Journal: International Journal of Molecular Sciences

Article Title: GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer

doi: 10.3390/ijms24032512

Figure Lengend Snippet: GRHL2 expression in breast cancer. ( A , B ) The expression of GRHL2 mRNA ( A ) and protein ( B ) in different subtypes of breast cancer based on analyzing data from TCGA, GTEx, and CPATC databases. TPM, transcripts per million. * Indicates p < 0.01. Red and gray blocks in ( A ) represent tumor and normal samples, respectively. Dots show full distribution of all samples in the given group. ( C , D ) Association of GRHL2 expression with overall survival based on analyzing data from KM plotter database.

Article Snippet: The following antibodies were used: GRHL2 (Atlas-Antibodies, hpa004820; Bio-connect BV, Huissen NL), GAPDH (SantaCruz, sc-32233; Bio-connect BV, Huissen NL), Vimentin (Abcam, ab8069; Abcam BV, Amsterdam NL), N-cadherin (BD Biosciences, Vianen NL, 610920), E-cadherin (Abcam, ab76055; Abcam BV, Amsterdam NL), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch, 111-035-003; SANBIO BV, Uden NL), and Peroxidase AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch, 115-035-003; SANBIO BV, Uden NL).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer

doi: 10.3390/ijms24032512

Figure Lengend Snippet: GRHL2 expression in a panel of human breast cancer cell lines representing different subtypes. ( A , B ) GRHL2 expression in a panel of >50 human breast cancer cell lines covering luminal, basal A, and basal B subtypes extracted from RNA-seq data. * indicates p < 0.05; NS, not significant. ( C , D ) Western blot analysis ( C ) and qRT-PCR ( D ) showing loss of GRHL2 and its target gene CDH1 in basal B subtype breast cancer. Color codes refer to ( B ).

Article Snippet: The following antibodies were used: GRHL2 (Atlas-Antibodies, hpa004820; Bio-connect BV, Huissen NL), GAPDH (SantaCruz, sc-32233; Bio-connect BV, Huissen NL), Vimentin (Abcam, ab8069; Abcam BV, Amsterdam NL), N-cadherin (BD Biosciences, Vianen NL, 610920), E-cadherin (Abcam, ab76055; Abcam BV, Amsterdam NL), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch, 111-035-003; SANBIO BV, Uden NL), and Peroxidase AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch, 115-035-003; SANBIO BV, Uden NL).

Techniques: Expressing, RNA Sequencing, Western Blot, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer

doi: 10.3390/ijms24032512

Figure Lengend Snippet: ChIP-seq analysis of GRHL2 target genes in luminal and basal A cells. ( A ) Venn diagrams showing overlap of GRHL2 binding sites among the indicated luminal and basal A cell lines (top panels) and overlap between shared GRHL2 binding sites in luminal and basal A cell lines (bottom panel). ( B ) GRHL2 DNA-binding motifs identified in the indicated cell lines. ( C ) Enriched GO terms associated with the indicated functions for GRHL2-occupied genes shared between all luminal (left panel) or all basal A cell lines (right panel). Color coding according to padj values in the legend. X-axis shows the number of genes involved. ( D ) Hub genes calculated by degree algorithm using Cytoscape (cytoHubba) software from the indicated GO terms for luminal and basal A cells. Color coding from red to yellow indicates the rank of the genes from top to low as assigned by cytoHubba.

Article Snippet: The following antibodies were used: GRHL2 (Atlas-Antibodies, hpa004820; Bio-connect BV, Huissen NL), GAPDH (SantaCruz, sc-32233; Bio-connect BV, Huissen NL), Vimentin (Abcam, ab8069; Abcam BV, Amsterdam NL), N-cadherin (BD Biosciences, Vianen NL, 610920), E-cadherin (Abcam, ab76055; Abcam BV, Amsterdam NL), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch, 111-035-003; SANBIO BV, Uden NL), and Peroxidase AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch, 115-035-003; SANBIO BV, Uden NL).

