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    Cell Signaling Technology Inc egfr
    <t>EGFR</t> inhibition decreases tumor progression of human OS cells 143b after orthotopic injection A Western blot analysis showing EGFR and <t>c‐Fos</t> protein levels in human OS cell lines cultured under standard conditions. B Cell viability of 143b or LM7 cells cultured for 24 h in medium (+10% FCS) with DMSO (1:2,000) or erlotinib (10 μM; n = 3, representative result from three independent experiments, shown as fold change, normalized to DMSO). C Cell viability of 143b or LM7 cells cultured for 24 h in medium (+0.5% FCS) containing 1× PBS (contr., 1:200) or EGF (50 ng/ml; n = 3, representative result from three independent experiments, shown as fold change, normalized to control). D Treatment scheme: 143b OS cells (10 6 in 25 μl PBS) were intratibially injected; mice were treated for 14 days with vehicle or erlotinib (50 mg/kg) 7 days after injection, when the tumors started to grow. E 143b xenograft growth curve during therapeutic regime ( n = 8 vehicle, 6 erlotinib; two independent experiments). F 143b tumor weight at endpoint ( n = 8 vehicle, 6 erlotinib; two independent experiments). G Treatment scheme: LM7 OS cells (10 6 in 25 μl PBS) were intratibially injected; mice were treated for 21 days with vehicle or erlotinib (50 mg/kg) 42 days after injection, when the tumors started to grow. H LM7 xenograft growth curve during therapeutic regime ( n = 7 vehicle, 5 erlotinib; two independent experiments). I LM7 tumor weight at endpoint ( n = 7 vehicle, 5 erlotinib, two independent experiments). J IHC analysis of PCNA and cleaved caspase‐3 in 143b‐derived tumors ( n = 4). Scale bars: 50 μm. K IHC analysis of pCREB and c‐Fos in 143b‐derived tumors ( n = 5 for pCREB and n = 4 for c‐Fos analysis). Scale bars: 50 μm. L Western blot analysis of pCREB/CREB and p‐c‐Fos/c‐Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or erlotinib treatment. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test (B, C, F, I, J, and K) or by two‐way ANOVA followed by Bonferroni multiple comparison test (E and H). Source data are available online for this figure.
    Egfr, supplied by Cell Signaling Technology Inc, 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/egfr/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egfr - by Bioz Stars, 2021-06
    86/100 stars
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    86
    Santa Cruz Biotechnology egfr
    <t>miR-138</t> and miR-204 regulate <t>EGFR</t> expression via post-transcriptional level in SGC7901 and MGC803 cells. (A) Suppressed EGFR protein expression after transfection with miR-138 or miR-204 mimics using western blotting and the correlated quantitative analysis in SGC7901 cells (n=3). (B) Relative level of EGFR mRNA after transfection with miR-138 or miR-204 mimics, inhibitors, and the relevant negative control in SGC7901 cells (n=3). (C) Suppressed EGFR protein expression after transfection with miR-138 or miR-204 mimics using western blotting and the correlated quantitative analysis in MGC803 cells (n=3). (D) Relative level of EGFR mRNA after transfection with miR-138 or miR-204 mimics, inhibitors, and the relevant negative control in MGC803 cells (n=3). **P
    Egfr, supplied by Santa Cruz Biotechnology, 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/egfr/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egfr - by Bioz Stars, 2021-06
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    99
    Millipore a549 cells
    LXA4 restored ENaC subunits' mRNA expressions in <t>A549</t> cells challenged by LPS. (a) LXA4 rescued ENaC-α subunit mRNA expression in A549 cells treated with LPS. The mRNA levels were determined by real-time PCR analysis. The treated groups were Control, LPS (1 μg/ml), LXA4 (100 nmol/L), and in combination. * P = 0.006, LPS versus LPS + LXA4. (b) LXA4 restored EnaC-γ subunit mRNA expression in A549 cells treated with LPS. † P = 0.026, LPS versus LPS + LXA4. LXA4: Lipoxin A4; LPS: Lipopolysaccharide; NDRG1: N-myc downstream-regulated gene 1; ENaC-α: Epithelial sodium channel α subunit; ENaC-γ: Epithelial sodium channel γ subunit; PCR: Polymerase chain reaction.
    A549 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a549 cells - by Bioz Stars, 2021-06
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    86
    Cell Signaling Technology Inc anti egfr
    Wnt signalling activation affects <t>EGFR–ERK</t> signalling dynamics through regulating expression of multiple molecules. a A volcano plot depicting the fold changes in gene expression levels between normal (ENR) and CHIR99021-treated organoids (ENR + CHIR), and statistical significance of the changes. b – e Enrichment plots from gene set enrichment analysis (GSEA). GESA plots for genes downregulated after Apc knockout ( b ), genes upregulated after Apc knockout through Myc ( c ), genes downregulated in colorectal adenoma ( d ), and genes upregulated in colorectal adenoma ( e ) are shown. f , g RT-PCR analysis revealed that Egfl6, Flna, and Troy were upregulated, and that Lrig3 was downregulated in both CHIR99021-treated and adenoma-derived organoids. The relative mRNA levels of indicated genes in normal (ENR) versus CHIR99021-treated organoids (ENR + CHIR) ( f ), and those in normal versus adenoma-derived organoids ( g ) are shown ( n = 3 samples containing more than ten organoids). h , i Adenoma-derived organoids expressing the ERK biosensor were infected with lentiviruses expressing control vector (control), Lrig3 (Lrig3), or shRNAs for Egfl6 (shEgfl6), Flna (shFlna), or Troy (shTroy). h Bee swarm plots of ERK activity in organoids before (Pre) and after (Post) EGFR inhibitor treatment under each condition ( n = 80 cells pooled from two organoids). i The frequency of ERK activity pulses under each condition. Time-lapse imaging was performed for 90 min ( n = 50 cells). j Schematic representation of ERK activity dynamics generated by kinase activity of EGFR and <t>ErbB2</t> in the normal and Wnt signalling-activated intestinal epithelia. Red lines represent mean. Error bars represent s.e.m. Welch’s t test ( f , g ), Mann–Whitney U -test ( h ), and Steel–Dwass test ( i ) were used for comparison.* P
    Anti Egfr, supplied by Cell Signaling Technology Inc, 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/anti egfr/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti egfr - by Bioz Stars, 2021-06
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    N/A
    EGFR Cytoplasmic Mouse Monoclonal
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    N/A
    Applications This cell line has been used for in vivo studies of molecular targeting of EGFR in synegeneic Fischer Rats using either the ligand EGF ref or monoclonal antibodies directed
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    N/A
    EGFR inhibitor is a cell permeable 4 6 disubstituted pyrimidine compound that selectively inhibits the EGFR kinase with an IC value of 21 nM in vitro and blocks receptor autophosphorylation
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    N/A
    Purified recombinant EGFR protein
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    Image Search Results


