ap 2α c83e10  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ap 2α c83e10
    Ap 2α C83e10, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ap 2α c83e10  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ap 2α c83e10
    Ap 2α C83e10, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    protein 2 ap2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc protein 2 ap2
    Protein 2 Ap2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antitfap2a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antitfap2a
    Antitfap2a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ap 2α  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ap 2α
    Involvement of RUNX3 in VPA-induced upregulation of TrkC. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or 1 mM VPA. Nuclear extracts and cytosolic fractions were prepared and analyzed for RUNX3, phospho-Ser249-AML1 (pAML1), <t>AML1,</t> <t>AP-2α,</t> HDAC1 and GAPDH levels. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y cells transfected with either control siRNA or RUNX3 siRNA duplexes were incubated for 24 h with either vehicle, 0.6 mM VPA (VPA 0.6), or 1 mM VPA (VPA 1.0). Cell lysates were analyzed for RUNX3 and TrkC expression. Values are the mean ± SEM of four independent experiments. *** p < 0.001 vs. control (control siRNA + vehicle). # p < 0.05, ### p < 0.001 vs the corresponding value in control siRNA-treated cells. ( C ) SH-SY5Y cells were incubated for 72 h in growth medium with either vehicle, 1 µM 3-deazaneplanocin A (DZNep), or 1 µM tazemetostat (TZM). Cell lysates were analyzed for TrkC and RUNX3 levels. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. the corresponding control value (vehicle) by ANOVA followed by Tukey’s test.
    Ap 2α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid"

    Article Title: The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22157790

    Involvement of RUNX3 in VPA-induced upregulation of TrkC. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or 1 mM VPA. Nuclear extracts and cytosolic fractions were prepared and analyzed for RUNX3, phospho-Ser249-AML1 (pAML1), AML1, AP-2α, HDAC1 and GAPDH levels. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y cells transfected with either control siRNA or RUNX3 siRNA duplexes were incubated for 24 h with either vehicle, 0.6 mM VPA (VPA 0.6), or 1 mM VPA (VPA 1.0). Cell lysates were analyzed for RUNX3 and TrkC expression. Values are the mean ± SEM of four independent experiments. *** p < 0.001 vs. control (control siRNA + vehicle). # p < 0.05, ### p < 0.001 vs the corresponding value in control siRNA-treated cells. ( C ) SH-SY5Y cells were incubated for 72 h in growth medium with either vehicle, 1 µM 3-deazaneplanocin A (DZNep), or 1 µM tazemetostat (TZM). Cell lysates were analyzed for TrkC and RUNX3 levels. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. the corresponding control value (vehicle) by ANOVA followed by Tukey’s test.
    Figure Legend Snippet: Involvement of RUNX3 in VPA-induced upregulation of TrkC. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or 1 mM VPA. Nuclear extracts and cytosolic fractions were prepared and analyzed for RUNX3, phospho-Ser249-AML1 (pAML1), AML1, AP-2α, HDAC1 and GAPDH levels. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y cells transfected with either control siRNA or RUNX3 siRNA duplexes were incubated for 24 h with either vehicle, 0.6 mM VPA (VPA 0.6), or 1 mM VPA (VPA 1.0). Cell lysates were analyzed for RUNX3 and TrkC expression. Values are the mean ± SEM of four independent experiments. *** p < 0.001 vs. control (control siRNA + vehicle). # p < 0.05, ### p < 0.001 vs the corresponding value in control siRNA-treated cells. ( C ) SH-SY5Y cells were incubated for 72 h in growth medium with either vehicle, 1 µM 3-deazaneplanocin A (DZNep), or 1 µM tazemetostat (TZM). Cell lysates were analyzed for TrkC and RUNX3 levels. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. the corresponding control value (vehicle) by ANOVA followed by Tukey’s test.

    Techniques Used: Transfection, Incubation, Expressing

    anti hs tfap2a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti hs tfap2a
    a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of <t>tfap2a</t> and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).
    Anti Hs Tfap2a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma"

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    Journal: bioRxiv

    doi: 10.1101/2020.08.24.265140

    a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).
    Figure Legend Snippet: a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).

    Techniques Used: CRISPR, Labeling, Time-lapse Microscopy

    (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).
    Figure Legend Snippet: (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).

    Techniques Used: Expressing, RNA Sequencing Assay, CRISPR, Western Blot, Time-lapse Microscopy

    (Related to ) a . Analysis of TFAP2A in the Dependency Map (DepMap, CRISPR (Avana) Public 19Q3) dataset reveals a dependence of melanoma proliferation on TFAP2A. b-c . High TFAP2A mRNA expression in primary tumors predicts worse (b) melanoma specific survival in patients in the Leeds Melanoma Cohort (HR [95% CI]: 1.6 [1.2, 2.1], p=0.001 upper vs. lower half by Cox proportional hazards and (c) progression free survival in patients in the AVAST-M Melanoma Cohort (multivariate Cox regression model). d-g . Primary tumors with high PRO or low INV expression are associated with worse outcomes in patients in (d-e) the Leeds Melanoma Cohort and (f-g) the AVAST-M Melanoma Cohort. h . Human melanoma samples from The Cancer Genome Atlas (TCGA) melanoma (SKCM, n=472) cohort are plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. i . Individual human melanoma cells are plotted as PRO and INV scores calculated from single-cell RNA-seq and colored according to TFAP2A mRNA expression (re-analyzed from Jerby-Arnon et al. ). Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. j-k . (j) S100A1 and (k) S100B mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p-values by Wilcoxon rank sum test with Bonferroni correction). l . Human melanoma cell lines ranked by cluster forming ability (left to right: low to high) demonstrate negative correlation between TFAP2A mRNA expression by RNA-seq and clustering (Spearman correlation shown). m . Western blot analysis of TFAP2A expression in panels of (left) low-passage human melanoma cell lines and (right) human melanoma cell lines.
    Figure Legend Snippet: (Related to ) a . Analysis of TFAP2A in the Dependency Map (DepMap, CRISPR (Avana) Public 19Q3) dataset reveals a dependence of melanoma proliferation on TFAP2A. b-c . High TFAP2A mRNA expression in primary tumors predicts worse (b) melanoma specific survival in patients in the Leeds Melanoma Cohort (HR [95% CI]: 1.6 [1.2, 2.1], p=0.001 upper vs. lower half by Cox proportional hazards and (c) progression free survival in patients in the AVAST-M Melanoma Cohort (multivariate Cox regression model). d-g . Primary tumors with high PRO or low INV expression are associated with worse outcomes in patients in (d-e) the Leeds Melanoma Cohort and (f-g) the AVAST-M Melanoma Cohort. h . Human melanoma samples from The Cancer Genome Atlas (TCGA) melanoma (SKCM, n=472) cohort are plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. i . Individual human melanoma cells are plotted as PRO and INV scores calculated from single-cell RNA-seq and colored according to TFAP2A mRNA expression (re-analyzed from Jerby-Arnon et al. ). Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. j-k . (j) S100A1 and (k) S100B mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p-values by Wilcoxon rank sum test with Bonferroni correction). l . Human melanoma cell lines ranked by cluster forming ability (left to right: low to high) demonstrate negative correlation between TFAP2A mRNA expression by RNA-seq and clustering (Spearman correlation shown). m . Western blot analysis of TFAP2A expression in panels of (left) low-passage human melanoma cell lines and (right) human melanoma cell lines.

    Techniques Used: CRISPR, Expressing, RNA Sequencing Assay, Western Blot

    a . Human melanoma cell lines in the Cancer Cell Line Encyclopedia (CCLE, n=56) plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. b . TFAP2A mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p<0.001 by Wilcoxon rank sum test with Bonferroni correction). c . Low-passage human melanoma cell lines ranked by increased cluster forming ability (left to right) with TFAP2A expression quantified by immunofluorescence (plot and top; Spearman correlation shown; scale bar 20 μm) and clustering (bottom, scale bar 500 μm). d . GSAA was run using gene sets and GO gene sets with FDR < 0.05 from INV vs. PRO RNA-seq (n=39 gene sets; cyan points in and ). Bars show Normalized Association Score (NAS) for CRISPR (ZMEL1-PRO sg_ tfap2a/e vs. sg_scr) and INV vs. PRO for each gene set, with black outline representing FDR<0.05 for CRISPR experiment. e . Plot of Hoek et al. INV signature by GSAA for ZMEL1-PRO sg_ tfap2a/e vs. sg_scr RNA-seq. f . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). Asterisks (*) indicate genes with associated TFAP2A CUT&RUN peaks. Human ortholog gene names are used for clarity (see Figure S7a for zebrafish gene names).
    Figure Legend Snippet: a . Human melanoma cell lines in the Cancer Cell Line Encyclopedia (CCLE, n=56) plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. b . TFAP2A mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p<0.001 by Wilcoxon rank sum test with Bonferroni correction). c . Low-passage human melanoma cell lines ranked by increased cluster forming ability (left to right) with TFAP2A expression quantified by immunofluorescence (plot and top; Spearman correlation shown; scale bar 20 μm) and clustering (bottom, scale bar 500 μm). d . GSAA was run using gene sets and GO gene sets with FDR < 0.05 from INV vs. PRO RNA-seq (n=39 gene sets; cyan points in and ). Bars show Normalized Association Score (NAS) for CRISPR (ZMEL1-PRO sg_ tfap2a/e vs. sg_scr) and INV vs. PRO for each gene set, with black outline representing FDR<0.05 for CRISPR experiment. e . Plot of Hoek et al. INV signature by GSAA for ZMEL1-PRO sg_ tfap2a/e vs. sg_scr RNA-seq. f . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). Asterisks (*) indicate genes with associated TFAP2A CUT&RUN peaks. Human ortholog gene names are used for clarity (see Figure S7a for zebrafish gene names).

    Techniques Used: RNA Sequencing Assay, Expressing, Immunofluorescence, CRISPR

    (Related to ) a . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). As in , but with zebrafish gene names. b . Distribution of TFAP2A CUT&RUN peaks as annotated by ChIPSeeker. c-d . Overlap of TFAP2A CUT&RUN peaks with genes upregulated in ZMEL1-PRO following CRISPR/Cas9 with (c) sg_scr (p=0.7 by bootstrapping) and (d) sg_ tfap2a/e (p<0.001 by bootstrapping).
    Figure Legend Snippet: (Related to ) a . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). As in , but with zebrafish gene names. b . Distribution of TFAP2A CUT&RUN peaks as annotated by ChIPSeeker. c-d . Overlap of TFAP2A CUT&RUN peaks with genes upregulated in ZMEL1-PRO following CRISPR/Cas9 with (c) sg_scr (p=0.7 by bootstrapping) and (d) sg_ tfap2a/e (p<0.001 by bootstrapping).

    Techniques Used: CRISPR

    anti hs tfap2a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti hs tfap2a
    a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of <t>tfap2a</t> and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).
    Anti Hs Tfap2a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    1) Product Images from "Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma"

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    Journal: bioRxiv

    doi: 10.1101/2020.08.24.265140

    a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).
    Figure Legend Snippet: a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).

    Techniques Used: CRISPR, Labeling, Time-lapse Microscopy

    (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).
    Figure Legend Snippet: (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).

    Techniques Used: Expressing, RNA Sequencing Assay, CRISPR, Western Blot, Time-lapse Microscopy

    (Related to ) a . Analysis of TFAP2A in the Dependency Map (DepMap, CRISPR (Avana) Public 19Q3) dataset reveals a dependence of melanoma proliferation on TFAP2A. b-c . High TFAP2A mRNA expression in primary tumors predicts worse (b) melanoma specific survival in patients in the Leeds Melanoma Cohort (HR [95% CI]: 1.6 [1.2, 2.1], p=0.001 upper vs. lower half by Cox proportional hazards and (c) progression free survival in patients in the AVAST-M Melanoma Cohort (multivariate Cox regression model). d-g . Primary tumors with high PRO or low INV expression are associated with worse outcomes in patients in (d-e) the Leeds Melanoma Cohort and (f-g) the AVAST-M Melanoma Cohort. h . Human melanoma samples from The Cancer Genome Atlas (TCGA) melanoma (SKCM, n=472) cohort are plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. i . Individual human melanoma cells are plotted as PRO and INV scores calculated from single-cell RNA-seq and colored according to TFAP2A mRNA expression (re-analyzed from Jerby-Arnon et al. ). Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. j-k . (j) S100A1 and (k) S100B mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p-values by Wilcoxon rank sum test with Bonferroni correction). l . Human melanoma cell lines ranked by cluster forming ability (left to right: low to high) demonstrate negative correlation between TFAP2A mRNA expression by RNA-seq and clustering (Spearman correlation shown). m . Western blot analysis of TFAP2A expression in panels of (left) low-passage human melanoma cell lines and (right) human melanoma cell lines.
    Figure Legend Snippet: (Related to ) a . Analysis of TFAP2A in the Dependency Map (DepMap, CRISPR (Avana) Public 19Q3) dataset reveals a dependence of melanoma proliferation on TFAP2A. b-c . High TFAP2A mRNA expression in primary tumors predicts worse (b) melanoma specific survival in patients in the Leeds Melanoma Cohort (HR [95% CI]: 1.6 [1.2, 2.1], p=0.001 upper vs. lower half by Cox proportional hazards and (c) progression free survival in patients in the AVAST-M Melanoma Cohort (multivariate Cox regression model). d-g . Primary tumors with high PRO or low INV expression are associated with worse outcomes in patients in (d-e) the Leeds Melanoma Cohort and (f-g) the AVAST-M Melanoma Cohort. h . Human melanoma samples from The Cancer Genome Atlas (TCGA) melanoma (SKCM, n=472) cohort are plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. i . Individual human melanoma cells are plotted as PRO and INV scores calculated from single-cell RNA-seq and colored according to TFAP2A mRNA expression (re-analyzed from Jerby-Arnon et al. ). Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. j-k . (j) S100A1 and (k) S100B mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p-values by Wilcoxon rank sum test with Bonferroni correction). l . Human melanoma cell lines ranked by cluster forming ability (left to right: low to high) demonstrate negative correlation between TFAP2A mRNA expression by RNA-seq and clustering (Spearman correlation shown). m . Western blot analysis of TFAP2A expression in panels of (left) low-passage human melanoma cell lines and (right) human melanoma cell lines.

