intestinal epithelial ht 29 cell line  (ATCC)


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    ATCC intestinal epithelial ht 29 cell line
    Intestinal Epithelial Ht 29 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ht 29 cells  (ATCC)


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    ATCC ht 29 cells
    Ht 29 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ht 29  (ATCC)


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    ATCC ht 29
    Ht 29, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ht 29  (ATCC)


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    ATCC ht 29
    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and <t>HT-29</t> cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
    Ht 29, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons"

    Article Title: TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons

    Journal: Nature

    doi: 10.1038/s41586-023-06688-z

    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
    Figure Legend Snippet: a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).

    Techniques Used: DNA Methylation Assay, Expressing, RNA Sequencing Assay, shRNA, Quantitative RT-PCR

    ht 29  (ATCC)


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    ATCC ht 29
    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and <t>HT-29</t> cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
    Ht 29, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ht 29/product/ATCC
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    Images

    1) Product Images from "TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons"

    Article Title: TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons

    Journal: Nature

    doi: 10.1038/s41586-023-06688-z

    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
    Figure Legend Snippet: a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).

    Techniques Used: DNA Methylation Assay, Expressing, RNA Sequencing Assay, shRNA, Quantitative RT-PCR

    ht 29  (ATCC)


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    ATCC ht 29
    a Results after WCLs from <t>HT-29</t> cells incubated with or without purified His-tagged Amuc_1409 protein (Amuc_1409 * , 0.5 μg) for 1 h were subjected to immunoprecipitation using His-antibodies, followed by immunoblotting with indicated antibodies. b Schematics of the domain constructs of E-cadherin (upper panel); HEK293T cells were transfected with Strep-tagged E-cadherin domain constructs, and WCLs were subjected to a Strep pull-down assay. Immunoprecipitates were incubated with Amuc_1409 * (20 μg), followed by immunoblotting with indicated antibodies (lower panel). Data are representative of three independent experiments. Representative immunoblot images ( c ) and relative quantitative analysis for E-cadherin ( d , left panel) and β-catenin ( d , right panel) of the membrane and cytosolic fractions from HT-29 cells treated with Amuc_1409 * (16 nM) for 30 min. The quantification data are expressed as relative densitometer units with respect to the control group of each fraction (RDU/C). Epidermal growth factor receptor (EGFR) and GAPDH were used as loading controls for the membrane and cytosol, respectively. The blots shown are representative of three independent experiments. e Representative images of IF staining for E-cadherin and β-catenin in HT-29 cells treated with Amuc_1409 * (16 nM) for 30 min. E-cadherin (red); β-catenin (green), DAPI (nuclei, blue). Scale bar, 10 μm. Immunoblot analysis of active and total β-catenin ( f ) and relative mRNA expression of Wnt/β-catenin target genes ( g ) in young mIOs treated with Amuc_1409 * (16 nM) for 30 min before harvest. In ( g ), a different symbol indicates a data point representing each biological replicate from independently established organoid lines derived from distinct mouse litters ( n = 3 biologically independent mice). Each biological replicate includes two or three technical replicates. All data are presented as the mean ± SEM. Data shown are representative of two independent experiments, each with similar results unless otherwise stated. Statistical analyses were performed using two-tailed Student’s t test ( d, g ) (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs control group). Source data, including the exact p values and uncropped western blot images, are provided as a file.
    Ht 29, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The secreted protein Amuc_1409 from Akkermansia muciniphila improves gut health through intestinal stem cell regulation"

    Article Title: The secreted protein Amuc_1409 from Akkermansia muciniphila improves gut health through intestinal stem cell regulation

    Journal: Nature Communications

    doi: 10.1038/s41467-024-47275-8

    a Results after WCLs from HT-29 cells incubated with or without purified His-tagged Amuc_1409 protein (Amuc_1409 * , 0.5 μg) for 1 h were subjected to immunoprecipitation using His-antibodies, followed by immunoblotting with indicated antibodies. b Schematics of the domain constructs of E-cadherin (upper panel); HEK293T cells were transfected with Strep-tagged E-cadherin domain constructs, and WCLs were subjected to a Strep pull-down assay. Immunoprecipitates were incubated with Amuc_1409 * (20 μg), followed by immunoblotting with indicated antibodies (lower panel). Data are representative of three independent experiments. Representative immunoblot images ( c ) and relative quantitative analysis for E-cadherin ( d , left panel) and β-catenin ( d , right panel) of the membrane and cytosolic fractions from HT-29 cells treated with Amuc_1409 * (16 nM) for 30 min. The quantification data are expressed as relative densitometer units with respect to the control group of each fraction (RDU/C). Epidermal growth factor receptor (EGFR) and GAPDH were used as loading controls for the membrane and cytosol, respectively. The blots shown are representative of three independent experiments. e Representative images of IF staining for E-cadherin and β-catenin in HT-29 cells treated with Amuc_1409 * (16 nM) for 30 min. E-cadherin (red); β-catenin (green), DAPI (nuclei, blue). Scale bar, 10 μm. Immunoblot analysis of active and total β-catenin ( f ) and relative mRNA expression of Wnt/β-catenin target genes ( g ) in young mIOs treated with Amuc_1409 * (16 nM) for 30 min before harvest. In ( g ), a different symbol indicates a data point representing each biological replicate from independently established organoid lines derived from distinct mouse litters ( n = 3 biologically independent mice). Each biological replicate includes two or three technical replicates. All data are presented as the mean ± SEM. Data shown are representative of two independent experiments, each with similar results unless otherwise stated. Statistical analyses were performed using two-tailed Student’s t test ( d, g ) (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs control group). Source data, including the exact p values and uncropped western blot images, are provided as a file.
    Figure Legend Snippet: a Results after WCLs from HT-29 cells incubated with or without purified His-tagged Amuc_1409 protein (Amuc_1409 * , 0.5 μg) for 1 h were subjected to immunoprecipitation using His-antibodies, followed by immunoblotting with indicated antibodies. b Schematics of the domain constructs of E-cadherin (upper panel); HEK293T cells were transfected with Strep-tagged E-cadherin domain constructs, and WCLs were subjected to a Strep pull-down assay. Immunoprecipitates were incubated with Amuc_1409 * (20 μg), followed by immunoblotting with indicated antibodies (lower panel). Data are representative of three independent experiments. Representative immunoblot images ( c ) and relative quantitative analysis for E-cadherin ( d , left panel) and β-catenin ( d , right panel) of the membrane and cytosolic fractions from HT-29 cells treated with Amuc_1409 * (16 nM) for 30 min. The quantification data are expressed as relative densitometer units with respect to the control group of each fraction (RDU/C). Epidermal growth factor receptor (EGFR) and GAPDH were used as loading controls for the membrane and cytosol, respectively. The blots shown are representative of three independent experiments. e Representative images of IF staining for E-cadherin and β-catenin in HT-29 cells treated with Amuc_1409 * (16 nM) for 30 min. E-cadherin (red); β-catenin (green), DAPI (nuclei, blue). Scale bar, 10 μm. Immunoblot analysis of active and total β-catenin ( f ) and relative mRNA expression of Wnt/β-catenin target genes ( g ) in young mIOs treated with Amuc_1409 * (16 nM) for 30 min before harvest. In ( g ), a different symbol indicates a data point representing each biological replicate from independently established organoid lines derived from distinct mouse litters ( n = 3 biologically independent mice). Each biological replicate includes two or three technical replicates. All data are presented as the mean ± SEM. Data shown are representative of two independent experiments, each with similar results unless otherwise stated. Statistical analyses were performed using two-tailed Student’s t test ( d, g ) (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs control group). Source data, including the exact p values and uncropped western blot images, are provided as a file.

    Techniques Used: Incubation, Purification, Immunoprecipitation, Western Blot, Construct, Transfection, Pull Down Assay, Membrane, Staining, Expressing, Derivative Assay, Two Tailed Test

    ht 29 cell line  (ATCC)


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    ATCC ht 29 cell line
    Effect of α-PA combined with 5-FU treatment on <t>HT-29</t> cell viability and apoptosis. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Viability of HT-29 cells. (B) Morphological examination of HT-29 cells (×400 magnification). (C) Immunocytochemical staining with Annexin V (green) and PI (red) (×400 magnification). Quantitative analysis of the relative intensities of (D) Annexin V and (E) PI staining after treatment. Untreated cells and cells treated with 5 µM 5-FU or 250 mM α-PA for 72 h served as control groups. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.
    Ht 29 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "α‑Phellandrene enhances the apoptosis of HT‑29 cells induced by 5‑fluorouracil by modulating the mitochondria‑dependent pathway"

    Article Title: α‑Phellandrene enhances the apoptosis of HT‑29 cells induced by 5‑fluorouracil by modulating the mitochondria‑dependent pathway

    Journal: Oncology Reports

    doi: 10.3892/or.2024.8720

    Effect of α-PA combined with 5-FU treatment on HT-29 cell viability and apoptosis. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Viability of HT-29 cells. (B) Morphological examination of HT-29 cells (×400 magnification). (C) Immunocytochemical staining with Annexin V (green) and PI (red) (×400 magnification). Quantitative analysis of the relative intensities of (D) Annexin V and (E) PI staining after treatment. Untreated cells and cells treated with 5 µM 5-FU or 250 mM α-PA for 72 h served as control groups. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.
    Figure Legend Snippet: Effect of α-PA combined with 5-FU treatment on HT-29 cell viability and apoptosis. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Viability of HT-29 cells. (B) Morphological examination of HT-29 cells (×400 magnification). (C) Immunocytochemical staining with Annexin V (green) and PI (red) (×400 magnification). Quantitative analysis of the relative intensities of (D) Annexin V and (E) PI staining after treatment. Untreated cells and cells treated with 5 µM 5-FU or 250 mM α-PA for 72 h served as control groups. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.

    Techniques Used: Staining

    Effect of α-PA combined with 5-FU treatment on p53, Bax and Bcl-2 protein expression, and MMP levels in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. Western blotting was performed to determine the expression levels of (A) p53, (B) Bax and (C) Bcl-2 in HT-29 cells after treatment. (D) Quantitative analysis of (E) immunocytochemical staining with DiOC6 (green) and Hoechst 33342 (blue) to analyze the relative MMP levels (×400 magnification). Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene; MMP, mitochondrial membrane potential.
    Figure Legend Snippet: Effect of α-PA combined with 5-FU treatment on p53, Bax and Bcl-2 protein expression, and MMP levels in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. Western blotting was performed to determine the expression levels of (A) p53, (B) Bax and (C) Bcl-2 in HT-29 cells after treatment. (D) Quantitative analysis of (E) immunocytochemical staining with DiOC6 (green) and Hoechst 33342 (blue) to analyze the relative MMP levels (×400 magnification). Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene; MMP, mitochondrial membrane potential.