    EGFR inhibition decreases tumor progression of human OS cells 143b after orthotopic injection A Western blot analysis showing EGFR and c‐Fos protein levels in human OS cell lines cultured under standard conditions. B Cell viability of 143b or LM7 cells cultured for 24 h in medium (+10% FCS) with DMSO (1:2,000) or erlotinib (10 μM; n = 3, representative result from three independent experiments, shown as fold change, normalized to DMSO). C Cell viability of 143b or LM7 cells cultured for 24 h in medium (+0.5% FCS) containing 1× PBS (contr., 1:200) or EGF (50 ng/ml; n = 3, representative result from three independent experiments, shown as fold change, normalized to control). D Treatment scheme: 143b OS cells (10 6 in 25 μl PBS) were intratibially injected; mice were treated for 14 days with vehicle or erlotinib (50 mg/kg) 7 days after injection, when the tumors started to grow. E 143b xenograft growth curve during therapeutic regime ( n = 8 vehicle, 6 erlotinib; two independent experiments). F 143b tumor weight at endpoint ( n = 8 vehicle, 6 erlotinib; two independent experiments). G Treatment scheme: LM7 OS cells (10 6 in 25 μl PBS) were intratibially injected; mice were treated for 21 days with vehicle or erlotinib (50 mg/kg) 42 days after injection, when the tumors started to grow. H LM7 xenograft growth curve during therapeutic regime ( n = 7 vehicle, 5 erlotinib; two independent experiments). I LM7 tumor weight at endpoint ( n = 7 vehicle, 5 erlotinib, two independent experiments). J IHC analysis of PCNA and cleaved caspase‐3 in 143b‐derived tumors ( n = 4). Scale bars: 50 μm. K IHC analysis of pCREB and c‐Fos in 143b‐derived tumors ( n = 5 for pCREB and n = 4 for c‐Fos analysis). Scale bars: 50 μm. L Western blot analysis of pCREB/CREB and p‐c‐Fos/c‐Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or erlotinib treatment. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test (B, C, F, I, J, and K) or by two‐way ANOVA followed by Bonferroni multiple comparison test (E and H). Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: EGFR is required for FOS‐dependent bone tumor development via RSK2/CREB signaling

    doi: 10.15252/emmm.201809408

    Figure Lengend Snippet: EGFR inhibition decreases tumor progression of human OS cells 143b after orthotopic injection A Western blot analysis showing EGFR and c‐Fos protein levels in human OS cell lines cultured under standard conditions. B Cell viability of 143b or LM7 cells cultured for 24 h in medium (+10% FCS) with DMSO (1:2,000) or erlotinib (10 μM; n = 3, representative result from three independent experiments, shown as fold change, normalized to DMSO). C Cell viability of 143b or LM7 cells cultured for 24 h in medium (+0.5% FCS) containing 1× PBS (contr., 1:200) or EGF (50 ng/ml; n = 3, representative result from three independent experiments, shown as fold change, normalized to control). D Treatment scheme: 143b OS cells (10 6 in 25 μl PBS) were intratibially injected; mice were treated for 14 days with vehicle or erlotinib (50 mg/kg) 7 days after injection, when the tumors started to grow. E 143b xenograft growth curve during therapeutic regime ( n = 8 vehicle, 6 erlotinib; two independent experiments). F 143b tumor weight at endpoint ( n = 8 vehicle, 6 erlotinib; two independent experiments). G Treatment scheme: LM7 OS cells (10 6 in 25 μl PBS) were intratibially injected; mice were treated for 21 days with vehicle or erlotinib (50 mg/kg) 42 days after injection, when the tumors started to grow. H LM7 xenograft growth curve during therapeutic regime ( n = 7 vehicle, 5 erlotinib; two independent experiments). I LM7 tumor weight at endpoint ( n = 7 vehicle, 5 erlotinib, two independent experiments). J IHC analysis of PCNA and cleaved caspase‐3 in 143b‐derived tumors ( n = 4). Scale bars: 50 μm. K IHC analysis of pCREB and c‐Fos in 143b‐derived tumors ( n = 5 for pCREB and n = 4 for c‐Fos analysis). Scale bars: 50 μm. L Western blot analysis of pCREB/CREB and p‐c‐Fos/c‐Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or erlotinib treatment. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test (B, C, F, I, J, and K) or by two‐way ANOVA followed by Bonferroni multiple comparison test (E and H). Source data are available online for this figure.

    Article Snippet: For analysis of EGFR/c‐Fos expression in human OS, two consecutive sections from a commercially available TMA (Novus, NBP2‐30289) were stained with antibodies directed against EGFR (Cell Signaling Technology, #4267) and c‐Fos (Santa Cruz Biotechnology, SC‐52).

    Techniques: Inhibition, Injection, Western Blot, Cell Culture, Mouse Assay, Immunohistochemistry, Derivative Assay, Expressing, Isolation, Two Tailed Test

    EGFR signaling is essential for c‐Fos‐dependent bone tumor formation A X‐ray analysis of 6‐month‐old H2 ‐c‐fos LTR/Egfr wa/+ and H2 ‐c‐fos LTR/Egfr wa/wa littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 5–6 months of age ( n = 23 wa/+, 15 wa/wa). C Quantification of tumor size in tibiae at 5–6 months of age ( n = 23 wa/+, 15 wa/wa). D Alkaline phosphatase (ALP) levels in the serum at endpoint (age = 5–6 months; n = 16 wa/+, 11 wa/wa). E X‐ray analysis before (age = 2 months) and after (age = 7 months) vehicle or erlotinib treatment. Scale bars: 1 cm. F Quantification of tumor number during treatment ( n = 6 vehicle, 5 erlotinib). G Tumor size during treatment ( n = 6 vehicle, 5 erlotinib). H Analysis of serum ALP levels during treatment ( n = 5). I PET summation images (0–90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [ 11 C]erlotinib distribution in one H2‐ c‐fos LTR/ Egfr wt mouse (M125). Anatomical structures are labeled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1 cm. J Concentration–time curves of [ 11 C]erlotinib in bone tumors in right scapula of three H2‐ c‐fos LTR/ Egfr wt mice measured with PET. Broken line indicates threshold for in vitro effect of erlotinib (1 μM). Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test (B–D) or by two‐way ANOVA followed by Bonferroni multiple comparison test (F–H).

    Journal: EMBO Molecular Medicine

    Article Title: EGFR is required for FOS‐dependent bone tumor development via RSK2/CREB signaling

    doi: 10.15252/emmm.201809408

    Figure Lengend Snippet: EGFR signaling is essential for c‐Fos‐dependent bone tumor formation A X‐ray analysis of 6‐month‐old H2 ‐c‐fos LTR/Egfr wa/+ and H2 ‐c‐fos LTR/Egfr wa/wa littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 5–6 months of age ( n = 23 wa/+, 15 wa/wa). C Quantification of tumor size in tibiae at 5–6 months of age ( n = 23 wa/+, 15 wa/wa). D Alkaline phosphatase (ALP) levels in the serum at endpoint (age = 5–6 months; n = 16 wa/+, 11 wa/wa). E X‐ray analysis before (age = 2 months) and after (age = 7 months) vehicle or erlotinib treatment. Scale bars: 1 cm. F Quantification of tumor number during treatment ( n = 6 vehicle, 5 erlotinib). G Tumor size during treatment ( n = 6 vehicle, 5 erlotinib). H Analysis of serum ALP levels during treatment ( n = 5). I PET summation images (0–90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [ 11 C]erlotinib distribution in one H2‐ c‐fos LTR/ Egfr wt mouse (M125). Anatomical structures are labeled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1 cm. J Concentration–time curves of [ 11 C]erlotinib in bone tumors in right scapula of three H2‐ c‐fos LTR/ Egfr wt mice measured with PET. Broken line indicates threshold for in vitro effect of erlotinib (1 μM). Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test (B–D) or by two‐way ANOVA followed by Bonferroni multiple comparison test (F–H).