Techniques: ChIP-sequencing, Binding Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer

doi: 10.3390/ijms24032512

Figure Lengend Snippet: Response to GRHL2 knockout in luminal and basal A cells. ( A ) Western blot analysis showing loss of GRHL2 in KO cells. WT, wild type cells; CTR, sgCTR transduced cells; KO, sgGRHL2 transduced cells. ( B ) FACS profiles with associated quantification of cell cycle phase distribution in sgCTR (CTR) and sgGRHL2 transduced (KO) MCF7 and HCC1806 cells. Representative experiment from 2 and 3 biological replicates is shown for MCF7 and HCC1806, respectively. ( C ) Graphs showing results from SRB assay for wild type (WT) and sgCTR and sgGRHL2 transduced MCF7 and HCC1806 cells for the indicated time periods after 4 days doxycycline. Data are presented as mean ± SEM from 3 biological replicates. Data are statistically analyzed by t -test comparing CTR and KO to WT. * indicates p < 0.05. ( D ) Analysis of random migration assay showing the average path speed (y-axis) captured at the indicated timepoints during the assay (x-axis) for wild type (WT) and sgCTR and sgGRHL2 transduced MCF7 and HCC1806 cells 10 days post-doxycycline. Data are presented as mean ± SEM from 3 biological replicates relative to WT. Data are statistically analyzed by two-way ANOVA. * indicates p < 0.05; ** indicates p < 0.01.

Article Snippet: The following antibodies were used: GRHL2 (Atlas-Antibodies, hpa004820; Bio-connect BV, Huissen NL), GAPDH (SantaCruz, sc-32233; Bio-connect BV, Huissen NL), Vimentin (Abcam, ab8069; Abcam BV, Amsterdam NL), N-cadherin (BD Biosciences, Vianen NL, 610920), E-cadherin (Abcam, ab76055; Abcam BV, Amsterdam NL), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch, 111-035-003; SANBIO BV, Uden NL), and Peroxidase AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch, 115-035-003; SANBIO BV, Uden NL).

Techniques: Knock-Out, Western Blot, Sulforhodamine B Assay, Migration

Journal: International Journal of Molecular Sciences

Article Title: GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer

doi: 10.3390/ijms24032512

Figure Lengend Snippet: Occupation of EMT related genes by GRHL2. ( A ) ChIP tracks for the indicated genes in luminal and basal A breast cancer cell lines. The track height is scaled from 0 to the indicated number. The locus with its exon/intron structure is presented in red. ( B ) ChIP-qPCR validation of presence and absence of GRHL2 binding sites identified by ChIP-seq. Location of the qPCR primer set in the locus is indicated by * in ( A ). Graphs represent the efficiency of indicated genomic DNA co-precipitation with anti-GRHL2 Ab or IgG control Ab. Signals for IgG control and GRHL2 antibody pulldown samples were normalized to input DNA and are presented as % input with SEM from 3 technical replicates with the exception of CLDN4 due to depletion of input material. Data were statistically analyzed by t -test and * indicates p < 0.05.

Article Snippet: The following antibodies were used: GRHL2 (Atlas-Antibodies, hpa004820; Bio-connect BV, Huissen NL), GAPDH (SantaCruz, sc-32233; Bio-connect BV, Huissen NL), Vimentin (Abcam, ab8069; Abcam BV, Amsterdam NL), N-cadherin (BD Biosciences, Vianen NL, 610920), E-cadherin (Abcam, ab76055; Abcam BV, Amsterdam NL), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch, 111-035-003; SANBIO BV, Uden NL), and Peroxidase AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch, 115-035-003; SANBIO BV, Uden NL).

Techniques: ChIP-qPCR, Biomarker Discovery, Binding Assay, ChIP-sequencing, Control

Journal: International Journal of Molecular Sciences

Article Title: GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer

doi: 10.3390/ijms24032512

Figure Lengend Snippet: Regulation of EMT related genes by GRHL2. ( A ) Western blot analysis of the indicated proteins in wild type (WT) and sgCTR (CTR) and sgGRHL2 transduced (KO) MCF7 and HCC1806 cells after 10 days doxycycline-induction. ( B ) Immunofluorescence analysis of HOECHST (nuclei, blue) and GRHL2 in MCF7 and HCC1806 cells expressing control sgRNA or GRHL2 sgRNA. ( C ) Immunofluorescence analysis of HOECHST (nuclei, blue), E-cadherin and Vimentin (Alexa-488, green), and F-actin (Rhodamine-Phalloidin, red) in WT, CTR, and KO MCF7 and HCC1806 cells after 10 days doxycycline-induction. Scale bar, 100 µm.