    Techniques Used: CRISPR, Expressing, RNA Sequencing Assay, Western Blot

    a . Human melanoma cell lines in the Cancer Cell Line Encyclopedia (CCLE, n=56) plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. b . TFAP2A mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p<0.001 by Wilcoxon rank sum test with Bonferroni correction). c . Low-passage human melanoma cell lines ranked by increased cluster forming ability (left to right) with TFAP2A expression quantified by immunofluorescence (plot and top; Spearman correlation shown; scale bar 20 μm) and clustering (bottom, scale bar 500 μm). d . GSAA was run using gene sets and GO gene sets with FDR < 0.05 from INV vs. PRO RNA-seq (n=39 gene sets; cyan points in and ). Bars show Normalized Association Score (NAS) for CRISPR (ZMEL1-PRO sg_ tfap2a/e vs. sg_scr) and INV vs. PRO for each gene set, with black outline representing FDR<0.05 for CRISPR experiment. e . Plot of Hoek et al. INV signature by GSAA for ZMEL1-PRO sg_ tfap2a/e vs. sg_scr RNA-seq. f . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). Asterisks (*) indicate genes with associated TFAP2A CUT&RUN peaks. Human ortholog gene names are used for clarity (see Figure S7a for zebrafish gene names).
    Figure Legend Snippet: a . Human melanoma cell lines in the Cancer Cell Line Encyclopedia (CCLE, n=56) plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. b . TFAP2A mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p<0.001 by Wilcoxon rank sum test with Bonferroni correction). c . Low-passage human melanoma cell lines ranked by increased cluster forming ability (left to right) with TFAP2A expression quantified by immunofluorescence (plot and top; Spearman correlation shown; scale bar 20 μm) and clustering (bottom, scale bar 500 μm). d . GSAA was run using gene sets and GO gene sets with FDR < 0.05 from INV vs. PRO RNA-seq (n=39 gene sets; cyan points in and ). Bars show Normalized Association Score (NAS) for CRISPR (ZMEL1-PRO sg_ tfap2a/e vs. sg_scr) and INV vs. PRO for each gene set, with black outline representing FDR<0.05 for CRISPR experiment. e . Plot of Hoek et al. INV signature by GSAA for ZMEL1-PRO sg_ tfap2a/e vs. sg_scr RNA-seq. f . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). Asterisks (*) indicate genes with associated TFAP2A CUT&RUN peaks. Human ortholog gene names are used for clarity (see Figure S7a for zebrafish gene names).

    Techniques Used: RNA Sequencing Assay, Expressing, Immunofluorescence, CRISPR

    (Related to ) a . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). As in , but with zebrafish gene names. b . Distribution of TFAP2A CUT&RUN peaks as annotated by ChIPSeeker. c-d . Overlap of TFAP2A CUT&RUN peaks with genes upregulated in ZMEL1-PRO following CRISPR/Cas9 with (c) sg_scr (p=0.7 by bootstrapping) and (d) sg_ tfap2a/e (p<0.001 by bootstrapping).
    Figure Legend Snippet: (Related to ) a . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). As in , but with zebrafish gene names. b . Distribution of TFAP2A CUT&RUN peaks as annotated by ChIPSeeker. c-d . Overlap of TFAP2A CUT&RUN peaks with genes upregulated in ZMEL1-PRO following CRISPR/Cas9 with (c) sg_scr (p=0.7 by bootstrapping) and (d) sg_ tfap2a/e (p<0.001 by bootstrapping).

    Techniques Used: CRISPR

    anti dr tfap2e  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti dr tfap2e
    a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and <t>tfap2e</t> alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).
    Anti Dr Tfap2e, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti dr tfap2e/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti dr tfap2e - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma"

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    Journal: bioRxiv

    doi: 10.1101/2020.08.24.265140

    a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).
    Figure Legend Snippet: a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).

    Techniques Used: CRISPR, Labeling, Time-lapse Microscopy

    (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).
    Figure Legend Snippet: (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).

    Techniques Used: Expressing, RNA Sequencing Assay, CRISPR, Western Blot, Time-lapse Microscopy

    a . Schematic of experiment. Prior to flow cytometry and 10X single-cell RNA-seq, ZMEL1-PRO and -INV cells were either grown in vitro (individual or co-culture) or isolated from zebrafish orthotopically transplanted with a 1:1 mixture of the two subpopulations (primary tumors or metastases). b . Relative number of ZMEL1-PRO and -INV cells isolated and quantified by flow cytometry from primary tumors and metastases of fish transplanted with a 1:1 mixture of ZMEL1-PRO and -INV (primary tumors from n=6 fish; metastases from n=4 fish; p= 0.51 and p=0.031, respectively, by one-sample two-sided t-test with Bonferroni correction). c . Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction of 40,293 ZMEL1 cells sequenced as in (a). Individual culture (IND); co-culture (CO); primary tumors (PRI); metastases (MET). d . PRO and INV scores based on ZMEL1 bulk RNA-seq are plotted for all cells in gray. ZMEL1-PRO (purple) and ZMEL1-INV (green) for the indicated condition are colored. Diagonal line represents the classifier used in (e). e . Confusion matrices comparing initial cell identity with observed cell classification based on a linear classifier trained on in vitro individual culture samples. f . ZMEL1-INV cells plotted as in (d) colored according to tfap2e mRNA expression reveal re-activation of tfap2e upon metastatic dissemination.
    Figure Legend Snippet: a . Schematic of experiment. Prior to flow cytometry and 10X single-cell RNA-seq, ZMEL1-PRO and -INV cells were either grown in vitro (individual or co-culture) or isolated from zebrafish orthotopically transplanted with a 1:1 mixture of the two subpopulations (primary tumors or metastases). b . Relative number of ZMEL1-PRO and -INV cells isolated and quantified by flow cytometry from primary tumors and metastases of fish transplanted with a 1:1 mixture of ZMEL1-PRO and -INV (primary tumors from n=6 fish; metastases from n=4 fish; p= 0.51 and p=0.031, respectively, by one-sample two-sided t-test with Bonferroni correction). c . Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction of 40,293 ZMEL1 cells sequenced as in (a). Individual culture (IND); co-culture (CO); primary tumors (PRI); metastases (MET). d . PRO and INV scores based on ZMEL1 bulk RNA-seq are plotted for all cells in gray. ZMEL1-PRO (purple) and ZMEL1-INV (green) for the indicated condition are colored. Diagonal line represents the classifier used in (e). e . Confusion matrices comparing initial cell identity with observed cell classification based on a linear classifier trained on in vitro individual culture samples. f . ZMEL1-INV cells plotted as in (d) colored according to tfap2e mRNA expression reveal re-activation of tfap2e upon metastatic dissemination.

    Techniques Used: Flow Cytometry, RNA Sequencing Assay, In Vitro, Co-Culture Assay, Isolation, Expressing, Activation Assay

    (Related to ) a . Single-cell expression of tfap2e in ZMEL1-PRO and -INV cells. Individual culture (IND); co-culture (CO); primary tumors (PRI); metastases (MET).
    Figure Legend Snippet: (Related to ) a . Single-cell expression of tfap2e in ZMEL1-PRO and -INV cells. Individual culture (IND); co-culture (CO); primary tumors (PRI); metastases (MET).

    Techniques Used: Expressing, Co-Culture Assay

    anti ap 2α  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ap 2α
    <t>Ap-2α</t> transcriptionally promotes Elk-1 expression. a A schematic depiction of different mouse Elk-1 promoter regions that were cloned into the pGL4-basic vector. The constructs were designed as pE-1~8 with different lengths, as indicated. b Effects of MC-38 conditioned medium on the promoter activity of the different Elk-1 promoter constructs described in ( a ). Raw cells were transfected with the truncated reporter constructs, stimulated with MC-38 conditioned medium or control normal medium for 24 h, and then harvested for assays assessing luciferase activity. The results are expressed as the relative activity normalized to the activity of each control medium-treated group, which was arbitrarily defined as 1 ( n = 3). c Mutation of a <t>potential</t> <t>Ap-2α</t> binding site in the reporter construct pE-1. The sequence GCCTGC located at −1396/−1391 was mutated to GACTAC in the mutant reporter construct pE-1-mut. d Effects of Ap-2α on the activity of the Elk-1 promoter constructs pE-1 and pE-1-mut. RAW cells were cotransfected with pE-1 or Mut-1 plus a scrambled shRNA (sh-con) or an Ap-2α-specific shRNA (sh-Ap2α) for 24 h and then harvested for luciferase activity assays ( n = 3). e Binding activity between Ap-2α and Elk-1 DNA in normal medium- or MC-38 conditioned medium-stimulated macrophages, as measured by ChIP assays ( n = 4). f , g Stimulation of the Ap-2α-dependent mRNA expression of Elk-1 and Sirpα by MC-38 cells. RAW cells were transfected with sh-con or sh-Ap2α for 12 h and then treated with MC-38 conditioned medium or control medium for 24 h. Cells were harvested for real-time PCR analysis of Elk-1 ( f ) and Sirpα ( g ). The data in ( b , d – g ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)
    Anti Ap 2α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer"

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-0124-z

    Ap-2α transcriptionally promotes Elk-1 expression. a A schematic depiction of different mouse Elk-1 promoter regions that were cloned into the pGL4-basic vector. The constructs were designed as pE-1~8 with different lengths, as indicated. b Effects of MC-38 conditioned medium on the promoter activity of the different Elk-1 promoter constructs described in ( a ). Raw cells were transfected with the truncated reporter constructs, stimulated with MC-38 conditioned medium or control normal medium for 24 h, and then harvested for assays assessing luciferase activity. The results are expressed as the relative activity normalized to the activity of each control medium-treated group, which was arbitrarily defined as 1 ( n = 3). c Mutation of a potential Ap-2α binding site in the reporter construct pE-1. The sequence GCCTGC located at −1396/−1391 was mutated to GACTAC in the mutant reporter construct pE-1-mut. d Effects of Ap-2α on the activity of the Elk-1 promoter constructs pE-1 and pE-1-mut. RAW cells were cotransfected with pE-1 or Mut-1 plus a scrambled shRNA (sh-con) or an Ap-2α-specific shRNA (sh-Ap2α) for 24 h and then harvested for luciferase activity assays ( n = 3). e Binding activity between Ap-2α and Elk-1 DNA in normal medium- or MC-38 conditioned medium-stimulated macrophages, as measured by ChIP assays ( n = 4). f , g Stimulation of the Ap-2α-dependent mRNA expression of Elk-1 and Sirpα by MC-38 cells. RAW cells were transfected with sh-con or sh-Ap2α for 12 h and then treated with MC-38 conditioned medium or control medium for 24 h. Cells were harvested for real-time PCR analysis of Elk-1 ( f ) and Sirpα ( g ). The data in ( b , d – g ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)
    Figure Legend Snippet: Ap-2α transcriptionally promotes Elk-1 expression. a A schematic depiction of different mouse Elk-1 promoter regions that were cloned into the pGL4-basic vector. The constructs were designed as pE-1~8 with different lengths, as indicated. b Effects of MC-38 conditioned medium on the promoter activity of the different Elk-1 promoter constructs described in ( a ). Raw cells were transfected with the truncated reporter constructs, stimulated with MC-38 conditioned medium or control normal medium for 24 h, and then harvested for assays assessing luciferase activity. The results are expressed as the relative activity normalized to the activity of each control medium-treated group, which was arbitrarily defined as 1 ( n = 3). c Mutation of a potential Ap-2α binding site in the reporter construct pE-1. The sequence GCCTGC located at −1396/−1391 was mutated to GACTAC in the mutant reporter construct pE-1-mut. d Effects of Ap-2α on the activity of the Elk-1 promoter constructs pE-1 and pE-1-mut. RAW cells were cotransfected with pE-1 or Mut-1 plus a scrambled shRNA (sh-con) or an Ap-2α-specific shRNA (sh-Ap2α) for 24 h and then harvested for luciferase activity assays ( n = 3). e Binding activity between Ap-2α and Elk-1 DNA in normal medium- or MC-38 conditioned medium-stimulated macrophages, as measured by ChIP assays ( n = 4). f , g Stimulation of the Ap-2α-dependent mRNA expression of Elk-1 and Sirpα by MC-38 cells. RAW cells were transfected with sh-con or sh-Ap2α for 12 h and then treated with MC-38 conditioned medium or control medium for 24 h. Cells were harvested for real-time PCR analysis of Elk-1 ( f ) and Sirpα ( g ). The data in ( b , d – g ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Techniques Used: Expressing, Clone Assay, Plasmid Preparation, Construct, Activity Assay, Transfection, Luciferase, Mutagenesis, Binding Assay, Sequencing, shRNA, Real-time Polymerase Chain Reaction