    Article Snippet: For analysis of EGFR/c‐Fos expression in human OS, two consecutive sections from a commercially available TMA (Novus, NBP2‐30289) were stained with antibodies directed against EGFR (Cell Signaling Technology, #4267) and c‐Fos (Santa Cruz Biotechnology, SC‐52).

    Techniques: ALP Assay, Positron Emission Tomography, Labeling, Concentration Assay, Mouse Assay, In Vitro, Two Tailed Test

    EGFR controls c‐Fos via MAPK‐dependent CREB activation in primary OS cells A Western blot analysis of primary OS cells isolated from H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice. B c‐fos and transgenic c‐fos ( c fos tg ) mRNA expression levels in primary H2‐ c‐fos LTR OS cells after 4× in vitro passages, cultured under standard conditions ( n = 3 independent cell lines). C Western blot analysis of H2 ‐c‐fos LTR/Egfr wt OS cells treated for 24 h with erlotinib. D c‐fos and c‐fos tg mRNA expression levels in H2 ‐c‐fos LTR/Egfr wt OS cells treated for 24 h with erlotinib (10 μM) or DMSO as control ( n = 4 independent cell lines). E, F Western blot analysis of starved H2 ‐c‐fos LTR/Egfr wt OS cells, pre‐treated with DMSO (1:1,000), afatinib (5 μM), GSK2233470 (10 μM), rapamycin (10 nM), or U0126 (10 μM) for 30 min and stimulated with EGF (50 ng/ml) as indicated. G Western blot analysis of primary OS cells isolated from a p53 f/f Rb1 f/f Osx ‐Cre mouse after 24‐h erlotinib treatment. H c‐fos mRNA expression levels in p53 f/f Rb1 f/f Osx ‐Cre OS cells treated for 24 h with erlotinib (10 μM; n = 3). I Western blot analysis of starved p53 f/f Rb1 f/f Osx ‐Cre OS cells, pre‐treated with DMSO (1:1,000) or afatinib (5 μM) for 30 min and stimulated with EGF (50 ng/ml) as indicated. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: EGFR is required for FOS‐dependent bone tumor development via RSK2/CREB signaling

    doi: 10.15252/emmm.201809408

    Figure Lengend Snippet: EGFR controls c‐Fos via MAPK‐dependent CREB activation in primary OS cells A Western blot analysis of primary OS cells isolated from H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice. B c‐fos and transgenic c‐fos ( c fos tg ) mRNA expression levels in primary H2‐ c‐fos LTR OS cells after 4× in vitro passages, cultured under standard conditions ( n = 3 independent cell lines). C Western blot analysis of H2 ‐c‐fos LTR/Egfr wt OS cells treated for 24 h with erlotinib. D c‐fos and c‐fos tg mRNA expression levels in H2 ‐c‐fos LTR/Egfr wt OS cells treated for 24 h with erlotinib (10 μM) or DMSO as control ( n = 4 independent cell lines). E, F Western blot analysis of starved H2 ‐c‐fos LTR/Egfr wt OS cells, pre‐treated with DMSO (1:1,000), afatinib (5 μM), GSK2233470 (10 μM), rapamycin (10 nM), or U0126 (10 μM) for 30 min and stimulated with EGF (50 ng/ml) as indicated. G Western blot analysis of primary OS cells isolated from a p53 f/f Rb1 f/f Osx ‐Cre mouse after 24‐h erlotinib treatment. H c‐fos mRNA expression levels in p53 f/f Rb1 f/f Osx ‐Cre OS cells treated for 24 h with erlotinib (10 μM; n = 3). I Western blot analysis of starved p53 f/f Rb1 f/f Osx ‐Cre OS cells, pre‐treated with DMSO (1:1,000) or afatinib (5 μM) for 30 min and stimulated with EGF (50 ng/ml) as indicated. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test. Source data are available online for this figure.

    Article Snippet: For analysis of EGFR/c‐Fos expression in human OS, two consecutive sections from a commercially available TMA (Novus, NBP2‐30289) were stained with antibodies directed against EGFR (Cell Signaling Technology, #4267) and c‐Fos (Santa Cruz Biotechnology, SC‐52).

    Techniques: Activation Assay, Western Blot, Isolation, Mouse Assay, Transgenic Assay, Expressing, In Vitro, Cell Culture, Two Tailed Test

    Osteoblast‐specific EGFR deletion reduces c‐Fos‐driven OS formation A X‐ray analysis of 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 6–7 months of age ( n = 22 wt, 11 ΔOb). C Quantification of tumor size in tibiae ( n = 22 wt, 11 ΔOb). D ALP levels in the serum at 7‐month endpoint ( n = 17 wt, 8 ΔOb). E μPET/CT analysis of 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. F Standardized uptake values (SUV) of the μPET tracer Na[ 18 F]F in the pelvic OS of 4‐ and 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice. n = 6 wt, 4 ΔOb for 4‐month time‐point, n = 6 wt, 3 ΔOb for 7‐month time‐point. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test.

    Journal: EMBO Molecular Medicine

    Article Title: EGFR is required for FOS‐dependent bone tumor development via RSK2/CREB signaling

    doi: 10.15252/emmm.201809408

    Figure Lengend Snippet: Osteoblast‐specific EGFR deletion reduces c‐Fos‐driven OS formation A X‐ray analysis of 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 6–7 months of age ( n = 22 wt, 11 ΔOb). C Quantification of tumor size in tibiae ( n = 22 wt, 11 ΔOb). D ALP levels in the serum at 7‐month endpoint ( n = 17 wt, 8 ΔOb). E μPET/CT analysis of 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. F Standardized uptake values (SUV) of the μPET tracer Na[ 18 F]F in the pelvic OS of 4‐ and 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice. n = 6 wt, 4 ΔOb for 4‐month time‐point, n = 6 wt, 3 ΔOb for 7‐month time‐point. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test.

    Article Snippet: For analysis of EGFR/c‐Fos expression in human OS, two consecutive sections from a commercially available TMA (Novus, NBP2‐30289) were stained with antibodies directed against EGFR (Cell Signaling Technology, #4267) and c‐Fos (Santa Cruz Biotechnology, SC‐52).