    Techniques Used: Expressing, Western Blot, Staining, Membrane

    Effect of α-PA combined with 5-FU treatment on VDAC-1 and HK-2 protein expression in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Immunocytochemical staining was performed to determine the expression levels of HK-2 (green) and VDAC-1 (red) (Hoechst 33342 (blue) (×400 magnification). Quantitative analysis of the relative expression levels of (B) HK-2 and (C) VDAC-1 after treatment. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene; HK-2, hexokinase-2; VDAC, voltage-dependent anion channel.
    Figure Legend Snippet: Effect of α-PA combined with 5-FU treatment on VDAC-1 and HK-2 protein expression in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Immunocytochemical staining was performed to determine the expression levels of HK-2 (green) and VDAC-1 (red) (Hoechst 33342 (blue) (×400 magnification). Quantitative analysis of the relative expression levels of (B) HK-2 and (C) VDAC-1 after treatment. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene; HK-2, hexokinase-2; VDAC, voltage-dependent anion channel.

    Techniques Used: Expressing, Staining

    Effect of α-PA combined with 5-FU treatment on cytochrome c and caspase mRNA expression in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. Reverse transcription-quantitative polymerase chain reaction analysis of the mRNA expression levels of (A) caspase-8, (B) Bid, (C) cytochrome c , (D) caspase-9 and (E) caspase-3. T. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.
    Figure Legend Snippet: Effect of α-PA combined with 5-FU treatment on cytochrome c and caspase mRNA expression in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. Reverse transcription-quantitative polymerase chain reaction analysis of the mRNA expression levels of (A) caspase-8, (B) Bid, (C) cytochrome c , (D) caspase-9 and (E) caspase-3. T. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Possible mechanisms by which α-phellandrene combined with 5-FU treatment induces apoptosis in human colon cancer HT-29 cells by regulating the mitochondria-dependent pathway. 5-FU, 5-fluorouracil; HK-2, hexokinase-2; MMP, mitochondrial membrane potential; VDAC, voltage-dependent anion channel.
    Figure Legend Snippet: Possible mechanisms by which α-phellandrene combined with 5-FU treatment induces apoptosis in human colon cancer HT-29 cells by regulating the mitochondria-dependent pathway. 5-FU, 5-fluorouracil; HK-2, hexokinase-2; MMP, mitochondrial membrane potential; VDAC, voltage-dependent anion channel.

    Techniques Used: Membrane

    ht 29 colon cancer cells  (ATCC)


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    ATCC ht 29 colon cancer cells
    Freshly trypsinized <t>HT-29</t> cells were seeded into 96-well ULA plates, types A-F, at a density of 500 cells per well and then cultured for up to four days. Spheroid morphology was visualized daily using automated brightfield microscopy. a) Representative micrographs showing individual spheroids from day 1 to day 4 (d1-d4) in plate types A-F. Scalebars, 100 µm. b-c) Box plots depicting spheroid diameters (b) or eccentricity (c) as a function of plate type A-F. Data are from 3 experiments with ≥ 24 spheroids per experiment (mean ± SD, 25- and 75-percentiles are plotted). Complete significance analysis, see Fig. S4. * , values significantly different compared to all other plate types for samples from the same day.
    Ht 29 Colon Cancer Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A Multiparametric Analysis Reveals Differential Behavior of Spheroid Cultures on Distinct Ultra-Low Attachment Plates Types"

    Article Title: A Multiparametric Analysis Reveals Differential Behavior of Spheroid Cultures on Distinct Ultra-Low Attachment Plates Types

    Journal: bioRxiv

    doi: 10.1101/2024.03.26.586778

    Freshly trypsinized HT-29 cells were seeded into 96-well ULA plates, types A-F, at a density of 500 cells per well and then cultured for up to four days. Spheroid morphology was visualized daily using automated brightfield microscopy. a) Representative micrographs showing individual spheroids from day 1 to day 4 (d1-d4) in plate types A-F. Scalebars, 100 µm. b-c) Box plots depicting spheroid diameters (b) or eccentricity (c) as a function of plate type A-F. Data are from 3 experiments with ≥ 24 spheroids per experiment (mean ± SD, 25- and 75-percentiles are plotted). Complete significance analysis, see Fig. S4. * , values significantly different compared to all other plate types for samples from the same day.
    Figure Legend Snippet: Freshly trypsinized HT-29 cells were seeded into 96-well ULA plates, types A-F, at a density of 500 cells per well and then cultured for up to four days. Spheroid morphology was visualized daily using automated brightfield microscopy. a) Representative micrographs showing individual spheroids from day 1 to day 4 (d1-d4) in plate types A-F. Scalebars, 100 µm. b-c) Box plots depicting spheroid diameters (b) or eccentricity (c) as a function of plate type A-F. Data are from 3 experiments with ≥ 24 spheroids per experiment (mean ± SD, 25- and 75-percentiles are plotted). Complete significance analysis, see Fig. S4. * , values significantly different compared to all other plate types for samples from the same day.

    Techniques Used: Cell Culture, Microscopy

    Freshly trypsinized HT-29 cells were seeded into 96-well ULA plates, types A-F, at a density of 500 cells per well and then cultured for four days. On day 4, spheroids were fixed, cleared, and stained for nuclei and plasma membrane, for the proliferation marker, Ki-67, and YAP1. Wholemount confocal 3D microscopy and 3D-image segmentation were performed. a) Representative micrographs showing single optical sections through individual spheroids at their largest circumference, from plate types as indicated. Upper panels, DAPI nuclear signals (gray); middle panels, Ki-67 immunofluorescence signals (green); lower panels, overlays. Scalebars, 100 µm. b-e) Box plots depicting the spheroid volumes (b) , the number of nuclei per spheroid (c) , the percentage of Ki-67+ nuclei of all nuclei (d) , and the density of nuclei packing within spheroids (e). (f) Violin plot showing the size distribution of nuclear volumes in HT-29 spheroids as a function of plate type. Data in (b-f) are from 3 experiments with ≥ 24 spheroids per experiment (mean ± SD, 25- and 75-percentiles are plotted). * , values significantly different compared to all other plate types. Complete significance analysis, see Fig. S5. (g) Representative micrograph showing a single optical section through a spheroid wholemount at its largest circumference, from plate type F. Immunofluorescence signals of Ki-67, green; YAP1, cyan; plasma membrane, red. Scalebar, 100 µm. (h) Scatterplots showing values of all segmented cells (59,337-74,649 cells per plate type) for plate types A-F (indicated). Depicted are YAP1 mean intensity per cell as a function of YAP1 N/C ratio (upper row), YAP1 mean intensity per cell as a function of the cell’s distance to spheroid hull (middle row), YAP1 N/C ratio as a function of the cell’s distance to spheroid hull (lower row). Yellow and purple dots represent values of Ki-67- and Ki-67+ cells, respectively.
    Figure Legend Snippet: Freshly trypsinized HT-29 cells were seeded into 96-well ULA plates, types A-F, at a density of 500 cells per well and then cultured for four days. On day 4, spheroids were fixed, cleared, and stained for nuclei and plasma membrane, for the proliferation marker, Ki-67, and YAP1. Wholemount confocal 3D microscopy and 3D-image segmentation were performed. a) Representative micrographs showing single optical sections through individual spheroids at their largest circumference, from plate types as indicated. Upper panels, DAPI nuclear signals (gray); middle panels, Ki-67 immunofluorescence signals (green); lower panels, overlays. Scalebars, 100 µm. b-e) Box plots depicting the spheroid volumes (b) , the number of nuclei per spheroid (c) , the percentage of Ki-67+ nuclei of all nuclei (d) , and the density of nuclei packing within spheroids (e). (f) Violin plot showing the size distribution of nuclear volumes in HT-29 spheroids as a function of plate type. Data in (b-f) are from 3 experiments with ≥ 24 spheroids per experiment (mean ± SD, 25- and 75-percentiles are plotted). * , values significantly different compared to all other plate types. Complete significance analysis, see Fig. S5. (g) Representative micrograph showing a single optical section through a spheroid wholemount at its largest circumference, from plate type F. Immunofluorescence signals of Ki-67, green; YAP1, cyan; plasma membrane, red. Scalebar, 100 µm. (h) Scatterplots showing values of all segmented cells (59,337-74,649 cells per plate type) for plate types A-F (indicated). Depicted are YAP1 mean intensity per cell as a function of YAP1 N/C ratio (upper row), YAP1 mean intensity per cell as a function of the cell’s distance to spheroid hull (middle row), YAP1 N/C ratio as a function of the cell’s distance to spheroid hull (lower row). Yellow and purple dots represent values of Ki-67- and Ki-67+ cells, respectively.

    Techniques Used: Cell Culture, Staining, Membrane, Marker, Microscopy, Immunofluorescence

    human cc cell lines ht 29  (ATCC)


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    ATCC human cc cell lines ht 29
    ERα expression positively correlates with tumour promoter expression in colon cancer. Mean immuno-reactive score (IRS) for ( A ) Cysteinyl leukotriene receptor 1 (CysLT 1 R) and ( B ) nuclear β-catenin expression in colorectal cancer (CRC) patients ( n = 267) with negative and positive ERα expression. XY scatter plots of the mRNA levels of ( C ) ERα ( ESR1 ) and CysLT 1 R ( CYSLTR1 ) and ( D ) ERα ( ESR1 ) and β-catenin ( CTNNB1 ) in the GSE39582 public dataset ( n = 566) of CRC patients. Violin plots showing the mRNA expression of ( E ) CYSLTR1 and ( F ) CTNNB1 in CRC patients with low or high ESR1 expression. G Immunohistochemical (IHC) images of ERα expression in the colons of wild-type ( WT ) and Cysltr1 knockout ( Cysltr1 −/− ) mice in a colitis-associated colon cancer (CAC) mouse model (n = 5). Bar graph showing the IRS of ERα expression compared between the WT and Cysltr1 −/− mouse groups. H IHC images of ERα expression in the colons of WT and Apc Min/+ mice, n = 5. Bar graph showing the IRS of ERα expression in the WT and Cysltr1 −/− mouse groups. For both mouse models, four random regions of interest (ROIs; marked with dotted lines) in colon tissue were evaluated for each mouse. Representative images of one ROI are shown as insets. The scale bars represent 2 mm ( G ) and 500 μm ( H ) in the image of the whole colon and 50 μm in the zoomed insets. P values were calculated using an unpaired Student’s t test for the bar graphs in G and H. Relative mRNA expression levels of ESR1 , CYSLTR1 , and CTNNB1 in ( I ) <t>HT-29</t> and ( J ) Caco-2 CC cells after treatment with PPT (ERα specific agonist, 40 nM) or AZD9496 (ERα specific antagonist, 0.3 nM for 30 min) alone or in combination of PPT (40 nM) with AZD9496 (0.3 nM for 30 min before the PPT treatment). The data are presented as the mean ± SEM ( n = 3 independent experiments). P values < 0.5 were considered significant were calculated using an unpaired Student’s t test
    Human Cc Cell Lines Ht 29, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer"

    Article Title: High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-024-01582-1