    Macrophage Ap2α-Elk-1 axis regulates CRC progression. a MC-38 cells (1.0 × 10 6 cells per mouse) were subcutaneously inoculated into fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice, and tumor volume was monitored dynamically ( n = 10). b The survival of the mice described above in ( a ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). c The Elk-1 transgene prevented Ap-2α silencing-induced tumor phagocytosis. Six-week-old male fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice were subcutaneously engrafted with GFP-tagged MC-38 cells (1.0 × 10 6 cells per mouse). Two weeks later, tumors were recovered for the measurement of tumor phagocytosis by TAMs ( n = 3). d Volumes of tumors in WT mice inoculated with MC-38 cells mixed 2:1 with macrophages isolated from corresponding tumors grown in fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 or Ap2α-cKO + Tg Elk-1 mice are shown ( n = 10). e The survival of the mice described above in ( d ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). The data in ( a , c , d ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)
    Figure Legend Snippet: Macrophage Ap2α-Elk-1 axis regulates CRC progression. a MC-38 cells (1.0 × 10 6 cells per mouse) were subcutaneously inoculated into fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice, and tumor volume was monitored dynamically ( n = 10). b The survival of the mice described above in ( a ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). c The Elk-1 transgene prevented Ap-2α silencing-induced tumor phagocytosis. Six-week-old male fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice were subcutaneously engrafted with GFP-tagged MC-38 cells (1.0 × 10 6 cells per mouse). Two weeks later, tumors were recovered for the measurement of tumor phagocytosis by TAMs ( n = 3). d Volumes of tumors in WT mice inoculated with MC-38 cells mixed 2:1 with macrophages isolated from corresponding tumors grown in fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 or Ap2α-cKO + Tg Elk-1 mice are shown ( n = 10). e The survival of the mice described above in ( d ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). The data in ( a , c , d ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Techniques Used: Isolation

    Lactate induces TAM Ap-2α activity and CRC progression. a Nuclear translocation of Ap-2α in TAMs was evaluated. Six-week-old C57BL/6 mice were subcutaneously inoculated with MC-38 cells (1.0 × 10 6 cells per mouse). TAMs isolated at different time points were stained with anti-Ap-2α antibodies (green). Nuclei were visualized by DAPI staining (blue). The total Ap-2α protein in TAMs at different time points was measured by western blotting assays. The nuclear translocation rates of Ap-2α were calculated ( n = 4). b Metabolic factors induced the nuclear translocation of Ap-2α in macrophages. MC-38 conditioned medium was roughly fractionated into a protein fraction (CM-1, >3 kD) and a metabolic fraction (CM-2, <3 kD) by molecular size. Bone marrow-derived macrophages were treated with CM-1, CM-2 or control normal medium for 24 h and then stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). c Lactate stimulated the nuclear translocation of Ap-2α in macrophages. Bone marrow-derived macrophages were treated with lactate (10 mM) or PBS as a control for 24 h. Then, cells were stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). d , e The mRNA levels of Elk-1 ( d ) and Sirpα ( e ) in macrophages treated with lactate (10 mM) or PBS for 24 h were measured ( n = 4). f The phagocytosis rates of macrophages treated with lactate (10 mM) or PBS for 24 h were evaluated ( n = 3). g Six-week-old male C57BL/6 mice were subcutaneously engrafted with MC-38 cells (1.0 × 10 6 cells per mouse) and then subcutaneously treated with lactate (50 μmol in 100 μl of PBS per injection) or PBS on days 1, 3, and 5. Tumor volume was measured dynamically ( n = 10). h The survival of the mice described in ( g ) is shown ( n = 10, *** P < 0.005; Gehan–Breslow–Wilcoxon test). The data in ( a – g ) are reported as the mean ± s.e.m. (*** P < 0.005; Student’s t -test)
    Figure Legend Snippet: Lactate induces TAM Ap-2α activity and CRC progression. a Nuclear translocation of Ap-2α in TAMs was evaluated. Six-week-old C57BL/6 mice were subcutaneously inoculated with MC-38 cells (1.0 × 10 6 cells per mouse). TAMs isolated at different time points were stained with anti-Ap-2α antibodies (green). Nuclei were visualized by DAPI staining (blue). The total Ap-2α protein in TAMs at different time points was measured by western blotting assays. The nuclear translocation rates of Ap-2α were calculated ( n = 4). b Metabolic factors induced the nuclear translocation of Ap-2α in macrophages. MC-38 conditioned medium was roughly fractionated into a protein fraction (CM-1, >3 kD) and a metabolic fraction (CM-2, <3 kD) by molecular size. Bone marrow-derived macrophages were treated with CM-1, CM-2 or control normal medium for 24 h and then stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). c Lactate stimulated the nuclear translocation of Ap-2α in macrophages. Bone marrow-derived macrophages were treated with lactate (10 mM) or PBS as a control for 24 h. Then, cells were stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). d , e The mRNA levels of Elk-1 ( d ) and Sirpα ( e ) in macrophages treated with lactate (10 mM) or PBS for 24 h were measured ( n = 4). f The phagocytosis rates of macrophages treated with lactate (10 mM) or PBS for 24 h were evaluated ( n = 3). g Six-week-old male C57BL/6 mice were subcutaneously engrafted with MC-38 cells (1.0 × 10 6 cells per mouse) and then subcutaneously treated with lactate (50 μmol in 100 μl of PBS per injection) or PBS on days 1, 3, and 5. Tumor volume was measured dynamically ( n = 10). h The survival of the mice described in ( g ) is shown ( n = 10, *** P < 0.005; Gehan–Breslow–Wilcoxon test). The data in ( a – g ) are reported as the mean ± s.e.m. (*** P < 0.005; Student’s t -test)

    Techniques Used: Activity Assay, Translocation Assay, Isolation, Staining, Western Blot, Derivative Assay, Injection

    Macrophage Elk-1 and Sirpα levels increase with CRC progression. a – c mRNA levels of Ap-2α ( a ), Elk-1 ( b ) and Sirpα ( c ) in macrophages from adjacent normal and carcinoma tissue samples from CRC patients ( n = 15). d Positive correlation of S irpα expression with Elk-1 expression in macrophages from each individual sample. Linear regression was performed ( n = 42, P < 0.05, Pearson R = 0.377). e , f mRNA levels of Elk-1 and Sirpα in TAMs from CRC patients with different TNM stages. Samples were divided into 3 groups according to the TNM stage: T2-4N0M0 ( n = 15), T2-4N1-2M0 ( n = 15) and T2-4N0-2M1 ( n = 12). g – i Overall survival of CRC patients with differential expression of AP-2α ( g ), ELK-1 ( h ) and SIRPα ( i ). Patients were placed into two groups according to the mRNA expression of AP-2α, ELK-1 or SIRPα in TAMs. Patients with expression levels in the top 50% for TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α hi , ELK-1 hi or SIRPα hi groups, respectively. The remaining patients with low TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α lo , ELK-1 lo or SIRPα lo groups, respectively. The overall survival time of patients was determined during follow-up visits ( n = 58, * P < 0.05, *** P < 0.005, Gehan–Breslow–Wilcoxon test). The data in ( a – c and e , f ) represent the mean ± s.e.m. (* P < 0.05, ** P < 0.01 and *** P < 0.005; Student’s t -test)
    Figure Legend Snippet: Macrophage Elk-1 and Sirpα levels increase with CRC progression. a – c mRNA levels of Ap-2α ( a ), Elk-1 ( b ) and Sirpα ( c ) in macrophages from adjacent normal and carcinoma tissue samples from CRC patients ( n = 15). d Positive correlation of S irpα expression with Elk-1 expression in macrophages from each individual sample. Linear regression was performed ( n = 42, P < 0.05, Pearson R = 0.377). e , f mRNA levels of Elk-1 and Sirpα in TAMs from CRC patients with different TNM stages. Samples were divided into 3 groups according to the TNM stage: T2-4N0M0 ( n = 15), T2-4N1-2M0 ( n = 15) and T2-4N0-2M1 ( n = 12). g – i Overall survival of CRC patients with differential expression of AP-2α ( g ), ELK-1 ( h ) and SIRPα ( i ). Patients were placed into two groups according to the mRNA expression of AP-2α, ELK-1 or SIRPα in TAMs. Patients with expression levels in the top 50% for TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α hi , ELK-1 hi or SIRPα hi groups, respectively. The remaining patients with low TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α lo , ELK-1 lo or SIRPα lo groups, respectively. The overall survival time of patients was determined during follow-up visits ( n = 58, * P < 0.05, *** P < 0.005, Gehan–Breslow–Wilcoxon test). The data in ( a – c and e , f ) represent the mean ± s.e.m. (* P < 0.05, ** P < 0.01 and *** P < 0.005; Student’s t -test)

    Techniques Used: Expressing

    The working model for CRC evasion of innate immune surveillance. Without a cancer challenge, the interaction between macrophage SIRPα and CRC CD47 is interrupted, and macrophages exhibit normal phagocytic function. In the CRC microenvironment, CRC-derived lactate induces the activity of the transcription factor Ap-2α and the expression of its target Elk-1, which further promotes the expression of Sirpα in TAMs. The enhanced interaction between macrophage SIRPα and CRC CD47 reduces the phagocytic function of TAMs
    Figure Legend Snippet: The working model for CRC evasion of innate immune surveillance. Without a cancer challenge, the interaction between macrophage SIRPα and CRC CD47 is interrupted, and macrophages exhibit normal phagocytic function. In the CRC microenvironment, CRC-derived lactate induces the activity of the transcription factor Ap-2α and the expression of its target Elk-1, which further promotes the expression of Sirpα in TAMs. The enhanced interaction between macrophage SIRPα and CRC CD47 reduces the phagocytic function of TAMs

    Techniques Used: Derivative Assay, Activity Assay, Expressing

    anti ap 2α antibody  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
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    Structured Review

    Cell Signaling Technology Inc anti ap 2α antibody
    <t>Ap-2α</t> transcriptionally promotes Elk-1 expression. a A schematic depiction of different mouse Elk-1 promoter regions that were cloned into the pGL4-basic vector. The constructs were designed as pE-1~8 with different lengths, as indicated. b Effects of MC-38 conditioned medium on the promoter activity of the different Elk-1 promoter constructs described in ( a ). Raw cells were transfected with the truncated reporter constructs, stimulated with MC-38 conditioned medium or control normal medium for 24 h, and then harvested for assays assessing luciferase activity. The results are expressed as the relative activity normalized to the activity of each control medium-treated group, which was arbitrarily defined as 1 ( n = 3). c Mutation of a <t>potential</t> <t>Ap-2α</t> binding site in the reporter construct pE-1. The sequence GCCTGC located at −1396/−1391 was mutated to GACTAC in the mutant reporter construct pE-1-mut. d Effects of Ap-2α on the activity of the Elk-1 promoter constructs pE-1 and pE-1-mut. RAW cells were cotransfected with pE-1 or Mut-1 plus a scrambled shRNA (sh-con) or an Ap-2α-specific shRNA (sh-Ap2α) for 24 h and then harvested for luciferase activity assays ( n = 3). e Binding activity between Ap-2α and Elk-1 DNA in normal medium- or MC-38 conditioned medium-stimulated macrophages, as measured by ChIP assays ( n = 4). f , g Stimulation of the Ap-2α-dependent mRNA expression of Elk-1 and Sirpα by MC-38 cells. RAW cells were transfected with sh-con or sh-Ap2α for 12 h and then treated with MC-38 conditioned medium or control medium for 24 h. Cells were harvested for real-time PCR analysis of Elk-1 ( f ) and Sirpα ( g ). The data in ( b , d – g ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)
    Anti Ap 2α Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ap 2α antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ap 2α antibody - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer"