    Techniques: ALP Assay, Mouse Assay, Two Tailed Test

    EGFR is essential for proliferation, survival and c‐Fos protein and mRNA expression via RSK2/CREB phosphorylation A PCNA and cleaved caspase‐3 IHC staining and quantification shown as % positive cells (for PCNA) and as positive cells per mm 2 (for cleaved caspase‐3) in OS from H2 ‐c‐fos LTR/Egfr wt ( n = 6) and H2 ‐c‐fos LTR/Egfr ΔOb ( n = 5) mice. Scale bars: 100 μm. B Egfr , Ccnd1 , c‐fos and transgenic c‐fos ( c‐fos tg ) mRNA expression levels in tumors of H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice normalized to Tbp . n = 17 wt, 14 ΔOb ( Egfr, Ccnd1 ), n = 16 wt, 13 ΔOb ( c‐fos ), n = 16 wt, 14 ΔOb ( c‐fos tg ). C IHC staining and quantification showing pRSK2‐ ( n = 4), pCREB‐ ( n = 7 wt, 6 ΔOb), and c‐Fos ( n = 5)‐positive cells (%) in OS from H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. Scale bars: 100 μm. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test.

    Journal: EMBO Molecular Medicine

    Article Title: EGFR is required for FOS‐dependent bone tumor development via RSK2/CREB signaling

    doi: 10.15252/emmm.201809408

    Figure Lengend Snippet: EGFR is essential for proliferation, survival and c‐Fos protein and mRNA expression via RSK2/CREB phosphorylation A PCNA and cleaved caspase‐3 IHC staining and quantification shown as % positive cells (for PCNA) and as positive cells per mm 2 (for cleaved caspase‐3) in OS from H2 ‐c‐fos LTR/Egfr wt ( n = 6) and H2 ‐c‐fos LTR/Egfr ΔOb ( n = 5) mice. Scale bars: 100 μm. B Egfr , Ccnd1 , c‐fos and transgenic c‐fos ( c‐fos tg ) mRNA expression levels in tumors of H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice normalized to Tbp . n = 17 wt, 14 ΔOb ( Egfr, Ccnd1 ), n = 16 wt, 13 ΔOb ( c‐fos ), n = 16 wt, 14 ΔOb ( c‐fos tg ). C IHC staining and quantification showing pRSK2‐ ( n = 4), pCREB‐ ( n = 7 wt, 6 ΔOb), and c‐Fos ( n = 5)‐positive cells (%) in OS from H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. Scale bars: 100 μm. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test.

    Article Snippet: For analysis of EGFR/c‐Fos expression in human OS, two consecutive sections from a commercially available TMA (Novus, NBP2‐30289) were stained with antibodies directed against EGFR (Cell Signaling Technology, #4267) and c‐Fos (Santa Cruz Biotechnology, SC‐52).

    Techniques: Expressing, Immunohistochemistry, Staining, Mouse Assay, Transgenic Assay, Two Tailed Test

    Osteoblast‐specific overexpression of the EGFR ligand Amphiregulin accelerates tumor formation in H2 ‐c‐fos LTR mice A X‐ray analysis of 6‐month‐old H2 ‐c‐fos LTR and H2‐ c‐fos LTR/ ColAREG littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 5–6 months of age ( n = 22 wt, 13 ColAREG ). C Quantification of tumor size in tibiae at 5–6 months of age ( n = 22 wt, 13 ColAREG ). D Alkaline phosphatase (ALP) levels in the serum at endpoint (age = 5–6 months; n = 29 wt, 19 ColAREG ). E c‐fos mRNA expression levels in OSs of H2 ‐c‐fos LTR ( n = 11) and H2 ‐c‐fos LTR/ ColAREG mice ( n = 14). F, G Western blot analysis of bone tumor protein lysates from H2 ‐c‐fos LTR and H2 ‐c‐fos LTR/ ColAREG mice. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: EGFR is required for FOS‐dependent bone tumor development via RSK2/CREB signaling

    doi: 10.15252/emmm.201809408

    Figure Lengend Snippet: Osteoblast‐specific overexpression of the EGFR ligand Amphiregulin accelerates tumor formation in H2 ‐c‐fos LTR mice A X‐ray analysis of 6‐month‐old H2 ‐c‐fos LTR and H2‐ c‐fos LTR/ ColAREG littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 5–6 months of age ( n = 22 wt, 13 ColAREG ). C Quantification of tumor size in tibiae at 5–6 months of age ( n = 22 wt, 13 ColAREG ). D Alkaline phosphatase (ALP) levels in the serum at endpoint (age = 5–6 months; n = 29 wt, 19 ColAREG ). E c‐fos mRNA expression levels in OSs of H2 ‐c‐fos LTR ( n = 11) and H2 ‐c‐fos LTR/ ColAREG mice ( n = 14). F, G Western blot analysis of bone tumor protein lysates from H2 ‐c‐fos LTR and H2 ‐c‐fos LTR/ ColAREG mice. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test. Source data are available online for this figure.

    Article Snippet: For analysis of EGFR/c‐Fos expression in human OS, two consecutive sections from a commercially available TMA (Novus, NBP2‐30289) were stained with antibodies directed against EGFR (Cell Signaling Technology, #4267) and c‐Fos (Santa Cruz Biotechnology, SC‐52).

    Techniques: Over Expression, Mouse Assay, ALP Assay, Expressing, Western Blot, Two Tailed Test

    EGFR and c‐Fos co‐expression in human OS negatively correlates with patient survival A Representative images of OS biopsies stained with antibodies against EGFR and c‐Fos. Scale bars: 50 μm. B Kaplan–Meier survival curve comparing the survival of patients with EGFR and c‐Fos double‐positive OSs against patients without co‐expression of both proteins ( n = 52). C Gene expression correlation analysis of several cancer‐associated RTKs with FOS in human OS (data analyzed from publicly available dataset E‐GEOD‐39058). Data information: P ‐values were calculated by log‐rank (Mantel–Cox) test comparing the two Kaplan–Meier curves (B) or by unpaired, two‐tailed t ‐test (C).

    Journal: EMBO Molecular Medicine

    Article Title: EGFR is required for FOS‐dependent bone tumor development via RSK2/CREB signaling

    doi: 10.15252/emmm.201809408

    Figure Lengend Snippet: EGFR and c‐Fos co‐expression in human OS negatively correlates with patient survival A Representative images of OS biopsies stained with antibodies against EGFR and c‐Fos. Scale bars: 50 μm. B Kaplan–Meier survival curve comparing the survival of patients with EGFR and c‐Fos double‐positive OSs against patients without co‐expression of both proteins ( n = 52). C Gene expression correlation analysis of several cancer‐associated RTKs with FOS in human OS (data analyzed from publicly available dataset E‐GEOD‐39058). Data information: P ‐values were calculated by log‐rank (Mantel–Cox) test comparing the two Kaplan–Meier curves (B) or by unpaired, two‐tailed t ‐test (C).

    Article Snippet: For analysis of EGFR/c‐Fos expression in human OS, two consecutive sections from a commercially available TMA (Novus, NBP2‐30289) were stained with antibodies directed against EGFR (Cell Signaling Technology, #4267) and c‐Fos (Santa Cruz Biotechnology, SC‐52).