    ERα expression positively correlates with tumour promoter expression in colon cancer. Mean immuno-reactive score (IRS) for ( A ) Cysteinyl leukotriene receptor 1 (CysLT 1 R) and ( B ) nuclear β-catenin expression in colorectal cancer (CRC) patients ( n = 267) with negative and positive ERα expression. XY scatter plots of the mRNA levels of ( C ) ERα ( ESR1 ) and CysLT 1 R ( CYSLTR1 ) and ( D ) ERα ( ESR1 ) and β-catenin ( CTNNB1 ) in the GSE39582 public dataset ( n = 566) of CRC patients. Violin plots showing the mRNA expression of ( E ) CYSLTR1 and ( F ) CTNNB1 in CRC patients with low or high ESR1 expression. G Immunohistochemical (IHC) images of ERα expression in the colons of wild-type ( WT ) and Cysltr1 knockout ( Cysltr1 −/− ) mice in a colitis-associated colon cancer (CAC) mouse model (n = 5). Bar graph showing the IRS of ERα expression compared between the WT and Cysltr1 −/− mouse groups. H IHC images of ERα expression in the colons of WT and Apc Min/+ mice, n = 5. Bar graph showing the IRS of ERα expression in the WT and Cysltr1 −/− mouse groups. For both mouse models, four random regions of interest (ROIs; marked with dotted lines) in colon tissue were evaluated for each mouse. Representative images of one ROI are shown as insets. The scale bars represent 2 mm ( G ) and 500 μm ( H ) in the image of the whole colon and 50 μm in the zoomed insets. P values were calculated using an unpaired Student’s t test for the bar graphs in G and H. Relative mRNA expression levels of ESR1 , CYSLTR1 , and CTNNB1 in ( I ) HT-29 and ( J ) Caco-2 CC cells after treatment with PPT (ERα specific agonist, 40 nM) or AZD9496 (ERα specific antagonist, 0.3 nM for 30 min) alone or in combination of PPT (40 nM) with AZD9496 (0.3 nM for 30 min before the PPT treatment). The data are presented as the mean ± SEM ( n = 3 independent experiments). P values < 0.5 were considered significant were calculated using an unpaired Student’s t test
    Figure Legend Snippet: ERα expression positively correlates with tumour promoter expression in colon cancer. Mean immuno-reactive score (IRS) for ( A ) Cysteinyl leukotriene receptor 1 (CysLT 1 R) and ( B ) nuclear β-catenin expression in colorectal cancer (CRC) patients ( n = 267) with negative and positive ERα expression. XY scatter plots of the mRNA levels of ( C ) ERα ( ESR1 ) and CysLT 1 R ( CYSLTR1 ) and ( D ) ERα ( ESR1 ) and β-catenin ( CTNNB1 ) in the GSE39582 public dataset ( n = 566) of CRC patients. Violin plots showing the mRNA expression of ( E ) CYSLTR1 and ( F ) CTNNB1 in CRC patients with low or high ESR1 expression. G Immunohistochemical (IHC) images of ERα expression in the colons of wild-type ( WT ) and Cysltr1 knockout ( Cysltr1 −/− ) mice in a colitis-associated colon cancer (CAC) mouse model (n = 5). Bar graph showing the IRS of ERα expression compared between the WT and Cysltr1 −/− mouse groups. H IHC images of ERα expression in the colons of WT and Apc Min/+ mice, n = 5. Bar graph showing the IRS of ERα expression in the WT and Cysltr1 −/− mouse groups. For both mouse models, four random regions of interest (ROIs; marked with dotted lines) in colon tissue were evaluated for each mouse. Representative images of one ROI are shown as insets. The scale bars represent 2 mm ( G ) and 500 μm ( H ) in the image of the whole colon and 50 μm in the zoomed insets. P values were calculated using an unpaired Student’s t test for the bar graphs in G and H. Relative mRNA expression levels of ESR1 , CYSLTR1 , and CTNNB1 in ( I ) HT-29 and ( J ) Caco-2 CC cells after treatment with PPT (ERα specific agonist, 40 nM) or AZD9496 (ERα specific antagonist, 0.3 nM for 30 min) alone or in combination of PPT (40 nM) with AZD9496 (0.3 nM for 30 min before the PPT treatment). The data are presented as the mean ± SEM ( n = 3 independent experiments). P values < 0.5 were considered significant were calculated using an unpaired Student’s t test

    Techniques Used: Expressing, Immunohistochemical staining, Knock-Out

    ERα activation in colon cancer cells promotes survival. A Alterations in the colonies formed by HT-29 and Caco-2 colon cancer (CC) cells treated with PPT (40 nM) alone for 48 h or in combination with AZD9496 (0.3 nM, for 30 min before PPT treatment). Bar graphs show the percentage of survival and are representative of n = 3 independent experiments. B Western blots showing the protein levels of phospho-β-catenin (Ser33/37/Thr41), non-phospho (active)-β-catenin, total β-catenin, and ERα in HT-29 and Caco-2 cells untreated or treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, for 30 min). Graphs showing the densitometric analysis of alterations in phospho- and non-phospho (active)-β-catenin and ERα protein levels as percentages of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. C Alterations in the colonies formed by HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment for 48 h. The graphs show the percentage of survival in each group. D Western blots showing the protein levels of non-phospho (active)-β-catenin, total β-catenin, and ERα in both HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment. Graphs showing the densitometric analysis of alterations in ERα and non-phospho (active)-β-catenin protein levels as percentages of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. The data are presented as the means ± SEMs. P values < 0.5 were considered significant calculated using an unpaired Student’s t test
    Figure Legend Snippet: ERα activation in colon cancer cells promotes survival. A Alterations in the colonies formed by HT-29 and Caco-2 colon cancer (CC) cells treated with PPT (40 nM) alone for 48 h or in combination with AZD9496 (0.3 nM, for 30 min before PPT treatment). Bar graphs show the percentage of survival and are representative of n = 3 independent experiments. B Western blots showing the protein levels of phospho-β-catenin (Ser33/37/Thr41), non-phospho (active)-β-catenin, total β-catenin, and ERα in HT-29 and Caco-2 cells untreated or treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, for 30 min). Graphs showing the densitometric analysis of alterations in phospho- and non-phospho (active)-β-catenin and ERα protein levels as percentages of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. C Alterations in the colonies formed by HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment for 48 h. The graphs show the percentage of survival in each group. D Western blots showing the protein levels of non-phospho (active)-β-catenin, total β-catenin, and ERα in both HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment. Graphs showing the densitometric analysis of alterations in ERα and non-phospho (active)-β-catenin protein levels as percentages of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. The data are presented as the means ± SEMs. P values < 0.5 were considered significant calculated using an unpaired Student’s t test

    Techniques Used: Activation Assay, Western Blot, Transfection

    Activation of ERα promotes colon cancer cell metastasis. An external dataset composed of data for CC patients with liver metastasis (GSE77955, n = 18) was used to analyse the correlations of CysLT 1 R ( CYSLTR1 ) and β-catenin ( CTNNB1 ) with ERα ( ESR1 ). The scatter plots show the positive correlations between ESR1 and both ( A ) CYSLTR1 and ( B ) CTNNB1 . C Schematic cartoon showing the zebrafish embryo-based colon cancer metastasis model. DiI-labelled HT-29 cells left untreated or treated with PPT alone or in combination with AZD9496 were injected into the perivitelline space of 2 dpf zebrafish embryos, and the embryos were incubated for 48 h. Images showing the metastatic spread of HT-29 cells in the tail veins of zebrafish embryos in each group (CTRL, n = 30; PPT, n = 30; AZD9496 + PPT, n = 43). Scale bars: full-size images; 10 μm, insets; 2 μm. The insets show the regions enclosed in the dotted lines in the full-size tail images. The arrows point to the metastatic foci and transendothelial migration of cancer cells. D Graphs showing the number of embryos with (M1, mets) or without (M0, no mets) metastasis in each group and D′, quantification of tail vein metastasis using the mean fluorescence intensity (MFI) of the embryos with metastasis. E Western blots showing the expression of the tight junction protein ZO-1 in HT-29 and Caco-2 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Graph showing the densitometric analysis of alterations in protein expression as a percentage of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. For the bar graphs, unpaired t-test was used. F Immunofluorescence analysis of ZO-1 and Occludin expression in HT-29 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Greyscale images (insets) showing a representative region of interest (dotted line) for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 (CTRL, n = 116; PPT, n = 105, AZD9496 + PPT, n = 107) and Occludin in random cell-cell junctions (CTRL, n = 103; PPT, n = 110, AZD9496 + PPT, n = 108). P values were calculated with unpaired Student’s t test. The arrows indicate gaps in ZO-1 expression. G Immunofluorescence analysis of ZO-1 and Occludin expression in Caco-2 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 (CTRL, n = 108; PPT, n = 116, AZD9496 + PPT, n = 105) and Occludin (CTRL, n = 105; PPT, n = 115, AZD9496 + PPT, n = 116) in random cell-cell junctions. The arrows indicate gaps in ZO-1 or Occludin expression. H Immunofluorescence analysis of ZO-1 in HT-29 cell-derived colonospheres treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Scale bars: 10 μm. Violin plot showing the mean fluorescence intensity of ZO-1 in random colonospheres (CTRL, n = 31; PPT, n = 28, AZD9496 + PPT, n = 30). The arrows indicate ZO-1 expression in the disseminated cells from the colonospheres. I Immunofluorescence analysis of ZO-1 in Caco-2 CC cell-derived colonospheres treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Scale bars: 10 μm. Violin plot showing the mean fluorescence intensity of ZO-1 in random colonospheres (CTRL, n = 29; PPT, n = 26, AZD9496 + PPT, n = 28). The data are presented as the mean ± SEM of three experiments. P values < 0.5 were considered significant were calculated using the chi-square test in D and an unpaired Student’s t test in D′-I
    Figure Legend Snippet: Activation of ERα promotes colon cancer cell metastasis. An external dataset composed of data for CC patients with liver metastasis (GSE77955, n = 18) was used to analyse the correlations of CysLT 1 R ( CYSLTR1 ) and β-catenin ( CTNNB1 ) with ERα ( ESR1 ). The scatter plots show the positive correlations between ESR1 and both ( A ) CYSLTR1 and ( B ) CTNNB1 . C Schematic cartoon showing the zebrafish embryo-based colon cancer metastasis model. DiI-labelled HT-29 cells left untreated or treated with PPT alone or in combination with AZD9496 were injected into the perivitelline space of 2 dpf zebrafish embryos, and the embryos were incubated for 48 h. Images showing the metastatic spread of HT-29 cells in the tail veins of zebrafish embryos in each group (CTRL, n = 30; PPT, n = 30; AZD9496 + PPT, n = 43). Scale bars: full-size images; 10 μm, insets; 2 μm. The insets show the regions enclosed in the dotted lines in the full-size tail images. The arrows point to the metastatic foci and transendothelial migration of cancer cells. D Graphs showing the number of embryos with (M1, mets) or without (M0, no mets) metastasis in each group and D′, quantification of tail vein metastasis using the mean fluorescence intensity (MFI) of the embryos with metastasis. E Western blots showing the expression of the tight junction protein ZO-1 in HT-29 and Caco-2 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Graph showing the densitometric analysis of alterations in protein expression as a percentage of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. For the bar graphs, unpaired t-test was used. F Immunofluorescence analysis of ZO-1 and Occludin expression in HT-29 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Greyscale images (insets) showing a representative region of interest (dotted line) for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 (CTRL, n = 116; PPT, n = 105, AZD9496 + PPT, n = 107) and Occludin in random cell-cell junctions (CTRL, n = 103; PPT, n = 110, AZD9496 + PPT, n = 108). P values were calculated with unpaired Student’s t test. The arrows indicate gaps in ZO-1 expression. G Immunofluorescence analysis of ZO-1 and Occludin expression in Caco-2 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 (CTRL, n = 108; PPT, n = 116, AZD9496 + PPT, n = 105) and Occludin (CTRL, n = 105; PPT, n = 115, AZD9496 + PPT, n = 116) in random cell-cell junctions. The arrows indicate gaps in ZO-1 or Occludin expression. H Immunofluorescence analysis of ZO-1 in HT-29 cell-derived colonospheres treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Scale bars: 10 μm. Violin plot showing the mean fluorescence intensity of ZO-1 in random colonospheres (CTRL, n = 31; PPT, n = 28, AZD9496 + PPT, n = 30). The arrows indicate ZO-1 expression in the disseminated cells from the colonospheres. I Immunofluorescence analysis of ZO-1 in Caco-2 CC cell-derived colonospheres treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Scale bars: 10 μm. Violin plot showing the mean fluorescence intensity of ZO-1 in random colonospheres (CTRL, n = 29; PPT, n = 26, AZD9496 + PPT, n = 28). The data are presented as the mean ± SEM of three experiments. P values < 0.5 were considered significant were calculated using the chi-square test in D and an unpaired Student’s t test in D′-I

