich, HPA004820; 1:100) and KDM4C (Novus Biologicals, NBP1-49600; 1:100) were applied for 1 h at room temperature.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Derivative Assay, Binding Assay, Flow Cytometry, Comparison, Activation 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: 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