    Techniques: Expressing, Staining, Two Tailed Test

    miR-138 and miR-204 regulate EGFR expression via post-transcriptional level in SGC7901 and MGC803 cells. (A) Suppressed EGFR protein expression after transfection with miR-138 or miR-204 mimics using western blotting and the correlated quantitative analysis in SGC7901 cells (n=3). (B) Relative level of EGFR mRNA after transfection with miR-138 or miR-204 mimics, inhibitors, and the relevant negative control in SGC7901 cells (n=3). (C) Suppressed EGFR protein expression after transfection with miR-138 or miR-204 mimics using western blotting and the correlated quantitative analysis in MGC803 cells (n=3). (D) Relative level of EGFR mRNA after transfection with miR-138 or miR-204 mimics, inhibitors, and the relevant negative control in MGC803 cells (n=3). **P

    Journal: Oncology Reports

    Article Title: Effects of miR-138-5p and miR-204-5p on the migration and proliferation of gastric cancer cells by targeting EGFR

    doi: 10.3892/or.2018.6389

    Figure Lengend Snippet: miR-138 and miR-204 regulate EGFR expression via post-transcriptional level in SGC7901 and MGC803 cells. (A) Suppressed EGFR protein expression after transfection with miR-138 or miR-204 mimics using western blotting and the correlated quantitative analysis in SGC7901 cells (n=3). (B) Relative level of EGFR mRNA after transfection with miR-138 or miR-204 mimics, inhibitors, and the relevant negative control in SGC7901 cells (n=3). (C) Suppressed EGFR protein expression after transfection with miR-138 or miR-204 mimics using western blotting and the correlated quantitative analysis in MGC803 cells (n=3). (D) Relative level of EGFR mRNA after transfection with miR-138 or miR-204 mimics, inhibitors, and the relevant negative control in MGC803 cells (n=3). **P

    Article Snippet: The miR mimics promoted the expression of miRs, and in contrast, miR inhibitors displayed anti-miR effects. siRNA was used to suppress the expression of EGFR (cat. no. sc-29301; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Expressing, Transfection, Western Blot, Negative Control

    Rescue experiment further demonstrates the specificity of miR-138 and miR-204 to EGFR. (A) The co-transfection of Si.EGFR plus miR-138 or miR-204 inhibitors could rescue the inhibitory effect of Si.EGFR via western blotting (n=3). (B) Quantification of A (n=3). (C) The EdU assay was used to verify the proliferation ability of the co-transfection of Si.EGFR and miR-138 or miR-204 inhibitors (n=3). (D) Quantification of C (n=3). (E) To further verify the migration ability of MGC803 cells after co-transfection of Si.EGFR and miR-138 or miR-204 inhibitors a wound healing assay was performed (n=3). Si.NC is the negative control of the siRNA of EGFR. **P

    Journal: Oncology Reports

    Article Title: Effects of miR-138-5p and miR-204-5p on the migration and proliferation of gastric cancer cells by targeting EGFR

    doi: 10.3892/or.2018.6389

    Figure Lengend Snippet: Rescue experiment further demonstrates the specificity of miR-138 and miR-204 to EGFR. (A) The co-transfection of Si.EGFR plus miR-138 or miR-204 inhibitors could rescue the inhibitory effect of Si.EGFR via western blotting (n=3). (B) Quantification of A (n=3). (C) The EdU assay was used to verify the proliferation ability of the co-transfection of Si.EGFR and miR-138 or miR-204 inhibitors (n=3). (D) Quantification of C (n=3). (E) To further verify the migration ability of MGC803 cells after co-transfection of Si.EGFR and miR-138 or miR-204 inhibitors a wound healing assay was performed (n=3). Si.NC is the negative control of the siRNA of EGFR. **P

    Article Snippet: The miR mimics promoted the expression of miRs, and in contrast, miR inhibitors displayed anti-miR effects. siRNA was used to suppress the expression of EGFR (cat. no. sc-29301; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Cotransfection, Western Blot, EdU Assay, Migration, Wound Healing Assay, Negative Control

    EGFR significantly promotes the proliferation and migration of SGC7901 cells. (A) Overexpression of EGFR significantly enhanced the proliferation ability of SGC7901 cells, whereas silencing of EGFR inhibited cell proliferation as demonstrated by EDU assay (n=3). (B) Quantification of A (n=3). (C) The Transwell assay demonstrated that overexpression of EGFR significantly promoted cell migration compared with silencing of EGFR (n=3). (D) Quantification of C (n=3). (E) To further verify the migration ability of SGC7901 cells after overexpression or silencing of EGFR a wound healing assay was performed (n=3). Si.NC is the negative control of the siRNA of EGFR. OE.NC is the negative control of the EGFR overexpression lentivirus. **P

    Journal: Oncology Reports

    Article Title: Effects of miR-138-5p and miR-204-5p on the migration and proliferation of gastric cancer cells by targeting EGFR

    doi: 10.3892/or.2018.6389

    Figure Lengend Snippet: EGFR significantly promotes the proliferation and migration of SGC7901 cells. (A) Overexpression of EGFR significantly enhanced the proliferation ability of SGC7901 cells, whereas silencing of EGFR inhibited cell proliferation as demonstrated by EDU assay (n=3). (B) Quantification of A (n=3). (C) The Transwell assay demonstrated that overexpression of EGFR significantly promoted cell migration compared with silencing of EGFR (n=3). (D) Quantification of C (n=3). (E) To further verify the migration ability of SGC7901 cells after overexpression or silencing of EGFR a wound healing assay was performed (n=3). Si.NC is the negative control of the siRNA of EGFR. OE.NC is the negative control of the EGFR overexpression lentivirus. **P

    Article Snippet: The miR mimics promoted the expression of miRs, and in contrast, miR inhibitors displayed anti-miR effects. siRNA was used to suppress the expression of EGFR (cat. no. sc-29301; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Migration, Over Expression, EdU Assay, Transwell Assay, Wound Healing Assay, Negative Control

    EGFR is a direct target of miR-138 and miR-204 in GC. (A) The base-pairing interaction between miR-138 or miR-204 and EGFR mRNA. (B) Relative luciferase activities were analyzed in 293T cells to validate the direct targeting connection between miR-138 or miR-204 and EGFR (n=3). **P

    Journal: Oncology Reports

    Article Title: Effects of miR-138-5p and miR-204-5p on the migration and proliferation of gastric cancer cells by targeting EGFR

    doi: 10.3892/or.2018.6389

    Figure Lengend Snippet: EGFR is a direct target of miR-138 and miR-204 in GC. (A) The base-pairing interaction between miR-138 or miR-204 and EGFR mRNA. (B) Relative luciferase activities were analyzed in 293T cells to validate the direct targeting connection between miR-138 or miR-204 and EGFR (n=3). **P

    Article Snippet: The miR mimics promoted the expression of miRs, and in contrast, miR inhibitors displayed anti-miR effects. siRNA was used to suppress the expression of EGFR (cat. no. sc-29301; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Luciferase

    Inverse correlation between EGFR and miR-138 or miR-204. (A) Increased EGFR protein expression in GC tissues compared with adjacent non-cancerous tissues using western blotting (n=15). (B) The correlated quantitative analysis of A (n=15). (C) Relative level of EGFR mRNA in GC tissues (n=15). (D) Immunohistochemistry of the paraffin-embedded human GC tissues and adjacent non-cancerous tissues (n=15). (E) miR-138 and miR-204 potentially targeted the 3′UTR of EGFR mRNA. (F) Number of copies of miR-138 and miR-204 in GC tissues and adjacent non-cancerous tissues according to the results of high-throughput sequencing (n=150). **P