Article Snippet: The following antibodies were used: GRHL2 (Atlas-Antibodies, hpa004820; Bio-connect BV, Huissen NL), GAPDH (SantaCruz, sc-32233; Bio-connect BV, Huissen NL), Vimentin (Abcam, ab8069; Abcam BV, Amsterdam NL), N-cadherin (BD Biosciences, Vianen NL, 610920), E-cadherin (Abcam, ab76055; Abcam BV, Amsterdam NL), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch, 111-035-003; SANBIO BV, Uden NL), and Peroxidase AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch, 115-035-003; SANBIO BV, Uden NL).

Techniques: Western Blot, Immunofluorescence, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer

doi: 10.3390/ijms24032512

Figure Lengend Snippet: Effect of GRHL2 depletion in 4T1 basal A orthotopic transplantation model. ( A ) RT qPCR analysis of efficiency of GRHL2 depletion in 4T1 cells transduced with a control or 2 GRHL2 shRNA constructs. Mean and SEM of 3 biological replicates is shown. * p < 0.05. ( B ) FACS analysis of E cadherin surface expression in 4T1 variants described in A. Mean and SD of 3 experiments is determined and relative MFU (mean fluorescence units) compared to WT is shown. * p < 0.05. ( C ) Graph showing mean and SD for analysis of primary tumor growth after orthotopic transplantation of control and GRHL2 shRNA transduced 4T1 cells. At least 12 mice per condition in two experiments were analyzed. ( D ) Graph showing mean and SD for number of detected lung colonies for the experiment as in ( C ). NS, not significant; * p < 0.05. Right panel shows representative images of total lungs (top) and hematoxylin/eosin-stained lung sections derived from tumors of 4T1 cells transduced with a control or a GRHL2 shRNA construct.

Article Snippet: The following antibodies were used: GRHL2 (Atlas-Antibodies, hpa004820; Bio-connect BV, Huissen NL), GAPDH (SantaCruz, sc-32233; Bio-connect BV, Huissen NL), Vimentin (Abcam, ab8069; Abcam BV, Amsterdam NL), N-cadherin (BD Biosciences, Vianen NL, 610920), E-cadherin (Abcam, ab76055; Abcam BV, Amsterdam NL), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch, 111-035-003; SANBIO BV, Uden NL), and Peroxidase AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch, 115-035-003; SANBIO BV, Uden NL).

Techniques: Transplantation Assay, Quantitative RT-PCR, Transduction, Control, shRNA, Construct, Expressing, Fluorescence, Staining, Derivative Assay

Journal: Clinical chemistry

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

doi: 10.1373/clinchem.2014.225854

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

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

Techniques: Expressing, Immunostaining, Staining, Western Blot

Journal: 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: Carcinogenesis

Article Title: Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells

doi: 10.1093/carcin/bgw027

Figure Lengend Snippet: GRHL2 expression is enhanced in cells and tissues of OSCCs. (A) Western blotting was performed for GRHL2 using whole cell extracts from six different strains of NHOK, immortalized cells (HOK-16B) and OSCC cell lines (HOK-16B/BaP-T and SCC15). Another blot containing whole cell extracts from NHEK and OSCC cell lines, including SCC4 and those from UM-OSCCs were probed for GRHL2 protein levels. GAPDH was used as loading control. PD refers to population doublings. (B) qRT-PCR was performed with RNA extracted from NHOF, three strains of rapidly proliferating NHOK and the established cell lines (HOK-16B, HOK-16B/BaP-T and 1483). Bars indicate standard deviation and the asterisk (*) indicates statistical significance (P < 0.05), compared with the mean values of the control group (NHOK). (C) Epithelial tissues were excised from NHOM, preneoplastic oral lesions and OSCC tissues by laser-captured microdissection (LCM). mRNA levels of GRHL2, PCNA, hTERT and GRHL1 were determined from each sample by qRT-PCR. P values are shown in each group. (D) IHC was performed with NHOM, preneoplastic oral lesions and OSCC tissues using α-GRHL2 or α-K1 antibody. Representative staining results were shown for various OSCC samples (OSCC1–OSCC5). GRHL2 and K1 staining patterns appear to be mutually exclusive; tumor cells with strong GRHL2 appear to be completely lacking K1 staining (arrows). (E) In some tumors, GRHL2 expression pattern was found heterogeneous within the same tumor island; some cells exhibited strong GHRL2 staining while others completely lacked (arrowheads). Right-side panel shows higher magnification of the view field within the boxed area.