    Techniques Used: Activation Assay, Injection, Incubation, Migration, Fluorescence, Western Blot, Expressing, Immunofluorescence, Staining, Derivative Assay

    Functional absence of ERα inhibits colon cancer cell metastasis. DiI-labelled HT-29 cells transfected with either siCTRL or siESR1 and treated with or without PPT for 48 h were injected into the perivitelline space of 2 dpf zebrafish embryos, and the embryos were incubated for 48 h. A Images showing the metastatic spread of HT-29 cells in the tail veins of zebrafish embryos in each group ( siCTRL ; CTRL, n = 30; PPT, n = 30; siESR1 ; CTRL, n = 33, PPT, n = 32). Graphs showing A ’, the number of embryos with (M1, mets) or without metastasis (M0, no mets) in each group and A ”, the quantification of tail vein metastasis using the mean fluorescence intensity (MFI) of the embryos with metastasis (M1 group). Scale bars: full-size images; 10 μm, insets; 2 μm. The insets show the regions enclosed in the dotted lines in the full-size tail images. The arrows point to the metastatic foci and transendothelial migration of cancer cells. B Western blots showing the expression of the tight junction protein ZO-1 in HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment. Graphs showing the densitometric analysis of alterations in protein expression as a percentage of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. C Immunofluorescence analysis of ZO-1 and Occludin expression in HT-29 cells transfected with either siCTRL or siESR1 prior to treatment with the ERα agonist PPT (40 nM). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 ( siCTRL (CTRL, n = 105; PPT, n = 115), siESR1 (CTRL, n = 108; PPT, n = 116)) and Occludin ( siCTRL (CTRL, n = 105; PPT, n = 105), siESR1 (CTRL, n = 107; PPT, n = 102)) in random cell-cell junctions. The arrows indicate gaps in ZO-1 or Occludin expression. D Immunofluorescence analysis of ZO-1 and Occludin expression in Caco-2 cells transfected with either siCTRL or siESR1 prior to treatment with the ERα agonist PPT (40 nM). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 and Occludin in random cell junctions. For ZO-1 staining in the siCTRL -transfected group (CTRL, n = 105; PPT, n = 105) and in the siESR1 -transfected group (CTRL, n = 108; PPT, n = 105), random cell junctions were evaluated. For Occludin staining in the siCTRL -transfected group (CTRL, n = 105; PPT, n = 105) and in the siESR1 -transfected group (CTRL, n = 102; PPT, n = 108), random cell junctions were evaluated. The arrows indicate gaps in ZO-1 or Occludin expression. Immunofluorescence analysis of ZO-1 in colonospheres derived from either siCTRL or siESR1 transfected ( E ) HT-29 and ( F ) Caco-2 cells. Scale bars: 10 μm. Violin plots showing the mean fluorescence intensity of ZO-1 in random ( E’ ) HT-29 ( siCTRL , n = 30; siESR1 , n = 32) or ( F′ ) Caco-2 ( siCTRL , n = 28; siESR1 , n = 31) colonospheres. The MFIs of the indicated proteins were measured using ImageJ software (NIH, USA). G Graphical representation of the summary of the study. Upon binding to the agonist PPT, ERα dimerizes and shuttles into the nucleus. This upregulates the transcription of CYSLTR1 and CTNNB1 . In addition, it promotes metastasis by disrupting the tight junction proteins ZO-1 and Occludin. However, blocking the binding of PPT to ERα by employing an antagonist, AZD9496, prevents the activation and hence the dimerization of the receptor. This further leads to downregulation of CYSLTR1 and CTNNB1 and upregulation of the tight junction proteins ZO-1 and Occludin. The data are presented as the mean ± SEM of three experiments. P values were calculated with the chi-square test for A’ and unpaired Student’s t test for A”, B-F
    Figure Legend Snippet: Functional absence of ERα inhibits colon cancer cell metastasis. DiI-labelled HT-29 cells transfected with either siCTRL or siESR1 and treated with or without PPT for 48 h were injected into the perivitelline space of 2 dpf zebrafish embryos, and the embryos were incubated for 48 h. A Images showing the metastatic spread of HT-29 cells in the tail veins of zebrafish embryos in each group ( siCTRL ; CTRL, n = 30; PPT, n = 30; siESR1 ; CTRL, n = 33, PPT, n = 32). Graphs showing A ’, the number of embryos with (M1, mets) or without metastasis (M0, no mets) in each group and A ”, the quantification of tail vein metastasis using the mean fluorescence intensity (MFI) of the embryos with metastasis (M1 group). Scale bars: full-size images; 10 μm, insets; 2 μm. The insets show the regions enclosed in the dotted lines in the full-size tail images. The arrows point to the metastatic foci and transendothelial migration of cancer cells. B Western blots showing the expression of the tight junction protein ZO-1 in HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment. Graphs showing the densitometric analysis of alterations in protein expression as a percentage of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. C Immunofluorescence analysis of ZO-1 and Occludin expression in HT-29 cells transfected with either siCTRL or siESR1 prior to treatment with the ERα agonist PPT (40 nM). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 ( siCTRL (CTRL, n = 105; PPT, n = 115), siESR1 (CTRL, n = 108; PPT, n = 116)) and Occludin ( siCTRL (CTRL, n = 105; PPT, n = 105), siESR1 (CTRL, n = 107; PPT, n = 102)) in random cell-cell junctions. The arrows indicate gaps in ZO-1 or Occludin expression. D Immunofluorescence analysis of ZO-1 and Occludin expression in Caco-2 cells transfected with either siCTRL or siESR1 prior to treatment with the ERα agonist PPT (40 nM). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 and Occludin in random cell junctions. For ZO-1 staining in the siCTRL -transfected group (CTRL, n = 105; PPT, n = 105) and in the siESR1 -transfected group (CTRL, n = 108; PPT, n = 105), random cell junctions were evaluated. For Occludin staining in the siCTRL -transfected group (CTRL, n = 105; PPT, n = 105) and in the siESR1 -transfected group (CTRL, n = 102; PPT, n = 108), random cell junctions were evaluated. The arrows indicate gaps in ZO-1 or Occludin expression. Immunofluorescence analysis of ZO-1 in colonospheres derived from either siCTRL or siESR1 transfected ( E ) HT-29 and ( F ) Caco-2 cells. Scale bars: 10 μm. Violin plots showing the mean fluorescence intensity of ZO-1 in random ( E’ ) HT-29 ( siCTRL , n = 30; siESR1 , n = 32) or ( F′ ) Caco-2 ( siCTRL , n = 28; siESR1 , n = 31) colonospheres. The MFIs of the indicated proteins were measured using ImageJ software (NIH, USA). G Graphical representation of the summary of the study. Upon binding to the agonist PPT, ERα dimerizes and shuttles into the nucleus. This upregulates the transcription of CYSLTR1 and CTNNB1 . In addition, it promotes metastasis by disrupting the tight junction proteins ZO-1 and Occludin. However, blocking the binding of PPT to ERα by employing an antagonist, AZD9496, prevents the activation and hence the dimerization of the receptor. This further leads to downregulation of CYSLTR1 and CTNNB1 and upregulation of the tight junction proteins ZO-1 and Occludin. The data are presented as the mean ± SEM of three experiments. P values were calculated with the chi-square test for A’ and unpaired Student’s t test for A”, B-F

    Techniques Used: Functional Assay, Transfection, Injection, Incubation, Fluorescence, Migration, Western Blot, Expressing, Immunofluorescence, Staining, Derivative Assay, Software, Binding Assay, Blocking Assay, Activation Assay

    colorectal adenocarcinoma cell line ht 29  (ATCC)


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    Structured Review

    ATCC colorectal adenocarcinoma cell line ht 29
    Analysis of the ∆ sap mutant in adherence to and invasion of <t>HT-29</t> epithelial cells. Infection analyses were performed following bacterial subculture in LB media supplemented with glucose (glu), with or without bile salts (BS), as well as TSB media with or without bile salts. The average percent recoveries ± standard error of the mean from three biological replicates are plotted relative to the recovery rate for 2457T from the bile salts subcultures (set at 100%). ( a ) In adherence assays following subculture in either LB + glu ± BS or TSB ± BS, the adherence rates were induced for both wild type 2457T and the Δ sap mutant following bile salts exposure (**, p < 0.005). However, the Δ sap mutant had lower adherence following subculture in bile salts compared to 2457T in the same condition (ǂ, p < 0.005). There were no significant differences in adherence between 2457T or the Δ sap mutant following subculturing in media without bile salts. While there is a decrease in adherence in the Δ sap mutant relative to 2457T following TSB subculture without bile salts, the difference was not statistically significant. ( b ) For invasion, there were no significant differences between 2457T and the Δ sap mutant following subculture in LB + glu ± BS. Both strains had significant increases in invasion following exposure to bile salts (**, p < 0.005).
    Colorectal Adenocarcinoma Cell Line Ht 29, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Gastrointestinal signals in supplemented media reveal a role in adherence for the Shigella flexneri sap autotransporter gene"

    Article Title: Gastrointestinal signals in supplemented media reveal a role in adherence for the Shigella flexneri sap autotransporter gene