    Journal: Oncology Reports

    Article Title: Effects of miR-138-5p and miR-204-5p on the migration and proliferation of gastric cancer cells by targeting EGFR

    doi: 10.3892/or.2018.6389

    Figure Lengend Snippet: Inverse correlation between EGFR and miR-138 or miR-204. (A) Increased EGFR protein expression in GC tissues compared with adjacent non-cancerous tissues using western blotting (n=15). (B) The correlated quantitative analysis of A (n=15). (C) Relative level of EGFR mRNA in GC tissues (n=15). (D) Immunohistochemistry of the paraffin-embedded human GC tissues and adjacent non-cancerous tissues (n=15). (E) miR-138 and miR-204 potentially targeted the 3′UTR of EGFR mRNA. (F) Number of copies of miR-138 and miR-204 in GC tissues and adjacent non-cancerous tissues according to the results of high-throughput sequencing (n=150). **P

    Article Snippet: The miR mimics promoted the expression of miRs, and in contrast, miR inhibitors displayed anti-miR effects. siRNA was used to suppress the expression of EGFR (cat. no. sc-29301; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Expressing, Western Blot, Immunohistochemistry, Next-Generation Sequencing

    Overexpression and silencing of EGFR in SGC7901 cells. (A) Knockdown and overexpression of EGFR via siRNA and lentivirus are detected in protein levels using western blotting (n=3). (B) Quantification of A (n=3). (C) The relative levels of EGFR mRNA after transfection with EGFR-tagged lentivirus (n=3). (D) The relative levels of EGFR mRNA after transfection with the siRNA sequence targeting EGFR (n=3). Si.NC is the negative control of the siRNA of EGFR. OE.NC is the negative control of the EGFR overexpression lentivirus. **P

    Journal: Oncology Reports

    Article Title: Effects of miR-138-5p and miR-204-5p on the migration and proliferation of gastric cancer cells by targeting EGFR

    doi: 10.3892/or.2018.6389

    Figure Lengend Snippet: Overexpression and silencing of EGFR in SGC7901 cells. (A) Knockdown and overexpression of EGFR via siRNA and lentivirus are detected in protein levels using western blotting (n=3). (B) Quantification of A (n=3). (C) The relative levels of EGFR mRNA after transfection with EGFR-tagged lentivirus (n=3). (D) The relative levels of EGFR mRNA after transfection with the siRNA sequence targeting EGFR (n=3). Si.NC is the negative control of the siRNA of EGFR. OE.NC is the negative control of the EGFR overexpression lentivirus. **P

    Article Snippet: The miR mimics promoted the expression of miRs, and in contrast, miR inhibitors displayed anti-miR effects. siRNA was used to suppress the expression of EGFR (cat. no. sc-29301; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Over Expression, Western Blot, Transfection, Sequencing, Negative Control

    LXA4 restored ENaC subunits' mRNA expressions in A549 cells challenged by LPS. (a) LXA4 rescued ENaC-α subunit mRNA expression in A549 cells treated with LPS. The mRNA levels were determined by real-time PCR analysis. The treated groups were Control, LPS (1 μg/ml), LXA4 (100 nmol/L), and in combination. * P = 0.006, LPS versus LPS + LXA4. (b) LXA4 restored EnaC-γ subunit mRNA expression in A549 cells treated with LPS. † P = 0.026, LPS versus LPS + LXA4. LXA4: Lipoxin A4; LPS: Lipopolysaccharide; NDRG1: N-myc downstream-regulated gene 1; ENaC-α: Epithelial sodium channel α subunit; ENaC-γ: Epithelial sodium channel γ subunit; PCR: Polymerase chain reaction.

    Journal: Chinese Medical Journal

    Article Title: Lipoxin A4 Ameliorates Lipopolysaccharide-Induced A549 Cell Injury through Upregulation of N-myc Downstream-Regulated Gene-1

    doi: 10.4103/0366-6999.232788

    Figure Lengend Snippet: LXA4 restored ENaC subunits' mRNA expressions in A549 cells challenged by LPS. (a) LXA4 rescued ENaC-α subunit mRNA expression in A549 cells treated with LPS. The mRNA levels were determined by real-time PCR analysis. The treated groups were Control, LPS (1 μg/ml), LXA4 (100 nmol/L), and in combination. * P = 0.006, LPS versus LPS + LXA4. (b) LXA4 restored EnaC-γ subunit mRNA expression in A549 cells treated with LPS. † P = 0.026, LPS versus LPS + LXA4. LXA4: Lipoxin A4; LPS: Lipopolysaccharide; NDRG1: N-myc downstream-regulated gene 1; ENaC-α: Epithelial sodium channel α subunit; ENaC-γ: Epithelial sodium channel γ subunit; PCR: Polymerase chain reaction.

    Article Snippet: For experimental tests, A549 cells were inoculated into 10 cm dishes or 6-well plates and cultured in humidified atmosphere containing 5% CO2 at 37°C for 24 h. As cell confluence reached 80%, A549 cells were further cultured with FBS-free DMEM for 12 h. Then, A549 cells were treated with LPS (Sigma, USA), LXA4 (Sigma), LXA4 receptor (ALX) inhibitor (BOC-2, GenScript, USA), or PI3K inhibitor LY-294002 (Sigma) for 6 h or more time.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    LXA4 promoted NDRG1 expression in A549 cells. (a) LXA4 induced NDRG1 expression and alleviated inhibition of LPS. * P

    Journal: Chinese Medical Journal

    Article Title: Lipoxin A4 Ameliorates Lipopolysaccharide-Induced A549 Cell Injury through Upregulation of N-myc Downstream-Regulated Gene-1

    doi: 10.4103/0366-6999.232788

    Figure Lengend Snippet: LXA4 promoted NDRG1 expression in A549 cells. (a) LXA4 induced NDRG1 expression and alleviated inhibition of LPS. * P

    Article Snippet: For experimental tests, A549 cells were inoculated into 10 cm dishes or 6-well plates and cultured in humidified atmosphere containing 5% CO2 at 37°C for 24 h. As cell confluence reached 80%, A549 cells were further cultured with FBS-free DMEM for 12 h. Then, A549 cells were treated with LPS (Sigma, USA), LXA4 (Sigma), LXA4 receptor (ALX) inhibitor (BOC-2, GenScript, USA), or PI3K inhibitor LY-294002 (Sigma) for 6 h or more time.