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-0124-z

    Ap-2α transcriptionally promotes Elk-1 expression. a A schematic depiction of different mouse Elk-1 promoter regions that were cloned into the pGL4-basic vector. The constructs were designed as pE-1~8 with different lengths, as indicated. b Effects of MC-38 conditioned medium on the promoter activity of the different Elk-1 promoter constructs described in ( a ). Raw cells were transfected with the truncated reporter constructs, stimulated with MC-38 conditioned medium or control normal medium for 24 h, and then harvested for assays assessing luciferase activity. The results are expressed as the relative activity normalized to the activity of each control medium-treated group, which was arbitrarily defined as 1 ( n = 3). c Mutation of a potential Ap-2α binding site in the reporter construct pE-1. The sequence GCCTGC located at −1396/−1391 was mutated to GACTAC in the mutant reporter construct pE-1-mut. d Effects of Ap-2α on the activity of the Elk-1 promoter constructs pE-1 and pE-1-mut. RAW cells were cotransfected with pE-1 or Mut-1 plus a scrambled shRNA (sh-con) or an Ap-2α-specific shRNA (sh-Ap2α) for 24 h and then harvested for luciferase activity assays ( n = 3). e Binding activity between Ap-2α and Elk-1 DNA in normal medium- or MC-38 conditioned medium-stimulated macrophages, as measured by ChIP assays ( n = 4). f , g Stimulation of the Ap-2α-dependent mRNA expression of Elk-1 and Sirpα by MC-38 cells. RAW cells were transfected with sh-con or sh-Ap2α for 12 h and then treated with MC-38 conditioned medium or control medium for 24 h. Cells were harvested for real-time PCR analysis of Elk-1 ( f ) and Sirpα ( g ). The data in ( b , d – g ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)
    Figure Legend Snippet: Ap-2α transcriptionally promotes Elk-1 expression. a A schematic depiction of different mouse Elk-1 promoter regions that were cloned into the pGL4-basic vector. The constructs were designed as pE-1~8 with different lengths, as indicated. b Effects of MC-38 conditioned medium on the promoter activity of the different Elk-1 promoter constructs described in ( a ). Raw cells were transfected with the truncated reporter constructs, stimulated with MC-38 conditioned medium or control normal medium for 24 h, and then harvested for assays assessing luciferase activity. The results are expressed as the relative activity normalized to the activity of each control medium-treated group, which was arbitrarily defined as 1 ( n = 3). c Mutation of a potential Ap-2α binding site in the reporter construct pE-1. The sequence GCCTGC located at −1396/−1391 was mutated to GACTAC in the mutant reporter construct pE-1-mut. d Effects of Ap-2α on the activity of the Elk-1 promoter constructs pE-1 and pE-1-mut. RAW cells were cotransfected with pE-1 or Mut-1 plus a scrambled shRNA (sh-con) or an Ap-2α-specific shRNA (sh-Ap2α) for 24 h and then harvested for luciferase activity assays ( n = 3). e Binding activity between Ap-2α and Elk-1 DNA in normal medium- or MC-38 conditioned medium-stimulated macrophages, as measured by ChIP assays ( n = 4). f , g Stimulation of the Ap-2α-dependent mRNA expression of Elk-1 and Sirpα by MC-38 cells. RAW cells were transfected with sh-con or sh-Ap2α for 12 h and then treated with MC-38 conditioned medium or control medium for 24 h. Cells were harvested for real-time PCR analysis of Elk-1 ( f ) and Sirpα ( g ). The data in ( b , d – g ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Techniques Used: Expressing, Clone Assay, Plasmid Preparation, Construct, Activity Assay, Transfection, Luciferase, Mutagenesis, Binding Assay, Sequencing, shRNA, Real-time Polymerase Chain Reaction

    Macrophage Ap2α-Elk-1 axis regulates CRC progression. a MC-38 cells (1.0 × 10 6 cells per mouse) were subcutaneously inoculated into fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice, and tumor volume was monitored dynamically ( n = 10). b The survival of the mice described above in ( a ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). c The Elk-1 transgene prevented Ap-2α silencing-induced tumor phagocytosis. Six-week-old male fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice were subcutaneously engrafted with GFP-tagged MC-38 cells (1.0 × 10 6 cells per mouse). Two weeks later, tumors were recovered for the measurement of tumor phagocytosis by TAMs ( n = 3). d Volumes of tumors in WT mice inoculated with MC-38 cells mixed 2:1 with macrophages isolated from corresponding tumors grown in fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 or Ap2α-cKO + Tg Elk-1 mice are shown ( n = 10). e The survival of the mice described above in ( d ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). The data in ( a , c , d ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)
    Figure Legend Snippet: Macrophage Ap2α-Elk-1 axis regulates CRC progression. a MC-38 cells (1.0 × 10 6 cells per mouse) were subcutaneously inoculated into fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice, and tumor volume was monitored dynamically ( n = 10). b The survival of the mice described above in ( a ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). c The Elk-1 transgene prevented Ap-2α silencing-induced tumor phagocytosis. Six-week-old male fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice were subcutaneously engrafted with GFP-tagged MC-38 cells (1.0 × 10 6 cells per mouse). Two weeks later, tumors were recovered for the measurement of tumor phagocytosis by TAMs ( n = 3). d Volumes of tumors in WT mice inoculated with MC-38 cells mixed 2:1 with macrophages isolated from corresponding tumors grown in fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 or Ap2α-cKO + Tg Elk-1 mice are shown ( n = 10). e The survival of the mice described above in ( d ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). The data in ( a , c , d ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Techniques Used: Isolation

    Lactate induces TAM Ap-2α activity and CRC progression. a Nuclear translocation of Ap-2α in TAMs was evaluated. Six-week-old C57BL/6 mice were subcutaneously inoculated with MC-38 cells (1.0 × 10 6 cells per mouse). TAMs isolated at different time points were stained with anti-Ap-2α antibodies (green). Nuclei were visualized by DAPI staining (blue). The total Ap-2α protein in TAMs at different time points was measured by western blotting assays. The nuclear translocation rates of Ap-2α were calculated ( n = 4). b Metabolic factors induced the nuclear translocation of Ap-2α in macrophages. MC-38 conditioned medium was roughly fractionated into a protein fraction (CM-1, >3 kD) and a metabolic fraction (CM-2, <3 kD) by molecular size. Bone marrow-derived macrophages were treated with CM-1, CM-2 or control normal medium for 24 h and then stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). c Lactate stimulated the nuclear translocation of Ap-2α in macrophages. Bone marrow-derived macrophages were treated with lactate (10 mM) or PBS as a control for 24 h. Then, cells were stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). d , e The mRNA levels of Elk-1 ( d ) and Sirpα ( e ) in macrophages treated with lactate (10 mM) or PBS for 24 h were measured ( n = 4). f The phagocytosis rates of macrophages treated with lactate (10 mM) or PBS for 24 h were evaluated ( n = 3). g Six-week-old male C57BL/6 mice were subcutaneously engrafted with MC-38 cells (1.0 × 10 6 cells per mouse) and then subcutaneously treated with lactate (50 μmol in 100 μl of PBS per injection) or PBS on days 1, 3, and 5. Tumor volume was measured dynamically ( n = 10). h The survival of the mice described in ( g ) is shown ( n = 10, *** P < 0.005; Gehan–Breslow–Wilcoxon test). The data in ( a – g ) are reported as the mean ± s.e.m. (*** P < 0.005; Student’s t -test)
    Figure Legend Snippet: Lactate induces TAM Ap-2α activity and CRC progression. a Nuclear translocation of Ap-2α in TAMs was evaluated. Six-week-old C57BL/6 mice were subcutaneously inoculated with MC-38 cells (1.0 × 10 6 cells per mouse). TAMs isolated at different time points were stained with anti-Ap-2α antibodies (green). Nuclei were visualized by DAPI staining (blue). The total Ap-2α protein in TAMs at different time points was measured by western blotting assays. The nuclear translocation rates of Ap-2α were calculated ( n = 4). b Metabolic factors induced the nuclear translocation of Ap-2α in macrophages. MC-38 conditioned medium was roughly fractionated into a protein fraction (CM-1, >3 kD) and a metabolic fraction (CM-2, <3 kD) by molecular size. Bone marrow-derived macrophages were treated with CM-1, CM-2 or control normal medium for 24 h and then stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). c Lactate stimulated the nuclear translocation of Ap-2α in macrophages. Bone marrow-derived macrophages were treated with lactate (10 mM) or PBS as a control for 24 h. Then, cells were stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). d , e The mRNA levels of Elk-1 ( d ) and Sirpα ( e ) in macrophages treated with lactate (10 mM) or PBS for 24 h were measured ( n = 4). f The phagocytosis rates of macrophages treated with lactate (10 mM) or PBS for 24 h were evaluated ( n = 3). g Six-week-old male C57BL/6 mice were subcutaneously engrafted with MC-38 cells (1.0 × 10 6 cells per mouse) and then subcutaneously treated with lactate (50 μmol in 100 μl of PBS per injection) or PBS on days 1, 3, and 5. Tumor volume was measured dynamically ( n = 10). h The survival of the mice described in ( g ) is shown ( n = 10, *** P < 0.005; Gehan–Breslow–Wilcoxon test). The data in ( a – g ) are reported as the mean ± s.e.m. (*** P < 0.005; Student’s t -test)

    Techniques Used: Activity Assay, Translocation Assay, Isolation, Staining, Western Blot, Derivative Assay, Injection

    Macrophage Elk-1 and Sirpα levels increase with CRC progression. a – c mRNA levels of Ap-2α ( a ), Elk-1 ( b ) and Sirpα ( c ) in macrophages from adjacent normal and carcinoma tissue samples from CRC patients ( n = 15). d Positive correlation of S irpα expression with Elk-1 expression in macrophages from each individual sample. Linear regression was performed ( n = 42, P < 0.05, Pearson R = 0.377). e , f mRNA levels of Elk-1 and Sirpα in TAMs from CRC patients with different TNM stages. Samples were divided into 3 groups according to the TNM stage: T2-4N0M0 ( n = 15), T2-4N1-2M0 ( n = 15) and T2-4N0-2M1 ( n = 12). g – i Overall survival of CRC patients with differential expression of AP-2α ( g ), ELK-1 ( h ) and SIRPα ( i ). Patients were placed into two groups according to the mRNA expression of AP-2α, ELK-1 or SIRPα in TAMs. Patients with expression levels in the top 50% for TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α hi , ELK-1 hi or SIRPα hi groups, respectively. The remaining patients with low TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α lo , ELK-1 lo or SIRPα lo groups, respectively. The overall survival time of patients was determined during follow-up visits ( n = 58, * P < 0.05, *** P < 0.005, Gehan–Breslow–Wilcoxon test). The data in ( a – c and e , f ) represent the mean ± s.e.m. (* P < 0.05, ** P < 0.01 and *** P < 0.005; Student’s t -test)
    Figure Legend Snippet: Macrophage Elk-1 and Sirpα levels increase with CRC progression. a – c mRNA levels of Ap-2α ( a ), Elk-1 ( b ) and Sirpα ( c ) in macrophages from adjacent normal and carcinoma tissue samples from CRC patients ( n = 15). d Positive correlation of S irpα expression with Elk-1 expression in macrophages from each individual sample. Linear regression was performed ( n = 42, P < 0.05, Pearson R = 0.377). e , f mRNA levels of Elk-1 and Sirpα in TAMs from CRC patients with different TNM stages. Samples were divided into 3 groups according to the TNM stage: T2-4N0M0 ( n = 15), T2-4N1-2M0 ( n = 15) and T2-4N0-2M1 ( n = 12). g – i Overall survival of CRC patients with differential expression of AP-2α ( g ), ELK-1 ( h ) and SIRPα ( i ). Patients were placed into two groups according to the mRNA expression of AP-2α, ELK-1 or SIRPα in TAMs. Patients with expression levels in the top 50% for TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α hi , ELK-1 hi or SIRPα hi groups, respectively. The remaining patients with low TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α lo , ELK-1 lo or SIRPα lo groups, respectively. The overall survival time of patients was determined during follow-up visits ( n = 58, * P < 0.05, *** P < 0.005, Gehan–Breslow–Wilcoxon test). The data in ( a – c and e , f ) represent the mean ± s.e.m. (* P < 0.05, ** P < 0.01 and *** P < 0.005; Student’s t -test)

    Techniques Used: Expressing

    The working model for CRC evasion of innate immune surveillance. Without a cancer challenge, the interaction between macrophage SIRPα and CRC CD47 is interrupted, and macrophages exhibit normal phagocytic function. In the CRC microenvironment, CRC-derived lactate induces the activity of the transcription factor Ap-2α and the expression of its target Elk-1, which further promotes the expression of Sirpα in TAMs. The enhanced interaction between macrophage SIRPα and CRC CD47 reduces the phagocytic function of TAMs
    Figure Legend Snippet: The working model for CRC evasion of innate immune surveillance. Without a cancer challenge, the interaction between macrophage SIRPα and CRC CD47 is interrupted, and macrophages exhibit normal phagocytic function. In the CRC microenvironment, CRC-derived lactate induces the activity of the transcription factor Ap-2α and the expression of its target Elk-1, which further promotes the expression of Sirpα in TAMs. The enhanced interaction between macrophage SIRPα and CRC CD47 reduces the phagocytic function of TAMs

    Techniques Used: Derivative Assay, Activity Assay, Expressing

    rabbit ap2α monoclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit ap2α monoclonal antibody
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    Involvement of RUNX3 in VPA-induced upregulation of TrkC. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or 1 mM VPA. Nuclear extracts and cytosolic fractions were prepared and analyzed for RUNX3, phospho-Ser249-AML1 (pAML1), AML1, AP-2α, HDAC1 and GAPDH levels. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y cells transfected with either control siRNA or RUNX3 siRNA duplexes were incubated for 24 h with either vehicle, 0.6 mM VPA (VPA 0.6), or 1 mM VPA (VPA 1.0). Cell lysates were analyzed for RUNX3 and TrkC expression. Values are the mean ± SEM of four independent experiments. *** p < 0.001 vs. control (control siRNA + vehicle). # p < 0.05, ### p < 0.001 vs the corresponding value in control siRNA-treated cells. ( C ) SH-SY5Y cells were incubated for 72 h in growth medium with either vehicle, 1 µM 3-deazaneplanocin A (DZNep), or 1 µM tazemetostat (TZM). Cell lysates were analyzed for TrkC and RUNX3 levels. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. the corresponding control value (vehicle) by ANOVA followed by Tukey’s test.