    Journal: Gut Microbes

    doi: 10.1080/19490976.2024.2331985

    Analysis of the ∆ sap mutant in adherence to and invasion of HT-29 epithelial cells. Infection analyses were performed following bacterial subculture in LB media supplemented with glucose (glu), with or without bile salts (BS), as well as TSB media with or without bile salts. The average percent recoveries ± standard error of the mean from three biological replicates are plotted relative to the recovery rate for 2457T from the bile salts subcultures (set at 100%). ( a ) In adherence assays following subculture in either LB + glu ± BS or TSB ± BS, the adherence rates were induced for both wild type 2457T and the Δ sap mutant following bile salts exposure (**, p < 0.005). However, the Δ sap mutant had lower adherence following subculture in bile salts compared to 2457T in the same condition (ǂ, p < 0.005). There were no significant differences in adherence between 2457T or the Δ sap mutant following subculturing in media without bile salts. While there is a decrease in adherence in the Δ sap mutant relative to 2457T following TSB subculture without bile salts, the difference was not statistically significant. ( b ) For invasion, there were no significant differences between 2457T and the Δ sap mutant following subculture in LB + glu ± BS. Both strains had significant increases in invasion following exposure to bile salts (**, p < 0.005).
    Figure Legend Snippet: Analysis of the ∆ sap mutant in adherence to and invasion of HT-29 epithelial cells. Infection analyses were performed following bacterial subculture in LB media supplemented with glucose (glu), with or without bile salts (BS), as well as TSB media with or without bile salts. The average percent recoveries ± standard error of the mean from three biological replicates are plotted relative to the recovery rate for 2457T from the bile salts subcultures (set at 100%). ( a ) In adherence assays following subculture in either LB + glu ± BS or TSB ± BS, the adherence rates were induced for both wild type 2457T and the Δ sap mutant following bile salts exposure (**, p < 0.005). However, the Δ sap mutant had lower adherence following subculture in bile salts compared to 2457T in the same condition (ǂ, p < 0.005). There were no significant differences in adherence between 2457T or the Δ sap mutant following subculturing in media without bile salts. While there is a decrease in adherence in the Δ sap mutant relative to 2457T following TSB subculture without bile salts, the difference was not statistically significant. ( b ) For invasion, there were no significant differences between 2457T and the Δ sap mutant following subculture in LB + glu ± BS. Both strains had significant increases in invasion following exposure to bile salts (**, p < 0.005).

    Techniques Used: Mutagenesis, Infection, Subculturing Assay

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    ATCC intestinal epithelial ht 29 cell line
    Intestinal Epithelial Ht 29 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC ht 29 cells
    Ht 29 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ht 29  (ATCC)
    86
    ATCC ht 29
    Ht 29, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC ht 29 cell line
    Effect of α-PA combined with 5-FU treatment on <t>HT-29</t> cell viability and apoptosis. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Viability of HT-29 cells. (B) Morphological examination of HT-29 cells (×400 magnification). (C) Immunocytochemical staining with Annexin V (green) and PI (red) (×400 magnification). Quantitative analysis of the relative intensities of (D) Annexin V and (E) PI staining after treatment. Untreated cells and cells treated with 5 µM 5-FU or 250 mM α-PA for 72 h served as control groups. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.
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    ATCC ht 29 colon cancer cells
    Freshly trypsinized <t>HT-29</t> cells were seeded into 96-well ULA plates, types A-F, at a density of 500 cells per well and then cultured for up to four days. Spheroid morphology was visualized daily using automated brightfield microscopy. a) Representative micrographs showing individual spheroids from day 1 to day 4 (d1-d4) in plate types A-F. Scalebars, 100 µm. b-c) Box plots depicting spheroid diameters (b) or eccentricity (c) as a function of plate type A-F. Data are from 3 experiments with ≥ 24 spheroids per experiment (mean ± SD, 25- and 75-percentiles are plotted). Complete significance analysis, see Fig. S4. * , values significantly different compared to all other plate types for samples from the same day.
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    ATCC human cc cell lines ht 29
    ERα expression positively correlates with tumour promoter expression in colon cancer. Mean immuno-reactive score (IRS) for ( A ) Cysteinyl leukotriene receptor 1 (CysLT 1 R) and ( B ) nuclear β-catenin expression in colorectal cancer (CRC) patients ( n = 267) with negative and positive ERα expression. XY scatter plots of the mRNA levels of ( C ) ERα ( ESR1 ) and CysLT 1 R ( CYSLTR1 ) and ( D ) ERα ( ESR1 ) and β-catenin ( CTNNB1 ) in the GSE39582 public dataset ( n = 566) of CRC patients. Violin plots showing the mRNA expression of ( E ) CYSLTR1 and ( F ) CTNNB1 in CRC patients with low or high ESR1 expression. G Immunohistochemical (IHC) images of ERα expression in the colons of wild-type ( WT ) and Cysltr1 knockout ( Cysltr1 −/− ) mice in a colitis-associated colon cancer (CAC) mouse model (n = 5). Bar graph showing the IRS of ERα expression compared between the WT and Cysltr1 −/− mouse groups. H IHC images of ERα expression in the colons of WT and Apc Min/+ mice, n = 5. Bar graph showing the IRS of ERα expression in the WT and Cysltr1 −/− mouse groups. For both mouse models, four random regions of interest (ROIs; marked with dotted lines) in colon tissue were evaluated for each mouse. Representative images of one ROI are shown as insets. The scale bars represent 2 mm ( G ) and 500 μm ( H ) in the image of the whole colon and 50 μm in the zoomed insets. P values were calculated using an unpaired Student’s t test for the bar graphs in G and H. Relative mRNA expression levels of ESR1 , CYSLTR1 , and CTNNB1 in ( I ) <t>HT-29</t> and ( J ) Caco-2 CC cells after treatment with PPT (ERα specific agonist, 40 nM) or AZD9496 (ERα specific antagonist, 0.3 nM for 30 min) alone or in combination of PPT (40 nM) with AZD9496 (0.3 nM for 30 min before the PPT treatment). The data are presented as the mean ± SEM ( n = 3 independent experiments). P values < 0.5 were considered significant were calculated using an unpaired Student’s t test
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    ATCC colorectal adenocarcinoma cell line ht 29
    Analysis of the ∆ sap mutant in adherence to and invasion of <t>HT-29</t> epithelial cells. Infection analyses were performed following bacterial subculture in LB media supplemented with glucose (glu), with or without bile salts (BS), as well as TSB media with or without bile salts. The average percent recoveries ± standard error of the mean from three biological replicates are plotted relative to the recovery rate for 2457T from the bile salts subcultures (set at 100%). ( a ) In adherence assays following subculture in either LB + glu ± BS or TSB ± BS, the adherence rates were induced for both wild type 2457T and the Δ sap mutant following bile salts exposure (**, p < 0.005). However, the Δ sap mutant had lower adherence following subculture in bile salts compared to 2457T in the same condition (ǂ, p < 0.005). There were no significant differences in adherence between 2457T or the Δ sap mutant following subculturing in media without bile salts. While there is a decrease in adherence in the Δ sap mutant relative to 2457T following TSB subculture without bile salts, the difference was not statistically significant. ( b ) For invasion, there were no significant differences between 2457T and the Δ sap mutant following subculture in LB + glu ± BS. Both strains had significant increases in invasion following exposure to bile salts (**, p < 0.005).
    Colorectal Adenocarcinoma Cell Line Ht 29, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of α-PA combined with 5-FU treatment on HT-29 cell viability and apoptosis. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Viability of HT-29 cells. (B) Morphological examination of HT-29 cells (×400 magnification). (C) Immunocytochemical staining with Annexin V (green) and PI (red) (×400 magnification). Quantitative analysis of the relative intensities of (D) Annexin V and (E) PI staining after treatment. Untreated cells and cells treated with 5 µM 5-FU or 250 mM α-PA for 72 h served as control groups. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.

    Journal: Oncology Reports

    Article Title: α‑Phellandrene enhances the apoptosis of HT‑29 cells induced by 5‑fluorouracil by modulating the mitochondria‑dependent pathway

    doi: 10.3892/or.2024.8720

    Figure Lengend Snippet: Effect of α-PA combined with 5-FU treatment on HT-29 cell viability and apoptosis. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Viability of HT-29 cells. (B) Morphological examination of HT-29 cells (×400 magnification). (C) Immunocytochemical staining with Annexin V (green) and PI (red) (×400 magnification). Quantitative analysis of the relative intensities of (D) Annexin V and (E) PI staining after treatment. Untreated cells and cells treated with 5 µM 5-FU or 250 mM α-PA for 72 h served as control groups. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.

    Article Snippet: Searching the sample genotypes in the ATCC/DSMZ/JCRB/RIKEN/ECACC/ExPASy STR database, the test sample result matched the HT-29 cell line.

    Techniques: Staining

    Effect of α-PA combined with 5-FU treatment on p53, Bax and Bcl-2 protein expression, and MMP levels in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. Western blotting was performed to determine the expression levels of (A) p53, (B) Bax and (C) Bcl-2 in HT-29 cells after treatment. (D) Quantitative analysis of (E) immunocytochemical staining with DiOC6 (green) and Hoechst 33342 (blue) to analyze the relative MMP levels (×400 magnification). Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene; MMP, mitochondrial membrane potential.

    Journal: Oncology Reports

    Article Title: α‑Phellandrene enhances the apoptosis of HT‑29 cells induced by 5‑fluorouracil by modulating the mitochondria‑dependent pathway

    doi: 10.3892/or.2024.8720

    Figure Lengend Snippet: Effect of α-PA combined with 5-FU treatment on p53, Bax and Bcl-2 protein expression, and MMP levels in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. Western blotting was performed to determine the expression levels of (A) p53, (B) Bax and (C) Bcl-2 in HT-29 cells after treatment. (D) Quantitative analysis of (E) immunocytochemical staining with DiOC6 (green) and Hoechst 33342 (blue) to analyze the relative MMP levels (×400 magnification). Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene; MMP, mitochondrial membrane potential.

    Article Snippet: Searching the sample genotypes in the ATCC/DSMZ/JCRB/RIKEN/ECACC/ExPASy STR database, the test sample result matched the HT-29 cell line.

    Techniques: Expressing, Western Blot, Staining, Membrane

    Effect of α-PA combined with 5-FU treatment on VDAC-1 and HK-2 protein expression in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Immunocytochemical staining was performed to determine the expression levels of HK-2 (green) and VDAC-1 (red) (Hoechst 33342 (blue) (×400 magnification). Quantitative analysis of the relative expression levels of (B) HK-2 and (C) VDAC-1 after treatment. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene; HK-2, hexokinase-2; VDAC, voltage-dependent anion channel.

    Journal: Oncology Reports

    Article Title: α‑Phellandrene enhances the apoptosis of HT‑29 cells induced by 5‑fluorouracil by modulating the mitochondria‑dependent pathway

    doi: 10.3892/or.2024.8720

    Figure Lengend Snippet: Effect of α-PA combined with 5-FU treatment on VDAC-1 and HK-2 protein expression in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Immunocytochemical staining was performed to determine the expression levels of HK-2 (green) and VDAC-1 (red) (Hoechst 33342 (blue) (×400 magnification). Quantitative analysis of the relative expression levels of (B) HK-2 and (C) VDAC-1 after treatment. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene; HK-2, hexokinase-2; VDAC, voltage-dependent anion channel.