Article Snippet: The following primary antibodies were used in this study: GAPDH, ZEB1, E-Cadherin (E-Cad) and p63 from Santa Cruz Biotech. (Santa Cruz, CA); GRHL2 (Abnova, Taiwan); Keratin 1 from Covance (Emeryville, CA); N-Cadherin (N-Cad) from BD Biosciences (San Jose, CA); Fibronectin from Sigma-Aldrich and Oct-4, Nanog and c-Myc from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Standard Deviation, Laser Capture Microdissection, Staining

Journal: Carcinogenesis

Article Title: Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells

doi: 10.1093/carcin/bgw027

Figure Lengend Snippet: GRHL2 is required for the maintenance of transformed phenotype of OSCC cells. (A) Epithelial morphology was lost in SCC4/ShGRHL2 cells compared with the control (SCC4/EGFP) cells. GRHL2 expression was knocked down using lentiviral vector (LV-ShGRHL2) in SCC4. As control, SCC4 was infected with lentiviral vector expressing EGFP (LV-EGFP). (B) Anchorage-independent growth assay was performed with SCC4/EGFP and SCC4/ShGRHL2 in soft agar plates. Growth of colonies in soft agar was visualized under light microscopy. Representative colony sizes are shown here after 14 days post-seeding. (C) GRHL2 is associated with the tumorigenicity of OSCCs in vivo. SCC4/EGFP or SCC4/ShGRHL2 cells were injected subcutaneously at 5×106 per injection in dorsal flank of immunocompromised (nu/nu) mice (n = 5 per group and per time point). Tumor volume was measured once every 2–3 days and plotted against days post-injection. (D) Tumor nodules were harvested at 1, 2 and 3 weeks post-injection and examined the histological change via H & E staining. SCC4/ShGRHL2 tumor regressed rapidly within 10 days post-injection while the control cells (SCC4/EGFP) developed well-differentiated solid tumor.

Article Snippet: The following primary antibodies were used in this study: GAPDH, ZEB1, E-Cadherin (E-Cad) and p63 from Santa Cruz Biotech. (Santa Cruz, CA); GRHL2 (Abnova, Taiwan); Keratin 1 from Covance (Emeryville, CA); N-Cadherin (N-Cad) from BD Biosciences (San Jose, CA); Fibronectin from Sigma-Aldrich and Oct-4, Nanog and c-Myc from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Transformation Assay, Control, Expressing, Plasmid Preparation, Infection, Growth Assay, Light Microscopy, In Vivo, Injection, Staining

Journal: Carcinogenesis

Article Title: Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells

doi: 10.1093/carcin/bgw027

Figure Lengend Snippet: GRHL2 promotes cancer stem-like characteristics of OSCC cells. (A) Tumor spheroid assay was performed with SCC4/EGFP and SCC4/ShGRHL2 cells in suspension culture for 7 days. Shown here are two independent colonies (C8 and C10) of SCC4/ShGRHL2 cells after cell cloning, demonstrating that the altered tumor spheroid formation by GRHL2 knockdown is not clone-specific. (B) SCC4 cells were seeded in limiting dilution in ultra low attachment 96-well plates to form tumor spheroids to rule out the possibility that the tumor spheroids were cell aggregation rather than growth of a single cell. GRHL2 knockdown strongly reduced the size and number of colonies derived from a single cell. Number of spheroids formed in each well of the 96-well plates were counted for each cell type and plotted. P value is shown in comparison with the control group (SCC4/EGFP). (C) To determine self-renewal characteristics of SCC4/EGFP and SCC4/ShGRHL2 cells, the primary tumor spheroids were collected and disbursed into single cells, which were then plated to form secondary tumor spheroids. After the suspension culture for 7 days, tumor spheroid size and number were determined and plotted (D). P values are shown in comparison to the control group (SCC4/EGFP). (E) Expression levels of pluripotency genes, e.g. Nanog, Oct-4, KLF4, Lin28, Sox9 and EZH2, were determined by qRT-PCR in SCC4 cells with or without GRHL2 knockdown. Bars indicate standard deviation and asterisk (*) indicates statistical significance (P < 0.05), compared with the mean values of the control groups (SCC4/EGFP). (F) ChIP assay was performed with α-GRHL2 antibody in SCC4 cells, and binding in Oct-4 proximal promoter region was assessed by PCR. IVL promoter served as a positive control. TATA binding protein (TBP) also served as the positive control, while IgGs were negative controls. (G) Western blot analysis of Oct-4, Nanog and c-Myc expression in SCC4/ShGRHL2 cells compared with the control, SCC4/EGFP. (H) Western blotting was performed with whole cell extracts of SCC9 and NHOF for GRHL2 and Oct-4 after GRHL2 overexpression via retroviral vector (LXSN-GRHL2). Primary NHOK and SCC4 cells were included for comparison. GAPDH was used for loading control. (I) GRHL2 and Oct-4 protein levels were determined in P19 embryonic carcinoma cell line during retinoic acid (RA)-induced differentiation (100nM), and mouse embryoid body differentiation by Western blotting. GAPDH was used as a loading control.