    Journal: International Journal of Molecular Sciences

    Article Title: The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid

    doi: 10.3390/ijms22157790

    Figure Lengend Snippet: Involvement of RUNX3 in VPA-induced upregulation of TrkC. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or 1 mM VPA. Nuclear extracts and cytosolic fractions were prepared and analyzed for RUNX3, phospho-Ser249-AML1 (pAML1), AML1, AP-2α, HDAC1 and GAPDH levels. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y cells transfected with either control siRNA or RUNX3 siRNA duplexes were incubated for 24 h with either vehicle, 0.6 mM VPA (VPA 0.6), or 1 mM VPA (VPA 1.0). Cell lysates were analyzed for RUNX3 and TrkC expression. Values are the mean ± SEM of four independent experiments. *** p < 0.001 vs. control (control siRNA + vehicle). # p < 0.05, ### p < 0.001 vs the corresponding value in control siRNA-treated cells. ( C ) SH-SY5Y cells were incubated for 72 h in growth medium with either vehicle, 1 µM 3-deazaneplanocin A (DZNep), or 1 µM tazemetostat (TZM). Cell lysates were analyzed for TrkC and RUNX3 levels. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. the corresponding control value (vehicle) by ANOVA followed by Tukey’s test.

    Article Snippet: Membranes were blocked with 5% low-fat dry milk, washed and incubated overnight at 4 °C with one of the following primary antibodies: TrkC (cat. no. 3376, Cell Signaling Technology) (1:1000), TrkC-T1 (cat no. 600-401-993, Rockland, Limerick, PA, USA) (1:1000), p75NTR (cat no. 8238, Cell Signaling Technology) (1:1000), Jun N-terminal kinase (JNK) (sc-571, Santa Cruz Biotechnology) (1:2000), phospho-JNK (Thr183/Tyr185) (cat. no. 9912, Cell Signaling Technology) (1:1000), phospho-c-Jun (Ser73) (cat. no. 3270, Cell Signaling Technology) (1:1000), c-Jun (cat. no. 9165, Cell Signaling Technology) (1:1000), phospho-Akt (Thr308) (cat. no. 2965, Cell Signaling Technology) (1:5000), Akt1/2/3 (sc-8312, Santa Cruz Biotechnology) (1:1000), extracellular signal-regulated kinases 1 and 2 (ERK1/2) (cat no. 9102, Cell Signaling Technology), phospho-ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187) (cat no. RA15002, Neuromics, Nothfield, MN, USA) (1:5000), phospho-MEK1/2 (Ser217/221) (cat no. 9154, Cell Signaling Technology) (1:1000), MEK1/2 (sc-81504, Santa Cruz Biotechnology) (1:1000), phospho-Elk-1 (Ser383) (cat. no. 9181, Cell Signaling Technology) (1:1000), Elk-1 (sc-365876, Santa Cruz Biotechnology) (1:1000), RUNX3/AML2 (RUNX3) (cat no. 9647, Cell Signaling Technology) (1:1000), RUNX1/AML1 (AML1) (cat. no. 4336, Cell Signaling Technology) (1:1000), phospho-AML1 (Ser249) (cat. no. 4327, Cell Signaling Technology) (1:1000), AP-2α (cat. 3215, Cell Signaling Technology) (1:1000), Egr1 (cat. no. 4154, Cell Signaling Technology) (1:1000), Sp1 (cat. no. 9389, Cell Signaling Technology) (1:1000), p53 (sc-6243, Santa Cruz Biotechnology) (1:500), HDAC1 (sc-81598, Santa Cruz Biotechnology) (1:2000), poly(ADP-ribose)polymerase (PARP) (cat. no. 9542, Cell Signaling Technology) (1:1000), pan-cadherin (cat no. 4073, Cell Signaling Technology) (1:2000); acetylated tubulin (cat. no. T7451, Sigma-Aldrich) (1:1000), α-tubulin (sc-5286, Santa Cruz Biotechnology) (1:1000), histone H3 (acetyl Lys9, Lys14) (cat no. GTX122648, GeneTex Inc., Irvine, CA, USA) (1:1000), histone H3 (cat. no. GTX 122148, GeneTex Inc.) (1:2000), actin (cat. no. A2066, Sigma-Aldrich) (1:2000), glyceradheyde-3-phosphate-dehydrogenase (GAPDH) (cat no. 247002, Synaptic Systems GmbH, Gottingen, Germany) (1:10,000).

    Techniques: Transfection, Incubation, Expressing

    a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).

    Journal: bioRxiv

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    doi: 10.1101/2020.08.24.265140

    Figure Lengend Snippet: a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).

    Article Snippet: Antibodies: rabbit anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), mouse anti-hs_H3 (Cell Signaling, #14269, clone 1B1B2, lot 6).

    Techniques: CRISPR, Labeling, Time-lapse Microscopy

    (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).

    Journal: bioRxiv

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    doi: 10.1101/2020.08.24.265140

    Figure Lengend Snippet: (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).

    Article Snippet: Antibodies: rabbit anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), mouse anti-hs_H3 (Cell Signaling, #14269, clone 1B1B2, lot 6).

    Techniques: Expressing, RNA Sequencing Assay, CRISPR, Western Blot, Time-lapse Microscopy

    (Related to ) a . Analysis of TFAP2A in the Dependency Map (DepMap, CRISPR (Avana) Public 19Q3) dataset reveals a dependence of melanoma proliferation on TFAP2A. b-c . High TFAP2A mRNA expression in primary tumors predicts worse (b) melanoma specific survival in patients in the Leeds Melanoma Cohort (HR [95% CI]: 1.6 [1.2, 2.1], p=0.001 upper vs. lower half by Cox proportional hazards and (c) progression free survival in patients in the AVAST-M Melanoma Cohort (multivariate Cox regression model). d-g . Primary tumors with high PRO or low INV expression are associated with worse outcomes in patients in (d-e) the Leeds Melanoma Cohort and (f-g) the AVAST-M Melanoma Cohort. h . Human melanoma samples from The Cancer Genome Atlas (TCGA) melanoma (SKCM, n=472) cohort are plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. i . Individual human melanoma cells are plotted as PRO and INV scores calculated from single-cell RNA-seq and colored according to TFAP2A mRNA expression (re-analyzed from Jerby-Arnon et al. ). Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. j-k . (j) S100A1 and (k) S100B mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p-values by Wilcoxon rank sum test with Bonferroni correction). l . Human melanoma cell lines ranked by cluster forming ability (left to right: low to high) demonstrate negative correlation between TFAP2A mRNA expression by RNA-seq and clustering (Spearman correlation shown). m . Western blot analysis of TFAP2A expression in panels of (left) low-passage human melanoma cell lines and (right) human melanoma cell lines.

    Journal: bioRxiv

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    doi: 10.1101/2020.08.24.265140

    Figure Lengend Snippet: (Related to ) a . Analysis of TFAP2A in the Dependency Map (DepMap, CRISPR (Avana) Public 19Q3) dataset reveals a dependence of melanoma proliferation on TFAP2A. b-c . High TFAP2A mRNA expression in primary tumors predicts worse (b) melanoma specific survival in patients in the Leeds Melanoma Cohort (HR [95% CI]: 1.6 [1.2, 2.1], p=0.001 upper vs. lower half by Cox proportional hazards and (c) progression free survival in patients in the AVAST-M Melanoma Cohort (multivariate Cox regression model). d-g . Primary tumors with high PRO or low INV expression are associated with worse outcomes in patients in (d-e) the Leeds Melanoma Cohort and (f-g) the AVAST-M Melanoma Cohort. h . Human melanoma samples from The Cancer Genome Atlas (TCGA) melanoma (SKCM, n=472) cohort are plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. i . Individual human melanoma cells are plotted as PRO and INV scores calculated from single-cell RNA-seq and colored according to TFAP2A mRNA expression (re-analyzed from Jerby-Arnon et al. ). Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. j-k . (j) S100A1 and (k) S100B mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p-values by Wilcoxon rank sum test with Bonferroni correction). l . Human melanoma cell lines ranked by cluster forming ability (left to right: low to high) demonstrate negative correlation between TFAP2A mRNA expression by RNA-seq and clustering (Spearman correlation shown). m . Western blot analysis of TFAP2A expression in panels of (left) low-passage human melanoma cell lines and (right) human melanoma cell lines.

    Article Snippet: Antibodies: rabbit anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), mouse anti-hs_H3 (Cell Signaling, #14269, clone 1B1B2, lot 6).

    Techniques: CRISPR, Expressing, RNA Sequencing Assay, Western Blot

    a . Human melanoma cell lines in the Cancer Cell Line Encyclopedia (CCLE, n=56) plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. b . TFAP2A mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p<0.001 by Wilcoxon rank sum test with Bonferroni correction). c . Low-passage human melanoma cell lines ranked by increased cluster forming ability (left to right) with TFAP2A expression quantified by immunofluorescence (plot and top; Spearman correlation shown; scale bar 20 μm) and clustering (bottom, scale bar 500 μm). d . GSAA was run using gene sets and GO gene sets with FDR < 0.05 from INV vs. PRO RNA-seq (n=39 gene sets; cyan points in and ). Bars show Normalized Association Score (NAS) for CRISPR (ZMEL1-PRO sg_ tfap2a/e vs. sg_scr) and INV vs. PRO for each gene set, with black outline representing FDR<0.05 for CRISPR experiment. e . Plot of Hoek et al. INV signature by GSAA for ZMEL1-PRO sg_ tfap2a/e vs. sg_scr RNA-seq. f . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). Asterisks (*) indicate genes with associated TFAP2A CUT&RUN peaks. Human ortholog gene names are used for clarity (see Figure S7a for zebrafish gene names).

    Journal: bioRxiv

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    doi: 10.1101/2020.08.24.265140

    Figure Lengend Snippet: a . Human melanoma cell lines in the Cancer Cell Line Encyclopedia (CCLE, n=56) plotted as PRO and INV scores calculated from RNA-seq and colored according to TFAP2A mRNA expression. Pearson correlation coefficients between TFAP2A and PRO/INV scores are shown on axes. b . TFAP2A mRNA expression in The Cancer Genome Atlas (TCGA) melanoma (SKCM) cohort comparing primary tumors and metastases (p<0.001 by Wilcoxon rank sum test with Bonferroni correction). c . Low-passage human melanoma cell lines ranked by increased cluster forming ability (left to right) with TFAP2A expression quantified by immunofluorescence (plot and top; Spearman correlation shown; scale bar 20 μm) and clustering (bottom, scale bar 500 μm). d . GSAA was run using gene sets and GO gene sets with FDR < 0.05 from INV vs. PRO RNA-seq (n=39 gene sets; cyan points in and ). Bars show Normalized Association Score (NAS) for CRISPR (ZMEL1-PRO sg_ tfap2a/e vs. sg_scr) and INV vs. PRO for each gene set, with black outline representing FDR<0.05 for CRISPR experiment. e . Plot of Hoek et al. INV signature by GSAA for ZMEL1-PRO sg_ tfap2a/e vs. sg_scr RNA-seq. f . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). Asterisks (*) indicate genes with associated TFAP2A CUT&RUN peaks. Human ortholog gene names are used for clarity (see Figure S7a for zebrafish gene names).

    Article Snippet: Antibodies: rabbit anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), mouse anti-hs_H3 (Cell Signaling, #14269, clone 1B1B2, lot 6).

    Techniques: RNA Sequencing Assay, Expressing, Immunofluorescence, CRISPR

    (Related to ) a . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). As in , but with zebrafish gene names. b . Distribution of TFAP2A CUT&RUN peaks as annotated by ChIPSeeker. c-d . Overlap of TFAP2A CUT&RUN peaks with genes upregulated in ZMEL1-PRO following CRISPR/Cas9 with (c) sg_scr (p=0.7 by bootstrapping) and (d) sg_ tfap2a/e (p<0.001 by bootstrapping).

    Journal: bioRxiv

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    doi: 10.1101/2020.08.24.265140

    Figure Lengend Snippet: (Related to ) a . Heatmap of top genes in Hoek INV and GO Adhesion gene sets that are differentially expressed between ZMEL1-PRO sg_ tfap2a/e and sg_scr (log 2 fold change cutoff ± 0.5, p adj < 0.05). As in , but with zebrafish gene names. b . Distribution of TFAP2A CUT&RUN peaks as annotated by ChIPSeeker. c-d . Overlap of TFAP2A CUT&RUN peaks with genes upregulated in ZMEL1-PRO following CRISPR/Cas9 with (c) sg_scr (p=0.7 by bootstrapping) and (d) sg_ tfap2a/e (p<0.001 by bootstrapping).