    Article Snippet: Searching the sample genotypes in the ATCC/DSMZ/JCRB/RIKEN/ECACC/ExPASy STR database, the test sample result matched the HT-29 cell line.

    Techniques: Expressing, Staining

    Effect of α-PA combined with 5-FU treatment on cytochrome c and caspase mRNA expression in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. Reverse transcription-quantitative polymerase chain reaction analysis of the mRNA expression levels of (A) caspase-8, (B) Bid, (C) cytochrome c , (D) caspase-9 and (E) caspase-3. T. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.

    Journal: Oncology Reports

    Article Title: α‑Phellandrene enhances the apoptosis of HT‑29 cells induced by 5‑fluorouracil by modulating the mitochondria‑dependent pathway

    doi: 10.3892/or.2024.8720

    Figure Lengend Snippet: Effect of α-PA combined with 5-FU treatment on cytochrome c and caspase mRNA expression in HT-29 cells. HT-29 cells (1.0×10 5 cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. Reverse transcription-quantitative polymerase chain reaction analysis of the mRNA expression levels of (A) caspase-8, (B) Bid, (C) cytochrome c , (D) caspase-9 and (E) caspase-3. T. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; # P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.

    Article Snippet: Searching the sample genotypes in the ATCC/DSMZ/JCRB/RIKEN/ECACC/ExPASy STR database, the test sample result matched the HT-29 cell line.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Possible mechanisms by which α-phellandrene combined with 5-FU treatment induces apoptosis in human colon cancer HT-29 cells by regulating the mitochondria-dependent pathway. 5-FU, 5-fluorouracil; HK-2, hexokinase-2; MMP, mitochondrial membrane potential; VDAC, voltage-dependent anion channel.

    Journal: Oncology Reports

    Article Title: α‑Phellandrene enhances the apoptosis of HT‑29 cells induced by 5‑fluorouracil by modulating the mitochondria‑dependent pathway

    doi: 10.3892/or.2024.8720

    Figure Lengend Snippet: Possible mechanisms by which α-phellandrene combined with 5-FU treatment induces apoptosis in human colon cancer HT-29 cells by regulating the mitochondria-dependent pathway. 5-FU, 5-fluorouracil; HK-2, hexokinase-2; MMP, mitochondrial membrane potential; VDAC, voltage-dependent anion channel.

    Article Snippet: Searching the sample genotypes in the ATCC/DSMZ/JCRB/RIKEN/ECACC/ExPASy STR database, the test sample result matched the HT-29 cell line.

    Techniques: Membrane

    Freshly trypsinized HT-29 cells were seeded into 96-well ULA plates, types A-F, at a density of 500 cells per well and then cultured for up to four days. Spheroid morphology was visualized daily using automated brightfield microscopy. a) Representative micrographs showing individual spheroids from day 1 to day 4 (d1-d4) in plate types A-F. Scalebars, 100 µm. b-c) Box plots depicting spheroid diameters (b) or eccentricity (c) as a function of plate type A-F. Data are from 3 experiments with ≥ 24 spheroids per experiment (mean ± SD, 25- and 75-percentiles are plotted). Complete significance analysis, see Fig. S4. * , values significantly different compared to all other plate types for samples from the same day.

    Journal: bioRxiv

    Article Title: A Multiparametric Analysis Reveals Differential Behavior of Spheroid Cultures on Distinct Ultra-Low Attachment Plates Types

    doi: 10.1101/2024.03.26.586778

    Figure Lengend Snippet: Freshly trypsinized HT-29 cells were seeded into 96-well ULA plates, types A-F, at a density of 500 cells per well and then cultured for up to four days. Spheroid morphology was visualized daily using automated brightfield microscopy. a) Representative micrographs showing individual spheroids from day 1 to day 4 (d1-d4) in plate types A-F. Scalebars, 100 µm. b-c) Box plots depicting spheroid diameters (b) or eccentricity (c) as a function of plate type A-F. Data are from 3 experiments with ≥ 24 spheroids per experiment (mean ± SD, 25- and 75-percentiles are plotted). Complete significance analysis, see Fig. S4. * , values significantly different compared to all other plate types for samples from the same day.

    Article Snippet: HT-29 colon cancer cells (ATCC) were cultured in McCoy’s 5A medium (Capricorn) supplemented with 10 % FBS and 1 % Pen/Strep.

    Techniques: Cell Culture, Microscopy

    Freshly trypsinized HT-29 cells were seeded into 96-well ULA plates, types A-F, at a density of 500 cells per well and then cultured for four days. On day 4, spheroids were fixed, cleared, and stained for nuclei and plasma membrane, for the proliferation marker, Ki-67, and YAP1. Wholemount confocal 3D microscopy and 3D-image segmentation were performed. a) Representative micrographs showing single optical sections through individual spheroids at their largest circumference, from plate types as indicated. Upper panels, DAPI nuclear signals (gray); middle panels, Ki-67 immunofluorescence signals (green); lower panels, overlays. Scalebars, 100 µm. b-e) Box plots depicting the spheroid volumes (b) , the number of nuclei per spheroid (c) , the percentage of Ki-67+ nuclei of all nuclei (d) , and the density of nuclei packing within spheroids (e). (f) Violin plot showing the size distribution of nuclear volumes in HT-29 spheroids as a function of plate type. Data in (b-f) are from 3 experiments with ≥ 24 spheroids per experiment (mean ± SD, 25- and 75-percentiles are plotted). * , values significantly different compared to all other plate types. Complete significance analysis, see Fig. S5. (g) Representative micrograph showing a single optical section through a spheroid wholemount at its largest circumference, from plate type F. Immunofluorescence signals of Ki-67, green; YAP1, cyan; plasma membrane, red. Scalebar, 100 µm. (h) Scatterplots showing values of all segmented cells (59,337-74,649 cells per plate type) for plate types A-F (indicated). Depicted are YAP1 mean intensity per cell as a function of YAP1 N/C ratio (upper row), YAP1 mean intensity per cell as a function of the cell’s distance to spheroid hull (middle row), YAP1 N/C ratio as a function of the cell’s distance to spheroid hull (lower row). Yellow and purple dots represent values of Ki-67- and Ki-67+ cells, respectively.

    Journal: bioRxiv

    Article Title: A Multiparametric Analysis Reveals Differential Behavior of Spheroid Cultures on Distinct Ultra-Low Attachment Plates Types

    doi: 10.1101/2024.03.26.586778

    Figure Lengend Snippet: Freshly trypsinized HT-29 cells were seeded into 96-well ULA plates, types A-F, at a density of 500 cells per well and then cultured for four days. On day 4, spheroids were fixed, cleared, and stained for nuclei and plasma membrane, for the proliferation marker, Ki-67, and YAP1. Wholemount confocal 3D microscopy and 3D-image segmentation were performed. a) Representative micrographs showing single optical sections through individual spheroids at their largest circumference, from plate types as indicated. Upper panels, DAPI nuclear signals (gray); middle panels, Ki-67 immunofluorescence signals (green); lower panels, overlays. Scalebars, 100 µm. b-e) Box plots depicting the spheroid volumes (b) , the number of nuclei per spheroid (c) , the percentage of Ki-67+ nuclei of all nuclei (d) , and the density of nuclei packing within spheroids (e). (f) Violin plot showing the size distribution of nuclear volumes in HT-29 spheroids as a function of plate type. Data in (b-f) are from 3 experiments with ≥ 24 spheroids per experiment (mean ± SD, 25- and 75-percentiles are plotted). * , values significantly different compared to all other plate types. Complete significance analysis, see Fig. S5. (g) Representative micrograph showing a single optical section through a spheroid wholemount at its largest circumference, from plate type F. Immunofluorescence signals of Ki-67, green; YAP1, cyan; plasma membrane, red. Scalebar, 100 µm. (h) Scatterplots showing values of all segmented cells (59,337-74,649 cells per plate type) for plate types A-F (indicated). Depicted are YAP1 mean intensity per cell as a function of YAP1 N/C ratio (upper row), YAP1 mean intensity per cell as a function of the cell’s distance to spheroid hull (middle row), YAP1 N/C ratio as a function of the cell’s distance to spheroid hull (lower row). Yellow and purple dots represent values of Ki-67- and Ki-67+ cells, respectively.

    Article Snippet: HT-29 colon cancer cells (ATCC) were cultured in McCoy’s 5A medium (Capricorn) supplemented with 10 % FBS and 1 % Pen/Strep.

    Techniques: Cell Culture, Staining, Membrane, Marker, Microscopy, Immunofluorescence

    ERα expression positively correlates with tumour promoter expression in colon cancer. Mean immuno-reactive score (IRS) for ( A ) Cysteinyl leukotriene receptor 1 (CysLT 1 R) and ( B ) nuclear β-catenin expression in colorectal cancer (CRC) patients ( n = 267) with negative and positive ERα expression. XY scatter plots of the mRNA levels of ( C ) ERα ( ESR1 ) and CysLT 1 R ( CYSLTR1 ) and ( D ) ERα ( ESR1 ) and β-catenin ( CTNNB1 ) in the GSE39582 public dataset ( n = 566) of CRC patients. Violin plots showing the mRNA expression of ( E ) CYSLTR1 and ( F ) CTNNB1 in CRC patients with low or high ESR1 expression. G Immunohistochemical (IHC) images of ERα expression in the colons of wild-type ( WT ) and Cysltr1 knockout ( Cysltr1 −/− ) mice in a colitis-associated colon cancer (CAC) mouse model (n = 5). Bar graph showing the IRS of ERα expression compared between the WT and Cysltr1 −/− mouse groups. H IHC images of ERα expression in the colons of WT and Apc Min/+ mice, n = 5. Bar graph showing the IRS of ERα expression in the WT and Cysltr1 −/− mouse groups. For both mouse models, four random regions of interest (ROIs; marked with dotted lines) in colon tissue were evaluated for each mouse. Representative images of one ROI are shown as insets. The scale bars represent 2 mm ( G ) and 500 μm ( H ) in the image of the whole colon and 50 μm in the zoomed insets. P values were calculated using an unpaired Student’s t test for the bar graphs in G and H. Relative mRNA expression levels of ESR1 , CYSLTR1 , and CTNNB1 in ( I ) HT-29 and ( J ) Caco-2 CC cells after treatment with PPT (ERα specific agonist, 40 nM) or AZD9496 (ERα specific antagonist, 0.3 nM for 30 min) alone or in combination of PPT (40 nM) with AZD9496 (0.3 nM for 30 min before the PPT treatment). The data are presented as the mean ± SEM ( n = 3 independent experiments). P values < 0.5 were considered significant were calculated using an unpaired Student’s t test