Article Snippet: The following primary antibodies were used in this study: GAPDH, ZEB1, E-Cadherin (E-Cad) and p63 from Santa Cruz Biotech. (Santa Cruz, CA); GRHL2 (Abnova, Taiwan); Keratin 1 from Covance (Emeryville, CA); N-Cadherin (N-Cad) from BD Biosciences (San Jose, CA); Fibronectin from Sigma-Aldrich and Oct-4, Nanog and c-Myc from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Suspension, Clone Assay, Knockdown, Derivative Assay, Comparison, Control, Expressing, Quantitative RT-PCR, Standard Deviation, Binding Assay, Positive Control, Western Blot, Over Expression, Retroviral, Plasmid Preparation

Journal: Carcinogenesis

Article Title: Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells

doi: 10.1093/carcin/bgw027

Figure Lengend Snippet: GRHL2 regulates epithelial plasticity in OSCC. (A) Immunofluorescent staining was performed in SCC9 cells with or without GRHL2 overexpression using the retroviral vector, LXSN-GRHL2. GRHL2 overexpression and repressed FN expression were confirmed in SCC9/GRHL2 cells after G418 selection compared with the empty vector (LXSN) control. (B) Immunofluorescent staining was performed in SCC4/EGFP and SCC4/ShGRHL2 cells for GRHL2, E-Cad, p63 and FN. GRHL2 knockdown led to loss of E-Cad and p63 levels, while FN was induced. Phase contrast views for each cell group were shown. (C) GRHL2 expression levels were altered in SCC9, SCC4 and SCC15 cell lines by either overexpression (in SCC9) or knockdown (in SCC4 and SCC15). Alteration of GRHL2 level led to corresponding changes in proteins involved in epithelial (e.g. E-Cad and p63) and mesenchymal (e.g. N-Cad, ZEB1 and FN) phenotypes. In SCC15 cells, GRHL2 was re-expressed by retroviral vector (LXSN-GRHL2) infection after lentivirus-mediated GRHL2 knockdown. GRHL2 re-expression partially restored the epithelial phenotype, e.g. loss of N-Cad and ZEB1. (D) Ectopic GRHL2 expression in NHOF and DPSC enhanced the expression of E-Cad and β-catenin, which make up the epithelial adherens complex. (E-G) Expression levels of E-Cad, ZEB1 and ZEB2 were determined by qRT-PCR in SCC9 with or without GRHL2 transduction by retroviral vector (LXSN-GRHL2). Bars indicate standard deviation and asterisk (*) indicates statistical significance (P < 0.05), compared with the mean values of their control cells.