    Article Snippet: Antibodies: rabbit anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), mouse anti-hs_H3 (Cell Signaling, #14269, clone 1B1B2, lot 6).

    Techniques: CRISPR

    a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).

    Journal: bioRxiv

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    doi: 10.1101/2020.08.24.265140

    Figure Lengend Snippet: a . HOMER de-novo motif analysis on genes upregulated in ZMEL1-PRO vs. -INV (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-INV in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of TSS). c . Cluster size after 2 days in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a and tfap2e alone or in combination (sg_ tfap2a/e ) versus control (sg_scr) (p-values by linear regression; N=3 independent experiments). d . Representative images of clusters formed after 2 days from ZMEL1-PRO with sg_scr vs. sg_ tfap2a/e . e . Growth of ZMEL1-PRO orthotopic primary tumors with sg_scr vs. sg_ tfap2a/e (p=0.011 by linear regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total). f . Representative image of extravasated (arrows) and partially extravasated (arrow-head) ZMEL1-PRO cells with sg_ tfap2a / e following intravenous transplant in casper fish with FLK-RFP transgene labeling the vasculature. g . Proportion of larval fish intravenously transplanted with ZMEL1-PRO with sg_scr or sg_ tfap2a/e with extravasated cells at 1 dpt, as quantified from confocal time lapse microscopy (OR [95% CI]: 2.20 [1.05, 4.61]; p=0.038 by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 20/20, 22/23, and 22/22 fish per group, respectively; n=129 fish total).

    Article Snippet: Antibodies: anti-hs_CDH1 (BD #610181, lot 8082613), anti-dr_Tfap2a (LifeSpan Biosciences, #LS-C87212, log 113877), anti-dr_Tfap2e (Fisher, #PA5-72631, lot UA2709682A), anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), anti-hs_cyclophilin B (Fisher #PA1-027A, lot SD248938).

    Techniques: CRISPR, Labeling, Time-lapse Microscopy

    (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).

    Journal: bioRxiv

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    doi: 10.1101/2020.08.24.265140

    Figure Lengend Snippet: (Related to ) a . Boxplots of tfap2a-e expression from RNA-seq of ZMEL1-PRO and -INV. b . HOMER de-novo motif analysis of genes differentially expressed between ZMEL1-PRO in 3D (clusters) vs. 2D (no clusters) (log 2 fold change cutoff ± 1.5, p adj < 0.05, ±500bp of transcription start site [TSS]). c . Cluster size in ZMEL1-PRO with CRISPR-Cas9 inactivation of tfap2a or tfap2e alone and in combination (p-values by linear regression; N=3 independent experiments). sgRNAs highlighted in purple (sg_scr) and orange (sg_ tfap2a/e 1/3) were used for further experiments ( and , ). d-e . Western blot confirmation of CRISPR inactivation of (d) tfap2a and (e) tfap2e with each sgRNA or combination of sgRNAs. f-i . Tracking of individual cells by time-lapse microscopy (N=3 independent experiments). f . ZMEL1-PRO with sg_ tfap2a/e has slowed growth versus sg_scr (p<0.001 by linear regression, model estimates ± 95% CI shown). g . Representative displacements of 500 tracks per sgRNA. h . Model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO sg_ tfap2a/e and sg_scr (p<0.001 by linear regression). Larger alpha indicates more persistent motion. i . Log-log plot of mean squared displacement (MSD) vs. lag time (tau) over the range of 5≤tau<100 minutes with model fits overlaid (see Methods for details). The slope (α) provides quantification of the persistence of motility, where a cell moving randomly will have α=1, and a cell moving along a straight line will have α=2 . Black line with α=1 is shown for comparison. j-k . ZMEL1-PRO orthotopic primary tumors with sg_ tfap2a/e do not seed (j) distant metastases and (k) caudal metastases in a significantly different proportion of zebrafish than with sg_scr control (p=0.44 and p=0.90, respectively, at 7 dpt by logistic regression; N=3 independent experiments with sg_scr/sg_tfap2a/e 24/22, 22/22, 24/24 fish per group, respectively; n=138 fish total).

    Article Snippet: Antibodies: anti-hs_CDH1 (BD #610181, lot 8082613), anti-dr_Tfap2a (LifeSpan Biosciences, #LS-C87212, log 113877), anti-dr_Tfap2e (Fisher, #PA5-72631, lot UA2709682A), anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), anti-hs_cyclophilin B (Fisher #PA1-027A, lot SD248938).

    Techniques: Expressing, RNA Sequencing Assay, CRISPR, Western Blot, Time-lapse Microscopy

    a . Schematic of experiment. Prior to flow cytometry and 10X single-cell RNA-seq, ZMEL1-PRO and -INV cells were either grown in vitro (individual or co-culture) or isolated from zebrafish orthotopically transplanted with a 1:1 mixture of the two subpopulations (primary tumors or metastases). b . Relative number of ZMEL1-PRO and -INV cells isolated and quantified by flow cytometry from primary tumors and metastases of fish transplanted with a 1:1 mixture of ZMEL1-PRO and -INV (primary tumors from n=6 fish; metastases from n=4 fish; p= 0.51 and p=0.031, respectively, by one-sample two-sided t-test with Bonferroni correction). c . Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction of 40,293 ZMEL1 cells sequenced as in (a). Individual culture (IND); co-culture (CO); primary tumors (PRI); metastases (MET). d . PRO and INV scores based on ZMEL1 bulk RNA-seq are plotted for all cells in gray. ZMEL1-PRO (purple) and ZMEL1-INV (green) for the indicated condition are colored. Diagonal line represents the classifier used in (e). e . Confusion matrices comparing initial cell identity with observed cell classification based on a linear classifier trained on in vitro individual culture samples. f . ZMEL1-INV cells plotted as in (d) colored according to tfap2e mRNA expression reveal re-activation of tfap2e upon metastatic dissemination.

    Journal: bioRxiv

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    doi: 10.1101/2020.08.24.265140

    Figure Lengend Snippet: a . Schematic of experiment. Prior to flow cytometry and 10X single-cell RNA-seq, ZMEL1-PRO and -INV cells were either grown in vitro (individual or co-culture) or isolated from zebrafish orthotopically transplanted with a 1:1 mixture of the two subpopulations (primary tumors or metastases). b . Relative number of ZMEL1-PRO and -INV cells isolated and quantified by flow cytometry from primary tumors and metastases of fish transplanted with a 1:1 mixture of ZMEL1-PRO and -INV (primary tumors from n=6 fish; metastases from n=4 fish; p= 0.51 and p=0.031, respectively, by one-sample two-sided t-test with Bonferroni correction). c . Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction of 40,293 ZMEL1 cells sequenced as in (a). Individual culture (IND); co-culture (CO); primary tumors (PRI); metastases (MET). d . PRO and INV scores based on ZMEL1 bulk RNA-seq are plotted for all cells in gray. ZMEL1-PRO (purple) and ZMEL1-INV (green) for the indicated condition are colored. Diagonal line represents the classifier used in (e). e . Confusion matrices comparing initial cell identity with observed cell classification based on a linear classifier trained on in vitro individual culture samples. f . ZMEL1-INV cells plotted as in (d) colored according to tfap2e mRNA expression reveal re-activation of tfap2e upon metastatic dissemination.

    Article Snippet: Antibodies: anti-hs_CDH1 (BD #610181, lot 8082613), anti-dr_Tfap2a (LifeSpan Biosciences, #LS-C87212, log 113877), anti-dr_Tfap2e (Fisher, #PA5-72631, lot UA2709682A), anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), anti-hs_cyclophilin B (Fisher #PA1-027A, lot SD248938).

    Techniques: Flow Cytometry, RNA Sequencing Assay, In Vitro, Co-Culture Assay, Isolation, Expressing, Activation Assay

    (Related to ) a . Single-cell expression of tfap2e in ZMEL1-PRO and -INV cells. Individual culture (IND); co-culture (CO); primary tumors (PRI); metastases (MET).

    Journal: bioRxiv

    Article Title: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma

    doi: 10.1101/2020.08.24.265140

    Figure Lengend Snippet: (Related to ) a . Single-cell expression of tfap2e in ZMEL1-PRO and -INV cells. Individual culture (IND); co-culture (CO); primary tumors (PRI); metastases (MET).

    Article Snippet: Antibodies: anti-hs_CDH1 (BD #610181, lot 8082613), anti-dr_Tfap2a (LifeSpan Biosciences, #LS-C87212, log 113877), anti-dr_Tfap2e (Fisher, #PA5-72631, lot UA2709682A), anti-hs_TFAP2A (Cell Signaling, #3215, clone C83E10, lot 2), anti-hs_cyclophilin B (Fisher #PA1-027A, lot SD248938).

    Techniques: Expressing, Co-Culture Assay

    Ap-2α transcriptionally promotes Elk-1 expression. a A schematic depiction of different mouse Elk-1 promoter regions that were cloned into the pGL4-basic vector. The constructs were designed as pE-1~8 with different lengths, as indicated. b Effects of MC-38 conditioned medium on the promoter activity of the different Elk-1 promoter constructs described in ( a ). Raw cells were transfected with the truncated reporter constructs, stimulated with MC-38 conditioned medium or control normal medium for 24 h, and then harvested for assays assessing luciferase activity. The results are expressed as the relative activity normalized to the activity of each control medium-treated group, which was arbitrarily defined as 1 ( n = 3). c Mutation of a potential Ap-2α binding site in the reporter construct pE-1. The sequence GCCTGC located at −1396/−1391 was mutated to GACTAC in the mutant reporter construct pE-1-mut. d Effects of Ap-2α on the activity of the Elk-1 promoter constructs pE-1 and pE-1-mut. RAW cells were cotransfected with pE-1 or Mut-1 plus a scrambled shRNA (sh-con) or an Ap-2α-specific shRNA (sh-Ap2α) for 24 h and then harvested for luciferase activity assays ( n = 3). e Binding activity between Ap-2α and Elk-1 DNA in normal medium- or MC-38 conditioned medium-stimulated macrophages, as measured by ChIP assays ( n = 4). f , g Stimulation of the Ap-2α-dependent mRNA expression of Elk-1 and Sirpα by MC-38 cells. RAW cells were transfected with sh-con or sh-Ap2α for 12 h and then treated with MC-38 conditioned medium or control medium for 24 h. Cells were harvested for real-time PCR analysis of Elk-1 ( f ) and Sirpα ( g ). The data in ( b , d – g ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    doi: 10.1038/s41392-020-0124-z

    Figure Lengend Snippet: Ap-2α transcriptionally promotes Elk-1 expression. a A schematic depiction of different mouse Elk-1 promoter regions that were cloned into the pGL4-basic vector. The constructs were designed as pE-1~8 with different lengths, as indicated. b Effects of MC-38 conditioned medium on the promoter activity of the different Elk-1 promoter constructs described in ( a ). Raw cells were transfected with the truncated reporter constructs, stimulated with MC-38 conditioned medium or control normal medium for 24 h, and then harvested for assays assessing luciferase activity. The results are expressed as the relative activity normalized to the activity of each control medium-treated group, which was arbitrarily defined as 1 ( n = 3). c Mutation of a potential Ap-2α binding site in the reporter construct pE-1. The sequence GCCTGC located at −1396/−1391 was mutated to GACTAC in the mutant reporter construct pE-1-mut. d Effects of Ap-2α on the activity of the Elk-1 promoter constructs pE-1 and pE-1-mut. RAW cells were cotransfected with pE-1 or Mut-1 plus a scrambled shRNA (sh-con) or an Ap-2α-specific shRNA (sh-Ap2α) for 24 h and then harvested for luciferase activity assays ( n = 3). e Binding activity between Ap-2α and Elk-1 DNA in normal medium- or MC-38 conditioned medium-stimulated macrophages, as measured by ChIP assays ( n = 4). f , g Stimulation of the Ap-2α-dependent mRNA expression of Elk-1 and Sirpα by MC-38 cells. RAW cells were transfected with sh-con or sh-Ap2α for 12 h and then treated with MC-38 conditioned medium or control medium for 24 h. Cells were harvested for real-time PCR analysis of Elk-1 ( f ) and Sirpα ( g ). The data in ( b , d – g ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Article Snippet: The primary antibodies included anti-Ap-2α (#3215, Cell Signaling; the dilution ratio was 1:1000), anti-Elk-1 (#9182, Cell Signaling; the dilution ratio was 1:1000), anti-Sirpα (#13379, Cell Signaling; the dilution ratio was 1:1000) and anti-GAPDH (#5174, Cell Signaling; the dilution ratio was 1:2000) antibodies.