    Journal: Cell Communication and Signaling : CCS

    Article Title: High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer

    doi: 10.1186/s12964-024-01582-1

    Figure Lengend Snippet: ERα expression positively correlates with tumour promoter expression in colon cancer. Mean immuno-reactive score (IRS) for ( A ) Cysteinyl leukotriene receptor 1 (CysLT 1 R) and ( B ) nuclear β-catenin expression in colorectal cancer (CRC) patients ( n = 267) with negative and positive ERα expression. XY scatter plots of the mRNA levels of ( C ) ERα ( ESR1 ) and CysLT 1 R ( CYSLTR1 ) and ( D ) ERα ( ESR1 ) and β-catenin ( CTNNB1 ) in the GSE39582 public dataset ( n = 566) of CRC patients. Violin plots showing the mRNA expression of ( E ) CYSLTR1 and ( F ) CTNNB1 in CRC patients with low or high ESR1 expression. G Immunohistochemical (IHC) images of ERα expression in the colons of wild-type ( WT ) and Cysltr1 knockout ( Cysltr1 −/− ) mice in a colitis-associated colon cancer (CAC) mouse model (n = 5). Bar graph showing the IRS of ERα expression compared between the WT and Cysltr1 −/− mouse groups. H IHC images of ERα expression in the colons of WT and Apc Min/+ mice, n = 5. Bar graph showing the IRS of ERα expression in the WT and Cysltr1 −/− mouse groups. For both mouse models, four random regions of interest (ROIs; marked with dotted lines) in colon tissue were evaluated for each mouse. Representative images of one ROI are shown as insets. The scale bars represent 2 mm ( G ) and 500 μm ( H ) in the image of the whole colon and 50 μm in the zoomed insets. P values were calculated using an unpaired Student’s t test for the bar graphs in G and H. Relative mRNA expression levels of ESR1 , CYSLTR1 , and CTNNB1 in ( I ) HT-29 and ( J ) Caco-2 CC cells after treatment with PPT (ERα specific agonist, 40 nM) or AZD9496 (ERα specific antagonist, 0.3 nM for 30 min) alone or in combination of PPT (40 nM) with AZD9496 (0.3 nM for 30 min before the PPT treatment). The data are presented as the mean ± SEM ( n = 3 independent experiments). P values < 0.5 were considered significant were calculated using an unpaired Student’s t test

    Article Snippet: The human CC cell lines HT-29 and Caco-2 were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).

    Techniques: Expressing, Immunohistochemical staining, Knock-Out

    ERα activation in colon cancer cells promotes survival. A Alterations in the colonies formed by HT-29 and Caco-2 colon cancer (CC) cells treated with PPT (40 nM) alone for 48 h or in combination with AZD9496 (0.3 nM, for 30 min before PPT treatment). Bar graphs show the percentage of survival and are representative of n = 3 independent experiments. B Western blots showing the protein levels of phospho-β-catenin (Ser33/37/Thr41), non-phospho (active)-β-catenin, total β-catenin, and ERα in HT-29 and Caco-2 cells untreated or treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, for 30 min). Graphs showing the densitometric analysis of alterations in phospho- and non-phospho (active)-β-catenin and ERα protein levels as percentages of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. C Alterations in the colonies formed by HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment for 48 h. The graphs show the percentage of survival in each group. D Western blots showing the protein levels of non-phospho (active)-β-catenin, total β-catenin, and ERα in both HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment. Graphs showing the densitometric analysis of alterations in ERα and non-phospho (active)-β-catenin protein levels as percentages of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. The data are presented as the means ± SEMs. P values < 0.5 were considered significant calculated using an unpaired Student’s t test

    Journal: Cell Communication and Signaling : CCS

    Article Title: High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer

    doi: 10.1186/s12964-024-01582-1

    Figure Lengend Snippet: ERα activation in colon cancer cells promotes survival. A Alterations in the colonies formed by HT-29 and Caco-2 colon cancer (CC) cells treated with PPT (40 nM) alone for 48 h or in combination with AZD9496 (0.3 nM, for 30 min before PPT treatment). Bar graphs show the percentage of survival and are representative of n = 3 independent experiments. B Western blots showing the protein levels of phospho-β-catenin (Ser33/37/Thr41), non-phospho (active)-β-catenin, total β-catenin, and ERα in HT-29 and Caco-2 cells untreated or treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, for 30 min). Graphs showing the densitometric analysis of alterations in phospho- and non-phospho (active)-β-catenin and ERα protein levels as percentages of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. C Alterations in the colonies formed by HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment for 48 h. The graphs show the percentage of survival in each group. D Western blots showing the protein levels of non-phospho (active)-β-catenin, total β-catenin, and ERα in both HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment. Graphs showing the densitometric analysis of alterations in ERα and non-phospho (active)-β-catenin protein levels as percentages of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. The data are presented as the means ± SEMs. P values < 0.5 were considered significant calculated using an unpaired Student’s t test

    Article Snippet: The human CC cell lines HT-29 and Caco-2 were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).

    Techniques: Activation Assay, Western Blot, Transfection

    Activation of ERα promotes colon cancer cell metastasis. An external dataset composed of data for CC patients with liver metastasis (GSE77955, n = 18) was used to analyse the correlations of CysLT 1 R ( CYSLTR1 ) and β-catenin ( CTNNB1 ) with ERα ( ESR1 ). The scatter plots show the positive correlations between ESR1 and both ( A ) CYSLTR1 and ( B ) CTNNB1 . C Schematic cartoon showing the zebrafish embryo-based colon cancer metastasis model. DiI-labelled HT-29 cells left untreated or treated with PPT alone or in combination with AZD9496 were injected into the perivitelline space of 2 dpf zebrafish embryos, and the embryos were incubated for 48 h. Images showing the metastatic spread of HT-29 cells in the tail veins of zebrafish embryos in each group (CTRL, n = 30; PPT, n = 30; AZD9496 + PPT, n = 43). Scale bars: full-size images; 10 μm, insets; 2 μm. The insets show the regions enclosed in the dotted lines in the full-size tail images. The arrows point to the metastatic foci and transendothelial migration of cancer cells. D Graphs showing the number of embryos with (M1, mets) or without (M0, no mets) metastasis in each group and D′, quantification of tail vein metastasis using the mean fluorescence intensity (MFI) of the embryos with metastasis. E Western blots showing the expression of the tight junction protein ZO-1 in HT-29 and Caco-2 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Graph showing the densitometric analysis of alterations in protein expression as a percentage of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. For the bar graphs, unpaired t-test was used. F Immunofluorescence analysis of ZO-1 and Occludin expression in HT-29 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Greyscale images (insets) showing a representative region of interest (dotted line) for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 (CTRL, n = 116; PPT, n = 105, AZD9496 + PPT, n = 107) and Occludin in random cell-cell junctions (CTRL, n = 103; PPT, n = 110, AZD9496 + PPT, n = 108). P values were calculated with unpaired Student’s t test. The arrows indicate gaps in ZO-1 expression. G Immunofluorescence analysis of ZO-1 and Occludin expression in Caco-2 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 (CTRL, n = 108; PPT, n = 116, AZD9496 + PPT, n = 105) and Occludin (CTRL, n = 105; PPT, n = 115, AZD9496 + PPT, n = 116) in random cell-cell junctions. The arrows indicate gaps in ZO-1 or Occludin expression. H Immunofluorescence analysis of ZO-1 in HT-29 cell-derived colonospheres treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Scale bars: 10 μm. Violin plot showing the mean fluorescence intensity of ZO-1 in random colonospheres (CTRL, n = 31; PPT, n = 28, AZD9496 + PPT, n = 30). The arrows indicate ZO-1 expression in the disseminated cells from the colonospheres. I Immunofluorescence analysis of ZO-1 in Caco-2 CC cell-derived colonospheres treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Scale bars: 10 μm. Violin plot showing the mean fluorescence intensity of ZO-1 in random colonospheres (CTRL, n = 29; PPT, n = 26, AZD9496 + PPT, n = 28). The data are presented as the mean ± SEM of three experiments. P values < 0.5 were considered significant were calculated using the chi-square test in D and an unpaired Student’s t test in D′-I

    Journal: Cell Communication and Signaling : CCS

    Article Title: High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer

    doi: 10.1186/s12964-024-01582-1

    Figure Lengend Snippet: Activation of ERα promotes colon cancer cell metastasis. An external dataset composed of data for CC patients with liver metastasis (GSE77955, n = 18) was used to analyse the correlations of CysLT 1 R ( CYSLTR1 ) and β-catenin ( CTNNB1 ) with ERα ( ESR1 ). The scatter plots show the positive correlations between ESR1 and both ( A ) CYSLTR1 and ( B ) CTNNB1 . C Schematic cartoon showing the zebrafish embryo-based colon cancer metastasis model. DiI-labelled HT-29 cells left untreated or treated with PPT alone or in combination with AZD9496 were injected into the perivitelline space of 2 dpf zebrafish embryos, and the embryos were incubated for 48 h. Images showing the metastatic spread of HT-29 cells in the tail veins of zebrafish embryos in each group (CTRL, n = 30; PPT, n = 30; AZD9496 + PPT, n = 43). Scale bars: full-size images; 10 μm, insets; 2 μm. The insets show the regions enclosed in the dotted lines in the full-size tail images. The arrows point to the metastatic foci and transendothelial migration of cancer cells. D Graphs showing the number of embryos with (M1, mets) or without (M0, no mets) metastasis in each group and D′, quantification of tail vein metastasis using the mean fluorescence intensity (MFI) of the embryos with metastasis. E Western blots showing the expression of the tight junction protein ZO-1 in HT-29 and Caco-2 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Graph showing the densitometric analysis of alterations in protein expression as a percentage of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. For the bar graphs, unpaired t-test was used. F Immunofluorescence analysis of ZO-1 and Occludin expression in HT-29 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Greyscale images (insets) showing a representative region of interest (dotted line) for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 (CTRL, n = 116; PPT, n = 105, AZD9496 + PPT, n = 107) and Occludin in random cell-cell junctions (CTRL, n = 103; PPT, n = 110, AZD9496 + PPT, n = 108). P values were calculated with unpaired Student’s t test. The arrows indicate gaps in ZO-1 expression. G Immunofluorescence analysis of ZO-1 and Occludin expression in Caco-2 cells treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 (CTRL, n = 108; PPT, n = 116, AZD9496 + PPT, n = 105) and Occludin (CTRL, n = 105; PPT, n = 115, AZD9496 + PPT, n = 116) in random cell-cell junctions. The arrows indicate gaps in ZO-1 or Occludin expression. H Immunofluorescence analysis of ZO-1 in HT-29 cell-derived colonospheres treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Scale bars: 10 μm. Violin plot showing the mean fluorescence intensity of ZO-1 in random colonospheres (CTRL, n = 31; PPT, n = 28, AZD9496 + PPT, n = 30). The arrows indicate ZO-1 expression in the disseminated cells from the colonospheres. I Immunofluorescence analysis of ZO-1 in Caco-2 CC cell-derived colonospheres treated with PPT (40 nM) alone or in combination with AZD9496 (0.3 nM, 30 min). Scale bars: 10 μm. Violin plot showing the mean fluorescence intensity of ZO-1 in random colonospheres (CTRL, n = 29; PPT, n = 26, AZD9496 + PPT, n = 28). The data are presented as the mean ± SEM of three experiments. P values < 0.5 were considered significant were calculated using the chi-square test in D and an unpaired Student’s t test in D′-I

    Article Snippet: The human CC cell lines HT-29 and Caco-2 were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).