Article Snippet: The following primary antibodies were used in this study: GAPDH, ZEB1, E-Cadherin (E-Cad) and p63 from Santa Cruz Biotech. (Santa Cruz, CA); GRHL2 (Abnova, Taiwan); Keratin 1 from Covance (Emeryville, CA); N-Cadherin (N-Cad) from BD Biosciences (San Jose, CA); Fibronectin from Sigma-Aldrich and Oct-4, Nanog and c-Myc from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Staining, Over Expression, Retroviral, Plasmid Preparation, Expressing, Selection, Control, Knockdown, Infection, Quantitative RT-PCR, Transduction, Standard Deviation

Journal: Carcinogenesis

Article Title: Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells

doi: 10.1093/carcin/bgw027

Figure Lengend Snippet: GRHL2 regulates the expression of miR-200 family genes through direct promoter binding. (A) miR-200 family genes were downregulated in SCC4/ShGRHL2 compared with the control (SCC4/EGFP), as determined by qPCR. In the same samples, knockdown of GRHL2 reduced E-Cad mRNA level and enhanced ZEB1 and ZEB2 mRNA levels. (B) Diminution of miR-200 gene levels was confirmed in HaCaT cells after GRHL2 knockdown by LV-ShGRHL2 lentiviral vector compared with the empty vector control (LV-EGFP). Asterisk (*) indicates P < 0.05 compared with control group (LV-EGFP). (C) miR-200 gene promoter activity was compared in SCC4/EGFP and SCC4/ShGRHL2 cells by promoter-luciferase (firefly) reporter assay. Co-tranfection with Renilla luciferase reporter plasmid driven by SV40 promoter was used for normalization. (D) GRHL2 binding was assessed by ChIP assay using α-GRHL2 antibody or IgG (negative control) in SCC4/EGFP and SCC4/ShGRHL2 cells. Immunoprecipitates were used for amplification of the miR-200 promoter fragments (miR-200b and miR-200c representing the clusters C1 and C2, respectively) near the transcription start sites by qPCR. Enrichment of GRHL2 in hTERT promoter was used as a positive control. P values are shown in comparison to the control group (SCC4/EGFP).

Article Snippet: The following primary antibodies were used in this study: GAPDH, ZEB1, E-Cadherin (E-Cad) and p63 from Santa Cruz Biotech. (Santa Cruz, CA); GRHL2 (Abnova, Taiwan); Keratin 1 from Covance (Emeryville, CA); N-Cadherin (N-Cad) from BD Biosciences (San Jose, CA); Fibronectin from Sigma-Aldrich and Oct-4, Nanog and c-Myc from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Expressing, Binding Assay, Control, Knockdown, Plasmid Preparation, Activity Assay, Luciferase, Reporter Assay, Negative Control, Amplification, Positive Control, Comparison

Journal: Carcinogenesis

Article Title: Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells

doi: 10.1093/carcin/bgw027

Figure Lengend Snippet: Overexpression of miR-200 family genes partially reverses the phenotypic changes caused by GRHL2 knockdown in OSCC cells. (A) SCC4/ShGRHL2 cells were infected with the retroviral vectors expressing miR-200 cluster 1 (miR-200b, -200a, -429) named MSCV-miR-200C1 or miR-200 cluster 2 (miR-200c and -141) named MSCV-miR-200C2. After selection with puromycin (1 μg/ml), the cells were maintained in culture for 7 days to assess altered cell proliferation rate; cell numbers were plotted against time in culture. (B) Western blotting was performed for GRHL2 and epithelial/mesenchymal markers, e.g. E-Cad, N-Cad and p63, in SCC4/EGFP, SCC4/ShGRHL2/MSCV (empty vector), SCC4/ShGRHL2/miR-200C1 and SCC4/ShGRHL2/miR-200C2. (C) Four days after seeding the same cell numbers across the groups, photomicrographs were taken to reveal the altered cell confluence and changes in intercellular adhesion. (D) Tumor spheroid assay was performed with SCC4/EGFP, SCC4/ShGRHL2/MSCV and SCC4/ShGRHL2/miR-200C1. In addition, colony formation assay (E) and Matrigel invasion assay (F) were performed among the indicated cell groups to test the effects of miR-200 gene overexpression on reversing the phenotypic changes induced by GRHL2 knockdown in SCC4 cells.

Article Snippet: The following primary antibodies were used in this study: GAPDH, ZEB1, E-Cadherin (E-Cad) and p63 from Santa Cruz Biotech. (Santa Cruz, CA); GRHL2 (Abnova, Taiwan); Keratin 1 from Covance (Emeryville, CA); N-Cadherin (N-Cad) from BD Biosciences (San Jose, CA); Fibronectin from Sigma-Aldrich and Oct-4, Nanog and c-Myc from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Over Expression, Knockdown, Infection, Retroviral, Expressing, Selection, Western Blot, Plasmid Preparation, Colony Assay, Invasion Assay