    Techniques: Expressing, Clone Assay, Plasmid Preparation, Construct, Activity Assay, Transfection, Luciferase, Mutagenesis, Binding Assay, Sequencing, shRNA, Real-time Polymerase Chain Reaction

    Macrophage Ap2α-Elk-1 axis regulates CRC progression. a MC-38 cells (1.0 × 10 6 cells per mouse) were subcutaneously inoculated into fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice, and tumor volume was monitored dynamically ( n = 10). b The survival of the mice described above in ( a ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). c The Elk-1 transgene prevented Ap-2α silencing-induced tumor phagocytosis. Six-week-old male fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice were subcutaneously engrafted with GFP-tagged MC-38 cells (1.0 × 10 6 cells per mouse). Two weeks later, tumors were recovered for the measurement of tumor phagocytosis by TAMs ( n = 3). d Volumes of tumors in WT mice inoculated with MC-38 cells mixed 2:1 with macrophages isolated from corresponding tumors grown in fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 or Ap2α-cKO + Tg Elk-1 mice are shown ( n = 10). e The survival of the mice described above in ( d ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). The data in ( a , c , d ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    doi: 10.1038/s41392-020-0124-z

    Figure Lengend Snippet: Macrophage Ap2α-Elk-1 axis regulates CRC progression. a MC-38 cells (1.0 × 10 6 cells per mouse) were subcutaneously inoculated into fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice, and tumor volume was monitored dynamically ( n = 10). b The survival of the mice described above in ( a ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). c The Elk-1 transgene prevented Ap-2α silencing-induced tumor phagocytosis. Six-week-old male fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice were subcutaneously engrafted with GFP-tagged MC-38 cells (1.0 × 10 6 cells per mouse). Two weeks later, tumors were recovered for the measurement of tumor phagocytosis by TAMs ( n = 3). d Volumes of tumors in WT mice inoculated with MC-38 cells mixed 2:1 with macrophages isolated from corresponding tumors grown in fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 or Ap2α-cKO + Tg Elk-1 mice are shown ( n = 10). e The survival of the mice described above in ( d ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). The data in ( a , c , d ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Article Snippet: The primary antibodies included anti-Ap-2α (#3215, Cell Signaling; the dilution ratio was 1:1000), anti-Elk-1 (#9182, Cell Signaling; the dilution ratio was 1:1000), anti-Sirpα (#13379, Cell Signaling; the dilution ratio was 1:1000) and anti-GAPDH (#5174, Cell Signaling; the dilution ratio was 1:2000) antibodies.

    Techniques: Isolation

    Lactate induces TAM Ap-2α activity and CRC progression. a Nuclear translocation of Ap-2α in TAMs was evaluated. Six-week-old C57BL/6 mice were subcutaneously inoculated with MC-38 cells (1.0 × 10 6 cells per mouse). TAMs isolated at different time points were stained with anti-Ap-2α antibodies (green). Nuclei were visualized by DAPI staining (blue). The total Ap-2α protein in TAMs at different time points was measured by western blotting assays. The nuclear translocation rates of Ap-2α were calculated ( n = 4). b Metabolic factors induced the nuclear translocation of Ap-2α in macrophages. MC-38 conditioned medium was roughly fractionated into a protein fraction (CM-1, >3 kD) and a metabolic fraction (CM-2, <3 kD) by molecular size. Bone marrow-derived macrophages were treated with CM-1, CM-2 or control normal medium for 24 h and then stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). c Lactate stimulated the nuclear translocation of Ap-2α in macrophages. Bone marrow-derived macrophages were treated with lactate (10 mM) or PBS as a control for 24 h. Then, cells were stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). d , e The mRNA levels of Elk-1 ( d ) and Sirpα ( e ) in macrophages treated with lactate (10 mM) or PBS for 24 h were measured ( n = 4). f The phagocytosis rates of macrophages treated with lactate (10 mM) or PBS for 24 h were evaluated ( n = 3). g Six-week-old male C57BL/6 mice were subcutaneously engrafted with MC-38 cells (1.0 × 10 6 cells per mouse) and then subcutaneously treated with lactate (50 μmol in 100 μl of PBS per injection) or PBS on days 1, 3, and 5. Tumor volume was measured dynamically ( n = 10). h The survival of the mice described in ( g ) is shown ( n = 10, *** P < 0.005; Gehan–Breslow–Wilcoxon test). The data in ( a – g ) are reported as the mean ± s.e.m. (*** P < 0.005; Student’s t -test)

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    doi: 10.1038/s41392-020-0124-z

    Figure Lengend Snippet: Lactate induces TAM Ap-2α activity and CRC progression. a Nuclear translocation of Ap-2α in TAMs was evaluated. Six-week-old C57BL/6 mice were subcutaneously inoculated with MC-38 cells (1.0 × 10 6 cells per mouse). TAMs isolated at different time points were stained with anti-Ap-2α antibodies (green). Nuclei were visualized by DAPI staining (blue). The total Ap-2α protein in TAMs at different time points was measured by western blotting assays. The nuclear translocation rates of Ap-2α were calculated ( n = 4). b Metabolic factors induced the nuclear translocation of Ap-2α in macrophages. MC-38 conditioned medium was roughly fractionated into a protein fraction (CM-1, >3 kD) and a metabolic fraction (CM-2, <3 kD) by molecular size. Bone marrow-derived macrophages were treated with CM-1, CM-2 or control normal medium for 24 h and then stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). c Lactate stimulated the nuclear translocation of Ap-2α in macrophages. Bone marrow-derived macrophages were treated with lactate (10 mM) or PBS as a control for 24 h. Then, cells were stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). d , e The mRNA levels of Elk-1 ( d ) and Sirpα ( e ) in macrophages treated with lactate (10 mM) or PBS for 24 h were measured ( n = 4). f The phagocytosis rates of macrophages treated with lactate (10 mM) or PBS for 24 h were evaluated ( n = 3). g Six-week-old male C57BL/6 mice were subcutaneously engrafted with MC-38 cells (1.0 × 10 6 cells per mouse) and then subcutaneously treated with lactate (50 μmol in 100 μl of PBS per injection) or PBS on days 1, 3, and 5. Tumor volume was measured dynamically ( n = 10). h The survival of the mice described in ( g ) is shown ( n = 10, *** P < 0.005; Gehan–Breslow–Wilcoxon test). The data in ( a – g ) are reported as the mean ± s.e.m. (*** P < 0.005; Student’s t -test)

    Article Snippet: The primary antibodies included anti-Ap-2α (#3215, Cell Signaling; the dilution ratio was 1:1000), anti-Elk-1 (#9182, Cell Signaling; the dilution ratio was 1:1000), anti-Sirpα (#13379, Cell Signaling; the dilution ratio was 1:1000) and anti-GAPDH (#5174, Cell Signaling; the dilution ratio was 1:2000) antibodies.

    Techniques: Activity Assay, Translocation Assay, Isolation, Staining, Western Blot, Derivative Assay, Injection

    Macrophage Elk-1 and Sirpα levels increase with CRC progression. a – c mRNA levels of Ap-2α ( a ), Elk-1 ( b ) and Sirpα ( c ) in macrophages from adjacent normal and carcinoma tissue samples from CRC patients ( n = 15). d Positive correlation of S irpα expression with Elk-1 expression in macrophages from each individual sample. Linear regression was performed ( n = 42, P < 0.05, Pearson R = 0.377). e , f mRNA levels of Elk-1 and Sirpα in TAMs from CRC patients with different TNM stages. Samples were divided into 3 groups according to the TNM stage: T2-4N0M0 ( n = 15), T2-4N1-2M0 ( n = 15) and T2-4N0-2M1 ( n = 12). g – i Overall survival of CRC patients with differential expression of AP-2α ( g ), ELK-1 ( h ) and SIRPα ( i ). Patients were placed into two groups according to the mRNA expression of AP-2α, ELK-1 or SIRPα in TAMs. Patients with expression levels in the top 50% for TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α hi , ELK-1 hi or SIRPα hi groups, respectively. The remaining patients with low TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α lo , ELK-1 lo or SIRPα lo groups, respectively. The overall survival time of patients was determined during follow-up visits ( n = 58, * P < 0.05, *** P < 0.005, Gehan–Breslow–Wilcoxon test). The data in ( a – c and e , f ) represent the mean ± s.e.m. (* P < 0.05, ** P < 0.01 and *** P < 0.005; Student’s t -test)

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    doi: 10.1038/s41392-020-0124-z

    Figure Lengend Snippet: Macrophage Elk-1 and Sirpα levels increase with CRC progression. a – c mRNA levels of Ap-2α ( a ), Elk-1 ( b ) and Sirpα ( c ) in macrophages from adjacent normal and carcinoma tissue samples from CRC patients ( n = 15). d Positive correlation of S irpα expression with Elk-1 expression in macrophages from each individual sample. Linear regression was performed ( n = 42, P < 0.05, Pearson R = 0.377). e , f mRNA levels of Elk-1 and Sirpα in TAMs from CRC patients with different TNM stages. Samples were divided into 3 groups according to the TNM stage: T2-4N0M0 ( n = 15), T2-4N1-2M0 ( n = 15) and T2-4N0-2M1 ( n = 12). g – i Overall survival of CRC patients with differential expression of AP-2α ( g ), ELK-1 ( h ) and SIRPα ( i ). Patients were placed into two groups according to the mRNA expression of AP-2α, ELK-1 or SIRPα in TAMs. Patients with expression levels in the top 50% for TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α hi , ELK-1 hi or SIRPα hi groups, respectively. The remaining patients with low TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α lo , ELK-1 lo or SIRPα lo groups, respectively. The overall survival time of patients was determined during follow-up visits ( n = 58, * P < 0.05, *** P < 0.005, Gehan–Breslow–Wilcoxon test). The data in ( a – c and e , f ) represent the mean ± s.e.m. (* P < 0.05, ** P < 0.01 and *** P < 0.005; Student’s t -test)

    Article Snippet: The primary antibodies included anti-Ap-2α (#3215, Cell Signaling; the dilution ratio was 1:1000), anti-Elk-1 (#9182, Cell Signaling; the dilution ratio was 1:1000), anti-Sirpα (#13379, Cell Signaling; the dilution ratio was 1:1000) and anti-GAPDH (#5174, Cell Signaling; the dilution ratio was 1:2000) antibodies.

    Techniques: Expressing

    The working model for CRC evasion of innate immune surveillance. Without a cancer challenge, the interaction between macrophage SIRPα and CRC CD47 is interrupted, and macrophages exhibit normal phagocytic function. In the CRC microenvironment, CRC-derived lactate induces the activity of the transcription factor Ap-2α and the expression of its target Elk-1, which further promotes the expression of Sirpα in TAMs. The enhanced interaction between macrophage SIRPα and CRC CD47 reduces the phagocytic function of TAMs

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    doi: 10.1038/s41392-020-0124-z

    Figure Lengend Snippet: The working model for CRC evasion of innate immune surveillance. Without a cancer challenge, the interaction between macrophage SIRPα and CRC CD47 is interrupted, and macrophages exhibit normal phagocytic function. In the CRC microenvironment, CRC-derived lactate induces the activity of the transcription factor Ap-2α and the expression of its target Elk-1, which further promotes the expression of Sirpα in TAMs. The enhanced interaction between macrophage SIRPα and CRC CD47 reduces the phagocytic function of TAMs

    Article Snippet: The primary antibodies included anti-Ap-2α (#3215, Cell Signaling; the dilution ratio was 1:1000), anti-Elk-1 (#9182, Cell Signaling; the dilution ratio was 1:1000), anti-Sirpα (#13379, Cell Signaling; the dilution ratio was 1:1000) and anti-GAPDH (#5174, Cell Signaling; the dilution ratio was 1:2000) antibodies.

    Techniques: Derivative Assay, Activity Assay, Expressing

    Ap-2α transcriptionally promotes Elk-1 expression. a A schematic depiction of different mouse Elk-1 promoter regions that were cloned into the pGL4-basic vector. The constructs were designed as pE-1~8 with different lengths, as indicated. b Effects of MC-38 conditioned medium on the promoter activity of the different Elk-1 promoter constructs described in ( a ). Raw cells were transfected with the truncated reporter constructs, stimulated with MC-38 conditioned medium or control normal medium for 24 h, and then harvested for assays assessing luciferase activity. The results are expressed as the relative activity normalized to the activity of each control medium-treated group, which was arbitrarily defined as 1 ( n = 3). c Mutation of a potential Ap-2α binding site in the reporter construct pE-1. The sequence GCCTGC located at −1396/−1391 was mutated to GACTAC in the mutant reporter construct pE-1-mut. d Effects of Ap-2α on the activity of the Elk-1 promoter constructs pE-1 and pE-1-mut. RAW cells were cotransfected with pE-1 or Mut-1 plus a scrambled shRNA (sh-con) or an Ap-2α-specific shRNA (sh-Ap2α) for 24 h and then harvested for luciferase activity assays ( n = 3). e Binding activity between Ap-2α and Elk-1 DNA in normal medium- or MC-38 conditioned medium-stimulated macrophages, as measured by ChIP assays ( n = 4). f , g Stimulation of the Ap-2α-dependent mRNA expression of Elk-1 and Sirpα by MC-38 cells. RAW cells were transfected with sh-con or sh-Ap2α for 12 h and then treated with MC-38 conditioned medium or control medium for 24 h. Cells were harvested for real-time PCR analysis of Elk-1 ( f ) and Sirpα ( g ). The data in ( b , d – g ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    doi: 10.1038/s41392-020-0124-z

    Figure Lengend Snippet: Ap-2α transcriptionally promotes Elk-1 expression. a A schematic depiction of different mouse Elk-1 promoter regions that were cloned into the pGL4-basic vector. The constructs were designed as pE-1~8 with different lengths, as indicated. b Effects of MC-38 conditioned medium on the promoter activity of the different Elk-1 promoter constructs described in ( a ). Raw cells were transfected with the truncated reporter constructs, stimulated with MC-38 conditioned medium or control normal medium for 24 h, and then harvested for assays assessing luciferase activity. The results are expressed as the relative activity normalized to the activity of each control medium-treated group, which was arbitrarily defined as 1 ( n = 3). c Mutation of a potential Ap-2α binding site in the reporter construct pE-1. The sequence GCCTGC located at −1396/−1391 was mutated to GACTAC in the mutant reporter construct pE-1-mut. d Effects of Ap-2α on the activity of the Elk-1 promoter constructs pE-1 and pE-1-mut. RAW cells were cotransfected with pE-1 or Mut-1 plus a scrambled shRNA (sh-con) or an Ap-2α-specific shRNA (sh-Ap2α) for 24 h and then harvested for luciferase activity assays ( n = 3). e Binding activity between Ap-2α and Elk-1 DNA in normal medium- or MC-38 conditioned medium-stimulated macrophages, as measured by ChIP assays ( n = 4). f , g Stimulation of the Ap-2α-dependent mRNA expression of Elk-1 and Sirpα by MC-38 cells. RAW cells were transfected with sh-con or sh-Ap2α for 12 h and then treated with MC-38 conditioned medium or control medium for 24 h. Cells were harvested for real-time PCR analysis of Elk-1 ( f ) and Sirpα ( g ). The data in ( b , d – g ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Article Snippet: The cells on coverslips were stained with an anti-Ap-2α antibody (#3215, Cell Signaling, the dilution ratio was 1:500) at 37 °C for 1 h and then 4 °C overnight.