    Techniques: Expressing, Inhibition

    Wnt signalling activation affects EGFR–ERK signalling dynamics through regulating expression of multiple molecules. a A volcano plot depicting the fold changes in gene expression levels between normal (ENR) and CHIR99021-treated organoids (ENR + CHIR), and statistical significance of the changes. b – e Enrichment plots from gene set enrichment analysis (GSEA). GESA plots for genes downregulated after Apc knockout ( b ), genes upregulated after Apc knockout through Myc ( c ), genes downregulated in colorectal adenoma ( d ), and genes upregulated in colorectal adenoma ( e ) are shown. f , g RT-PCR analysis revealed that Egfl6, Flna, and Troy were upregulated, and that Lrig3 was downregulated in both CHIR99021-treated and adenoma-derived organoids. The relative mRNA levels of indicated genes in normal (ENR) versus CHIR99021-treated organoids (ENR + CHIR) ( f ), and those in normal versus adenoma-derived organoids ( g ) are shown ( n = 3 samples containing more than ten organoids). h , i Adenoma-derived organoids expressing the ERK biosensor were infected with lentiviruses expressing control vector (control), Lrig3 (Lrig3), or shRNAs for Egfl6 (shEgfl6), Flna (shFlna), or Troy (shTroy). h Bee swarm plots of ERK activity in organoids before (Pre) and after (Post) EGFR inhibitor treatment under each condition ( n = 80 cells pooled from two organoids). i The frequency of ERK activity pulses under each condition. Time-lapse imaging was performed for 90 min ( n = 50 cells). j Schematic representation of ERK activity dynamics generated by kinase activity of EGFR and ErbB2 in the normal and Wnt signalling-activated intestinal epithelia. Red lines represent mean. Error bars represent s.e.m. Welch’s t test ( f , g ), Mann–Whitney U -test ( h ), and Steel–Dwass test ( i ) were used for comparison.* P

    Journal: Nature Communications

    Article Title: Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine

    doi: 10.1038/s41467-018-04527-8

    Figure Lengend Snippet: Wnt signalling activation affects EGFR–ERK signalling dynamics through regulating expression of multiple molecules. a A volcano plot depicting the fold changes in gene expression levels between normal (ENR) and CHIR99021-treated organoids (ENR + CHIR), and statistical significance of the changes. b – e Enrichment plots from gene set enrichment analysis (GSEA). GESA plots for genes downregulated after Apc knockout ( b ), genes upregulated after Apc knockout through Myc ( c ), genes downregulated in colorectal adenoma ( d ), and genes upregulated in colorectal adenoma ( e ) are shown. f , g RT-PCR analysis revealed that Egfl6, Flna, and Troy were upregulated, and that Lrig3 was downregulated in both CHIR99021-treated and adenoma-derived organoids. The relative mRNA levels of indicated genes in normal (ENR) versus CHIR99021-treated organoids (ENR + CHIR) ( f ), and those in normal versus adenoma-derived organoids ( g ) are shown ( n = 3 samples containing more than ten organoids). h , i Adenoma-derived organoids expressing the ERK biosensor were infected with lentiviruses expressing control vector (control), Lrig3 (Lrig3), or shRNAs for Egfl6 (shEgfl6), Flna (shFlna), or Troy (shTroy). h Bee swarm plots of ERK activity in organoids before (Pre) and after (Post) EGFR inhibitor treatment under each condition ( n = 80 cells pooled from two organoids). i The frequency of ERK activity pulses under each condition. Time-lapse imaging was performed for 90 min ( n = 50 cells). j Schematic representation of ERK activity dynamics generated by kinase activity of EGFR and ErbB2 in the normal and Wnt signalling-activated intestinal epithelia. Red lines represent mean. Error bars represent s.e.m. Welch’s t test ( f , g ), Mann–Whitney U -test ( h ), and Steel–Dwass test ( i ) were used for comparison.* P

    Article Snippet: Sections were subjected to immunofluorescence staining with the following primary antibodies: anti-EGFR (Medical & Biological Laboratories, clone 6F1), anti-ErbB2 (Cell Signaling Technology, clone 29D8), anti-phospho-ErbB2 (Cell Signaling Technology, clone 6B12), anit-Ki67 (Abcam, ab15580), and anti-E-cadherin (Cell Signaling Technology, clone 24E10).

    Techniques: Activation Assay, Expressing, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Infection, Plasmid Preparation, Activity Assay, Imaging, Generated, MANN-WHITNEY

    EGFR signalling is augmented in adenomas and CHIR99021-treated mouse intestine in vivo. a Bee swarm plots of ERK activity in the vehicle- (Control) or CHIR99021-treated (CHIR) mouse intestinal epithelium before (Pre) and after (Post) treatment with an EGFR inhibitor, erlotinib. Mice were injected with vehicle or 20 mg kg −1 body weight of CHIR99021 for 3 days and then treated with 100 mg kg −1 body weight erlotinib for 30 min (Control: n = 48, CHIR: n = 61 cells from three crypts). b , c Immunofluorescence staining of the small intestine in the mice injected with vehicle (Control) or CHIR99021 for 3 days using anti-EGFR and anti-E-cadherin antibodies. b The ratio of EGFR and E-cadherin staining intensities is shown in the IMD mode according to the colour scale. Counterstaining was performed with Hoechst. Note that strong staining of stromal cells results from non-specific binding of the secondary antibodies used here (anti-mouse IgG). c Quantification of the EGFR/E-cadherin staining intensity ratio in each cell under vehicle or CHIR99021-treated condition ( n = 50 cells). d Immunofluorescence staining of an adenoma and the normal small intestine of Apc Δ716 mice with anti-EGFR and anti-E-cadherin antibodies. EGFR/E-cadherin staining intensity ratio is shown in the IMD mode. e Quantification of the EGFR/E-cadherin ratio in each cell located in the indicated regions ( n = 50 cells). f Immunofluorescence staining of normal and adenoma-derived organoids with anti-EGFR and anti-ErbB2 antibodies. EGFR/ErbB2 staining intensity ratio is shown in the IMD mode. g Quantification of the EGFR/ErbB2 ratio in each cell in the organoids ( n = 40 cells pooled from at least two organoids). h , i EdU staining of the small intestine of CHIR99021- and/or erlotinib-treated mice. Mice were injected with vehicle, 20 mg kg −1 body weight of CHIR99021, and/or 100 mg kg −1 body weight of erlotinib for 3 days. i Quantification of EdU + cells per crypt section (−/−: n = 46, CHIR/–: n = 42, −/Erlotinib: n = 54, and CHIR/Erlotinib: n = 45 crypts from three mice). Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U -test ( a , c , g ), and Steel–Dwass test ( e , i ) were used for comparison.* P