    Techniques: Activation Assay, Injection, Incubation, Migration, Fluorescence, Western Blot, Expressing, Immunofluorescence, Staining, Derivative Assay

    Functional absence of ERα inhibits colon cancer cell metastasis. DiI-labelled HT-29 cells transfected with either siCTRL or siESR1 and treated with or without PPT for 48 h were injected into the perivitelline space of 2 dpf zebrafish embryos, and the embryos were incubated for 48 h. A Images showing the metastatic spread of HT-29 cells in the tail veins of zebrafish embryos in each group ( siCTRL ; CTRL, n = 30; PPT, n = 30; siESR1 ; CTRL, n = 33, PPT, n = 32). Graphs showing A ’, the number of embryos with (M1, mets) or without metastasis (M0, no mets) in each group and A ”, the quantification of tail vein metastasis using the mean fluorescence intensity (MFI) of the embryos with metastasis (M1 group). Scale bars: full-size images; 10 μm, insets; 2 μm. The insets show the regions enclosed in the dotted lines in the full-size tail images. The arrows point to the metastatic foci and transendothelial migration of cancer cells. B Western blots showing the expression of the tight junction protein ZO-1 in HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment. Graphs showing the densitometric analysis of alterations in protein expression as a percentage of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. C Immunofluorescence analysis of ZO-1 and Occludin expression in HT-29 cells transfected with either siCTRL or siESR1 prior to treatment with the ERα agonist PPT (40 nM). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 ( siCTRL (CTRL, n = 105; PPT, n = 115), siESR1 (CTRL, n = 108; PPT, n = 116)) and Occludin ( siCTRL (CTRL, n = 105; PPT, n = 105), siESR1 (CTRL, n = 107; PPT, n = 102)) in random cell-cell junctions. The arrows indicate gaps in ZO-1 or Occludin expression. D Immunofluorescence analysis of ZO-1 and Occludin expression in Caco-2 cells transfected with either siCTRL or siESR1 prior to treatment with the ERα agonist PPT (40 nM). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 and Occludin in random cell junctions. For ZO-1 staining in the siCTRL -transfected group (CTRL, n = 105; PPT, n = 105) and in the siESR1 -transfected group (CTRL, n = 108; PPT, n = 105), random cell junctions were evaluated. For Occludin staining in the siCTRL -transfected group (CTRL, n = 105; PPT, n = 105) and in the siESR1 -transfected group (CTRL, n = 102; PPT, n = 108), random cell junctions were evaluated. The arrows indicate gaps in ZO-1 or Occludin expression. Immunofluorescence analysis of ZO-1 in colonospheres derived from either siCTRL or siESR1 transfected ( E ) HT-29 and ( F ) Caco-2 cells. Scale bars: 10 μm. Violin plots showing the mean fluorescence intensity of ZO-1 in random ( E’ ) HT-29 ( siCTRL , n = 30; siESR1 , n = 32) or ( F′ ) Caco-2 ( siCTRL , n = 28; siESR1 , n = 31) colonospheres. The MFIs of the indicated proteins were measured using ImageJ software (NIH, USA). G Graphical representation of the summary of the study. Upon binding to the agonist PPT, ERα dimerizes and shuttles into the nucleus. This upregulates the transcription of CYSLTR1 and CTNNB1 . In addition, it promotes metastasis by disrupting the tight junction proteins ZO-1 and Occludin. However, blocking the binding of PPT to ERα by employing an antagonist, AZD9496, prevents the activation and hence the dimerization of the receptor. This further leads to downregulation of CYSLTR1 and CTNNB1 and upregulation of the tight junction proteins ZO-1 and Occludin. The data are presented as the mean ± SEM of three experiments. P values were calculated with the chi-square test for A’ and unpaired Student’s t test for A”, B-F

    Journal: Cell Communication and Signaling : CCS

    Article Title: High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer

    doi: 10.1186/s12964-024-01582-1

    Figure Lengend Snippet: Functional absence of ERα inhibits colon cancer cell metastasis. DiI-labelled HT-29 cells transfected with either siCTRL or siESR1 and treated with or without PPT for 48 h were injected into the perivitelline space of 2 dpf zebrafish embryos, and the embryos were incubated for 48 h. A Images showing the metastatic spread of HT-29 cells in the tail veins of zebrafish embryos in each group ( siCTRL ; CTRL, n = 30; PPT, n = 30; siESR1 ; CTRL, n = 33, PPT, n = 32). Graphs showing A ’, the number of embryos with (M1, mets) or without metastasis (M0, no mets) in each group and A ”, the quantification of tail vein metastasis using the mean fluorescence intensity (MFI) of the embryos with metastasis (M1 group). Scale bars: full-size images; 10 μm, insets; 2 μm. The insets show the regions enclosed in the dotted lines in the full-size tail images. The arrows point to the metastatic foci and transendothelial migration of cancer cells. B Western blots showing the expression of the tight junction protein ZO-1 in HT-29 and Caco-2 cells transfected with either siCTRL or siESR1 prior to PPT (40 nM) treatment. Graphs showing the densitometric analysis of alterations in protein expression as a percentage of the loading control (α-tubulin). The blots are representative of n = 3 independent experiments. C Immunofluorescence analysis of ZO-1 and Occludin expression in HT-29 cells transfected with either siCTRL or siESR1 prior to treatment with the ERα agonist PPT (40 nM). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 ( siCTRL (CTRL, n = 105; PPT, n = 115), siESR1 (CTRL, n = 108; PPT, n = 116)) and Occludin ( siCTRL (CTRL, n = 105; PPT, n = 105), siESR1 (CTRL, n = 107; PPT, n = 102)) in random cell-cell junctions. The arrows indicate gaps in ZO-1 or Occludin expression. D Immunofluorescence analysis of ZO-1 and Occludin expression in Caco-2 cells transfected with either siCTRL or siESR1 prior to treatment with the ERα agonist PPT (40 nM). Greyscale images (insets) showing representative regions of interest for ZO-1 and Occludin staining. Scale bars: full-size images; 5 μm, insets; 1 μm. Violin plots showing the mean fluorescence intensity of ZO-1 and Occludin in random cell junctions. For ZO-1 staining in the siCTRL -transfected group (CTRL, n = 105; PPT, n = 105) and in the siESR1 -transfected group (CTRL, n = 108; PPT, n = 105), random cell junctions were evaluated. For Occludin staining in the siCTRL -transfected group (CTRL, n = 105; PPT, n = 105) and in the siESR1 -transfected group (CTRL, n = 102; PPT, n = 108), random cell junctions were evaluated. The arrows indicate gaps in ZO-1 or Occludin expression. Immunofluorescence analysis of ZO-1 in colonospheres derived from either siCTRL or siESR1 transfected ( E ) HT-29 and ( F ) Caco-2 cells. Scale bars: 10 μm. Violin plots showing the mean fluorescence intensity of ZO-1 in random ( E’ ) HT-29 ( siCTRL , n = 30; siESR1 , n = 32) or ( F′ ) Caco-2 ( siCTRL , n = 28; siESR1 , n = 31) colonospheres. The MFIs of the indicated proteins were measured using ImageJ software (NIH, USA). G Graphical representation of the summary of the study. Upon binding to the agonist PPT, ERα dimerizes and shuttles into the nucleus. This upregulates the transcription of CYSLTR1 and CTNNB1 . In addition, it promotes metastasis by disrupting the tight junction proteins ZO-1 and Occludin. However, blocking the binding of PPT to ERα by employing an antagonist, AZD9496, prevents the activation and hence the dimerization of the receptor. This further leads to downregulation of CYSLTR1 and CTNNB1 and upregulation of the tight junction proteins ZO-1 and Occludin. The data are presented as the mean ± SEM of three experiments. P values were calculated with the chi-square test for A’ and unpaired Student’s t test for A”, B-F

    Article Snippet: The human CC cell lines HT-29 and Caco-2 were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).

    Techniques: Functional Assay, Transfection, Injection, Incubation, Fluorescence, Migration, Western Blot, Expressing, Immunofluorescence, Staining, Derivative Assay, Software, Binding Assay, Blocking Assay, Activation Assay

    Analysis of the ∆ sap mutant in adherence to and invasion of HT-29 epithelial cells. Infection analyses were performed following bacterial subculture in LB media supplemented with glucose (glu), with or without bile salts (BS), as well as TSB media with or without bile salts. The average percent recoveries ± standard error of the mean from three biological replicates are plotted relative to the recovery rate for 2457T from the bile salts subcultures (set at 100%). ( a ) In adherence assays following subculture in either LB + glu ± BS or TSB ± BS, the adherence rates were induced for both wild type 2457T and the Δ sap mutant following bile salts exposure (**, p < 0.005). However, the Δ sap mutant had lower adherence following subculture in bile salts compared to 2457T in the same condition (ǂ, p < 0.005). There were no significant differences in adherence between 2457T or the Δ sap mutant following subculturing in media without bile salts. While there is a decrease in adherence in the Δ sap mutant relative to 2457T following TSB subculture without bile salts, the difference was not statistically significant. ( b ) For invasion, there were no significant differences between 2457T and the Δ sap mutant following subculture in LB + glu ± BS. Both strains had significant increases in invasion following exposure to bile salts (**, p < 0.005).

    Journal: Gut Microbes

    Article Title: Gastrointestinal signals in supplemented media reveal a role in adherence for the Shigella flexneri sap autotransporter gene

    doi: 10.1080/19490976.2024.2331985

    Figure Lengend Snippet: Analysis of the ∆ sap mutant in adherence to and invasion of HT-29 epithelial cells. Infection analyses were performed following bacterial subculture in LB media supplemented with glucose (glu), with or without bile salts (BS), as well as TSB media with or without bile salts. The average percent recoveries ± standard error of the mean from three biological replicates are plotted relative to the recovery rate for 2457T from the bile salts subcultures (set at 100%). ( a ) In adherence assays following subculture in either LB + glu ± BS or TSB ± BS, the adherence rates were induced for both wild type 2457T and the Δ sap mutant following bile salts exposure (**, p < 0.005). However, the Δ sap mutant had lower adherence following subculture in bile salts compared to 2457T in the same condition (ǂ, p < 0.005). There were no significant differences in adherence between 2457T or the Δ sap mutant following subculturing in media without bile salts. While there is a decrease in adherence in the Δ sap mutant relative to 2457T following TSB subculture without bile salts, the difference was not statistically significant. ( b ) For invasion, there were no significant differences between 2457T and the Δ sap mutant following subculture in LB + glu ± BS. Both strains had significant increases in invasion following exposure to bile salts (**, p < 0.005).

    Article Snippet: The role of the sap gene product in the adherence and invasion of S. flexneri was evaluated using the human colorectal adenocarcinoma cell line HT-29 (ATCC HTB-38) as previously described.

    Techniques: Mutagenesis, Infection, Subculturing Assay