    Techniques: Expressing, Clone Assay, Plasmid Preparation, Construct, Activity Assay, Transfection, Luciferase, Mutagenesis, Binding Assay, Sequencing, shRNA, Real-time Polymerase Chain Reaction

    Macrophage Ap2α-Elk-1 axis regulates CRC progression. a MC-38 cells (1.0 × 10 6 cells per mouse) were subcutaneously inoculated into fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice, and tumor volume was monitored dynamically ( n = 10). b The survival of the mice described above in ( a ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). c The Elk-1 transgene prevented Ap-2α silencing-induced tumor phagocytosis. Six-week-old male fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice were subcutaneously engrafted with GFP-tagged MC-38 cells (1.0 × 10 6 cells per mouse). Two weeks later, tumors were recovered for the measurement of tumor phagocytosis by TAMs ( n = 3). d Volumes of tumors in WT mice inoculated with MC-38 cells mixed 2:1 with macrophages isolated from corresponding tumors grown in fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 or Ap2α-cKO + Tg Elk-1 mice are shown ( n = 10). e The survival of the mice described above in ( d ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). The data in ( a , c , d ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    doi: 10.1038/s41392-020-0124-z

    Figure Lengend Snippet: Macrophage Ap2α-Elk-1 axis regulates CRC progression. a MC-38 cells (1.0 × 10 6 cells per mouse) were subcutaneously inoculated into fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice, and tumor volume was monitored dynamically ( n = 10). b The survival of the mice described above in ( a ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). c The Elk-1 transgene prevented Ap-2α silencing-induced tumor phagocytosis. Six-week-old male fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 , and Ap2α-cKO + Tg Elk-1 mice were subcutaneously engrafted with GFP-tagged MC-38 cells (1.0 × 10 6 cells per mouse). Two weeks later, tumors were recovered for the measurement of tumor phagocytosis by TAMs ( n = 3). d Volumes of tumors in WT mice inoculated with MC-38 cells mixed 2:1 with macrophages isolated from corresponding tumors grown in fl/fl, Ap2α-cKO, fl/fl + Tg Elk-1 or Ap2α-cKO + Tg Elk-1 mice are shown ( n = 10). e The survival of the mice described above in ( d ) is shown ( n = 10, *** P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). The data in ( a , c , d ) are shown as the mean ± s.e.m. (** P < 0.01, *** P < 0.005; ns, not significant; Student’s t -test)

    Article Snippet: The cells on coverslips were stained with an anti-Ap-2α antibody (#3215, Cell Signaling, the dilution ratio was 1:500) at 37 °C for 1 h and then 4 °C overnight.

    Techniques: Isolation

    Lactate induces TAM Ap-2α activity and CRC progression. a Nuclear translocation of Ap-2α in TAMs was evaluated. Six-week-old C57BL/6 mice were subcutaneously inoculated with MC-38 cells (1.0 × 10 6 cells per mouse). TAMs isolated at different time points were stained with anti-Ap-2α antibodies (green). Nuclei were visualized by DAPI staining (blue). The total Ap-2α protein in TAMs at different time points was measured by western blotting assays. The nuclear translocation rates of Ap-2α were calculated ( n = 4). b Metabolic factors induced the nuclear translocation of Ap-2α in macrophages. MC-38 conditioned medium was roughly fractionated into a protein fraction (CM-1, >3 kD) and a metabolic fraction (CM-2, <3 kD) by molecular size. Bone marrow-derived macrophages were treated with CM-1, CM-2 or control normal medium for 24 h and then stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). c Lactate stimulated the nuclear translocation of Ap-2α in macrophages. Bone marrow-derived macrophages were treated with lactate (10 mM) or PBS as a control for 24 h. Then, cells were stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). d , e The mRNA levels of Elk-1 ( d ) and Sirpα ( e ) in macrophages treated with lactate (10 mM) or PBS for 24 h were measured ( n = 4). f The phagocytosis rates of macrophages treated with lactate (10 mM) or PBS for 24 h were evaluated ( n = 3). g Six-week-old male C57BL/6 mice were subcutaneously engrafted with MC-38 cells (1.0 × 10 6 cells per mouse) and then subcutaneously treated with lactate (50 μmol in 100 μl of PBS per injection) or PBS on days 1, 3, and 5. Tumor volume was measured dynamically ( n = 10). h The survival of the mice described in ( g ) is shown ( n = 10, *** P < 0.005; Gehan–Breslow–Wilcoxon test). The data in ( a – g ) are reported as the mean ± s.e.m. (*** P < 0.005; Student’s t -test)

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    doi: 10.1038/s41392-020-0124-z

    Figure Lengend Snippet: Lactate induces TAM Ap-2α activity and CRC progression. a Nuclear translocation of Ap-2α in TAMs was evaluated. Six-week-old C57BL/6 mice were subcutaneously inoculated with MC-38 cells (1.0 × 10 6 cells per mouse). TAMs isolated at different time points were stained with anti-Ap-2α antibodies (green). Nuclei were visualized by DAPI staining (blue). The total Ap-2α protein in TAMs at different time points was measured by western blotting assays. The nuclear translocation rates of Ap-2α were calculated ( n = 4). b Metabolic factors induced the nuclear translocation of Ap-2α in macrophages. MC-38 conditioned medium was roughly fractionated into a protein fraction (CM-1, >3 kD) and a metabolic fraction (CM-2, <3 kD) by molecular size. Bone marrow-derived macrophages were treated with CM-1, CM-2 or control normal medium for 24 h and then stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). c Lactate stimulated the nuclear translocation of Ap-2α in macrophages. Bone marrow-derived macrophages were treated with lactate (10 mM) or PBS as a control for 24 h. Then, cells were stained with anti-Ap-2α antibodies as described in ( a ) ( n = 3). d , e The mRNA levels of Elk-1 ( d ) and Sirpα ( e ) in macrophages treated with lactate (10 mM) or PBS for 24 h were measured ( n = 4). f The phagocytosis rates of macrophages treated with lactate (10 mM) or PBS for 24 h were evaluated ( n = 3). g Six-week-old male C57BL/6 mice were subcutaneously engrafted with MC-38 cells (1.0 × 10 6 cells per mouse) and then subcutaneously treated with lactate (50 μmol in 100 μl of PBS per injection) or PBS on days 1, 3, and 5. Tumor volume was measured dynamically ( n = 10). h The survival of the mice described in ( g ) is shown ( n = 10, *** P < 0.005; Gehan–Breslow–Wilcoxon test). The data in ( a – g ) are reported as the mean ± s.e.m. (*** P < 0.005; Student’s t -test)

    Article Snippet: The cells on coverslips were stained with an anti-Ap-2α antibody (#3215, Cell Signaling, the dilution ratio was 1:500) at 37 °C for 1 h and then 4 °C overnight.

    Techniques: Activity Assay, Translocation Assay, Isolation, Staining, Western Blot, Derivative Assay, Injection

    Macrophage Elk-1 and Sirpα levels increase with CRC progression. a – c mRNA levels of Ap-2α ( a ), Elk-1 ( b ) and Sirpα ( c ) in macrophages from adjacent normal and carcinoma tissue samples from CRC patients ( n = 15). d Positive correlation of S irpα expression with Elk-1 expression in macrophages from each individual sample. Linear regression was performed ( n = 42, P < 0.05, Pearson R = 0.377). e , f mRNA levels of Elk-1 and Sirpα in TAMs from CRC patients with different TNM stages. Samples were divided into 3 groups according to the TNM stage: T2-4N0M0 ( n = 15), T2-4N1-2M0 ( n = 15) and T2-4N0-2M1 ( n = 12). g – i Overall survival of CRC patients with differential expression of AP-2α ( g ), ELK-1 ( h ) and SIRPα ( i ). Patients were placed into two groups according to the mRNA expression of AP-2α, ELK-1 or SIRPα in TAMs. Patients with expression levels in the top 50% for TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α hi , ELK-1 hi or SIRPα hi groups, respectively. The remaining patients with low TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α lo , ELK-1 lo or SIRPα lo groups, respectively. The overall survival time of patients was determined during follow-up visits ( n = 58, * P < 0.05, *** P < 0.005, Gehan–Breslow–Wilcoxon test). The data in ( a – c and e , f ) represent the mean ± s.e.m. (* P < 0.05, ** P < 0.01 and *** P < 0.005; Student’s t -test)

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    doi: 10.1038/s41392-020-0124-z

    Figure Lengend Snippet: Macrophage Elk-1 and Sirpα levels increase with CRC progression. a – c mRNA levels of Ap-2α ( a ), Elk-1 ( b ) and Sirpα ( c ) in macrophages from adjacent normal and carcinoma tissue samples from CRC patients ( n = 15). d Positive correlation of S irpα expression with Elk-1 expression in macrophages from each individual sample. Linear regression was performed ( n = 42, P < 0.05, Pearson R = 0.377). e , f mRNA levels of Elk-1 and Sirpα in TAMs from CRC patients with different TNM stages. Samples were divided into 3 groups according to the TNM stage: T2-4N0M0 ( n = 15), T2-4N1-2M0 ( n = 15) and T2-4N0-2M1 ( n = 12). g – i Overall survival of CRC patients with differential expression of AP-2α ( g ), ELK-1 ( h ) and SIRPα ( i ). Patients were placed into two groups according to the mRNA expression of AP-2α, ELK-1 or SIRPα in TAMs. Patients with expression levels in the top 50% for TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α hi , ELK-1 hi or SIRPα hi groups, respectively. The remaining patients with low TAM AP-2α, ELK-1 or SIRPα expression were assigned to the AP-2α lo , ELK-1 lo or SIRPα lo groups, respectively. The overall survival time of patients was determined during follow-up visits ( n = 58, * P < 0.05, *** P < 0.005, Gehan–Breslow–Wilcoxon test). The data in ( a – c and e , f ) represent the mean ± s.e.m. (* P < 0.05, ** P < 0.01 and *** P < 0.005; Student’s t -test)

    Article Snippet: The cells on coverslips were stained with an anti-Ap-2α antibody (#3215, Cell Signaling, the dilution ratio was 1:500) at 37 °C for 1 h and then 4 °C overnight.

    Techniques: Expressing

    The working model for CRC evasion of innate immune surveillance. Without a cancer challenge, the interaction between macrophage SIRPα and CRC CD47 is interrupted, and macrophages exhibit normal phagocytic function. In the CRC microenvironment, CRC-derived lactate induces the activity of the transcription factor Ap-2α and the expression of its target Elk-1, which further promotes the expression of Sirpα in TAMs. The enhanced interaction between macrophage SIRPα and CRC CD47 reduces the phagocytic function of TAMs

    Journal: Signal Transduction and Targeted Therapy

    Article Title: The Ap-2α/Elk-1 axis regulates Sirpα-dependent tumor phagocytosis by tumor-associated macrophages in colorectal cancer

    doi: 10.1038/s41392-020-0124-z

    Figure Lengend Snippet: The working model for CRC evasion of innate immune surveillance. Without a cancer challenge, the interaction between macrophage SIRPα and CRC CD47 is interrupted, and macrophages exhibit normal phagocytic function. In the CRC microenvironment, CRC-derived lactate induces the activity of the transcription factor Ap-2α and the expression of its target Elk-1, which further promotes the expression of Sirpα in TAMs. The enhanced interaction between macrophage SIRPα and CRC CD47 reduces the phagocytic function of TAMs

    Article Snippet: The cells on coverslips were stained with an anti-Ap-2α antibody (#3215, Cell Signaling, the dilution ratio was 1:500) at 37 °C for 1 h and then 4 °C overnight.

    Techniques: Derivative Assay, Activity Assay, Expressing