    Journal: Nature Communications

    Article Title: Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine

    doi: 10.1038/s41467-018-04527-8

    Figure Lengend Snippet: EGFR signalling is augmented in adenomas and CHIR99021-treated mouse intestine in vivo. a Bee swarm plots of ERK activity in the vehicle- (Control) or CHIR99021-treated (CHIR) mouse intestinal epithelium before (Pre) and after (Post) treatment with an EGFR inhibitor, erlotinib. Mice were injected with vehicle or 20 mg kg −1 body weight of CHIR99021 for 3 days and then treated with 100 mg kg −1 body weight erlotinib for 30 min (Control: n = 48, CHIR: n = 61 cells from three crypts). b , c Immunofluorescence staining of the small intestine in the mice injected with vehicle (Control) or CHIR99021 for 3 days using anti-EGFR and anti-E-cadherin antibodies. b The ratio of EGFR and E-cadherin staining intensities is shown in the IMD mode according to the colour scale. Counterstaining was performed with Hoechst. Note that strong staining of stromal cells results from non-specific binding of the secondary antibodies used here (anti-mouse IgG). c Quantification of the EGFR/E-cadherin staining intensity ratio in each cell under vehicle or CHIR99021-treated condition ( n = 50 cells). d Immunofluorescence staining of an adenoma and the normal small intestine of Apc Δ716 mice with anti-EGFR and anti-E-cadherin antibodies. EGFR/E-cadherin staining intensity ratio is shown in the IMD mode. e Quantification of the EGFR/E-cadherin ratio in each cell located in the indicated regions ( n = 50 cells). f Immunofluorescence staining of normal and adenoma-derived organoids with anti-EGFR and anti-ErbB2 antibodies. EGFR/ErbB2 staining intensity ratio is shown in the IMD mode. g Quantification of the EGFR/ErbB2 ratio in each cell in the organoids ( n = 40 cells pooled from at least two organoids). h , i EdU staining of the small intestine of CHIR99021- and/or erlotinib-treated mice. Mice were injected with vehicle, 20 mg kg −1 body weight of CHIR99021, and/or 100 mg kg −1 body weight of erlotinib for 3 days. i Quantification of EdU + cells per crypt section (−/−: n = 46, CHIR/–: n = 42, −/Erlotinib: n = 54, and CHIR/Erlotinib: n = 45 crypts from three mice). Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U -test ( a , c , g ), and Steel–Dwass test ( e , i ) were used for comparison.* P

    Article Snippet: Sections were subjected to immunofluorescence staining with the following primary antibodies: anti-EGFR (Medical & Biological Laboratories, clone 6F1), anti-ErbB2 (Cell Signaling Technology, clone 29D8), anti-phospho-ErbB2 (Cell Signaling Technology, clone 6B12), anit-Ki67 (Abcam, ab15580), and anti-E-cadherin (Cell Signaling Technology, clone 24E10).

    Techniques: In Vivo, Activity Assay, Mouse Assay, Injection, Immunofluorescence, Staining, Radial Immuno Diffusion, Binding Assay, Derivative Assay, MANN-WHITNEY

    EGFR and ErbB2 generate two distinct modes of ERK activity in intestinal organoids. a , b EKAREV-NLS organoids were treated with EGFRi (PD153035) (1 μM), and/or ErbB2i (CP-724714) (10 μM), at time point 0. a Time courses of average ERK activity under each condition (EGFRi: n = 70, ErbB2i: n = 62, EGFRi + ErbB2i: n = 57 cells). b Quantification of ERK activity before and after treatment with EGFRi or ErbB2i (EGFRi: n = 113, ErbB2i: n = 147 cells, pooled from two organoids). c Time course of average ERK activity in organoids that were cultured under EGF-starved condition (NR) for 24 h, treated with either EGFRi or ErbB2i for 60 min, and then stimulated with 50 ng ml −1 of EGF (EGFRi: n = 48, ErbB2i: n = 32 cells). d Quantification of ERK activity in organoids infected with a control lentivirus (control) or lentiviruses expressing either a dominant negative form of EGFR (dnEGFR) or that of ErbB2 (dnErbB2) (Control: n = 134, dnEGFR: n = 129, dnErbB2: n = 151 cells, pooled from five organoids cultured in the ENR medium). e , f Quantification of ERK activity pulses in EKAREV-NLS organoids cultured in the ENR medium and treated with EGFRi and/or ErbB2i (−/−: n = 71, EGFRi/−: n = 49, −/ErbB2i: n = 36, EGFRi/ ErbB2i: n = 53 cells). Organoids were treated with indicated inhibitors and imaged for 90 min. ERK activity data were smoothened by 6-min moving average, and fitted to flat lines or multi-peak functions. e The proportion of cells exhibiting the pulse-like ERK activation (ERK-pulse + ) under each condition. f Frequencies of ERK activity pulses under each condition. Duration ( g ) and frequencies ( h ) of ERK activity pulses in organoids cultured in the ENR medium and treated with 0, 10, 1, or 0.1 μM of a BRAF inhibitor (SB590885) for 90 min ( n = 55, 52, 29, and 65 cells, respectively). Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U -test ( b ) and Steel–Dwass test ( d , f – h ) were used for comparison. * P

    Journal: Nature Communications

    Article Title: Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine

    doi: 10.1038/s41467-018-04527-8

    Figure Lengend Snippet: EGFR and ErbB2 generate two distinct modes of ERK activity in intestinal organoids. a , b EKAREV-NLS organoids were treated with EGFRi (PD153035) (1 μM), and/or ErbB2i (CP-724714) (10 μM), at time point 0. a Time courses of average ERK activity under each condition (EGFRi: n = 70, ErbB2i: n = 62, EGFRi + ErbB2i: n = 57 cells). b Quantification of ERK activity before and after treatment with EGFRi or ErbB2i (EGFRi: n = 113, ErbB2i: n = 147 cells, pooled from two organoids). c Time course of average ERK activity in organoids that were cultured under EGF-starved condition (NR) for 24 h, treated with either EGFRi or ErbB2i for 60 min, and then stimulated with 50 ng ml −1 of EGF (EGFRi: n = 48, ErbB2i: n = 32 cells). d Quantification of ERK activity in organoids infected with a control lentivirus (control) or lentiviruses expressing either a dominant negative form of EGFR (dnEGFR) or that of ErbB2 (dnErbB2) (Control: n = 134, dnEGFR: n = 129, dnErbB2: n = 151 cells, pooled from five organoids cultured in the ENR medium). e , f Quantification of ERK activity pulses in EKAREV-NLS organoids cultured in the ENR medium and treated with EGFRi and/or ErbB2i (−/−: n = 71, EGFRi/−: n = 49, −/ErbB2i: n = 36, EGFRi/ ErbB2i: n = 53 cells). Organoids were treated with indicated inhibitors and imaged for 90 min. ERK activity data were smoothened by 6-min moving average, and fitted to flat lines or multi-peak functions. e The proportion of cells exhibiting the pulse-like ERK activation (ERK-pulse + ) under each condition. f Frequencies of ERK activity pulses under each condition. Duration ( g ) and frequencies ( h ) of ERK activity pulses in organoids cultured in the ENR medium and treated with 0, 10, 1, or 0.1 μM of a BRAF inhibitor (SB590885) for 90 min ( n = 55, 52, 29, and 65 cells, respectively). Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U -test ( b ) and Steel–Dwass test ( d , f – h ) were used for comparison. * P

    Article Snippet: Sections were subjected to immunofluorescence staining with the following primary antibodies: anti-EGFR (Medical & Biological Laboratories, clone 6F1), anti-ErbB2 (Cell Signaling Technology, clone 29D8), anti-phospho-ErbB2 (Cell Signaling Technology, clone 6B12), anit-Ki67 (Abcam, ab15580), and anti-E-cadherin (Cell Signaling Technology, clone 24E10).

    Techniques: Activity Assay, Cell Culture, Infection, Expressing, Dominant Negative Mutation, Activation Assay, MANN-WHITNEY