β4 Search Results


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  • 94
    Santa Cruz Biotechnology integrin β4
    <t>Integrin</t> <t>β4</t> promotes resistance of gastric cancer cell lines to gefitinib. a The expression of integrin β4 with integrin β4 siRNA or over-expressed vector was assayed by Western blot (n = 4) and real-time PCR (n = 4). b The proliferation of gastric cancer cell lines was assayed by MTT (n = 5). c Apoptosis rate was assayed by flow cytometry (n = 4). *P
    Integrin β4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore β4 integrin
    Determination of DHHC proteins responsible for <t>β4</t> <t>integrin</t> palmitoylation. a MDA-MB-231 cells were treated with 10 mg/ml of brefeldin A (Golgi inhibitor), 2 h prior to metabolic [ 3 H]palmitate labeling. After cell lysis in 1% Brij96 buffer, β4
    β4 Integrin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology β4 integrin
    Cell surface biomarker expression. a – d The MCF10A cell-formed acini in TMG and Matrigel were IF stained with either β1 or <t>β4</t> <t>integrin</t> (red), E-cadherin (green), and Hoechst (blue). Scale bars, 100 μm. e – g The expression of CD44 in MM231 cells cultured on the three types of hydrogels were examined using IF staining. CD44, red; Col I, green; nucleus/Hoechst, blue. Scale bars, 10 μm
    β4 Integrin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti β4
    Cell surface biomarker expression. a – d The MCF10A cell-formed acini in TMG and Matrigel were IF stained with either β1 or <t>β4</t> <t>integrin</t> (red), E-cadherin (green), and Hoechst (blue). Scale bars, 100 μm. e – g The expression of CD44 in MM231 cells cultured on the three types of hydrogels were examined using IF staining. CD44, red; Col I, green; nucleus/Hoechst, blue. Scale bars, 10 μm
    Anti β4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 95/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam integrin β4
    Matrix-deprived cells are susceptible to ferroptosis in the absence of the <t>α6β4-integrin.</t> (A) Control and <t>β4-depleted</t> cells were detached for 24 h in the presence of either DMSO or 2 µM ferrostatin-1, and the number of viable cells was quantified. (B) Control and β4-depleted Hs578t cells were detached for 24 h in the presence of either DMSO or 2 µM ferrostatin-1, and the number of viable cells was quantified. (C) Control and β4-depleted SUM-159 cells were detached for 24 h in the presence of either DMSO or 2 µM liproxstatin-1, and the number of viable cells was quantified. (D) Control and β4-depleted MCF-10A and SUM-159 cells were detached for 24 h in the presence of either DMSO, 100 µM DFO, 100 µM α-tocopherol, or 500 µM Trolox, and the number of viable cells was quantified. (E) MCF10-A or SUM-159 vector control and β4-depleted cells were assayed for LDH after 6 h of detachment with either DMSO or 2 µM ferrostatin-1. (F) Vector and β4-depleted cells were incubated for the times indicated with either DMSO or 2 µM ferrostatin-1, and viable cells were quantified. (G) Vector and β4-depleted cells were detached for 24 h in the presence of either DMSO, 2 µM ferrostatin-1, 25 µM Z-VAD-FMK, or ferrostatin-1 and Z-VAD-FMK, and the number of viable cells was quantified. All experiments were performed independently three times and a representative experiment is shown. The bars in graphs represent means ± SD. *, P
    Integrin β4, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson rat anti β4 integrin
    Immunostaining of basal and luminal markers after LC ablation and antibody permeability a – f , Confocal imaging of immunostaining for E-cadherin, K5 and tdTomato ( a – c ) or E-cadherin, K14 and tdTomato ( d – f ) in CTR ( a , d ) or K5CreER/tdTomato/K8rtTA/TetO-DTA mice after IDI of DOX and chased for 1 week ( b , e ) or 2 weeks ( c , f ) ( n = 3 mice per condition). g , Quantification of the proportion of hybrid K8 + K14 + tdTomato + cells and K8 + tdTomato + cells on the total K8 + tdTomato + cells at 1 week ( n = 7 mice), 2 weeks ( n = 4 mice), 4 weeks ( n = 3 mice) and 7 weeks ( n = 3 mice) after DOX treatment. The P value between 1 week and 7 weeks, derived from ANOVA followed by two-sided Dunnett’s test, is shown. h , i , Confocal imaging of immunostaining for K14, p63 and K8 ( h ) and quantification of p63 + cells in K8 + K14 + cells ( i ) in CTR or K5CreER/tdTomato/K8rtTA/TetO-DTA mice after DOX IDI and chased for 1 week. n = 3 mice per condition. j , k , Confocal imaging of immunostaining for K14, Foxa1 and K8 ( j ) and quantification of Foxa1 + cells in K8 + K14 + cells ( k ) in CTR or K5CreER/tdTomato/K8rtTA/TetO-DTA mice after DOX IDI and chased for 1 week. n = 3 mice per condition. l , Confocal imaging of immunostaining for K14 and anti-rat Alexa Fluor 488 (green) in CTR MG not injected or MG after IDI of rat <t>anti-β4</t> <t>integrin</t> (CD104) and chased for 2 days. n = 3 mice per condition. Data are mean ± s.e.m. For the immunofluorescence data, Hoechst nuclear staining is shown in blue. Scale bars, 5 μm.
    Rat Anti β4 Integrin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Alomone Labs anti bk β4
    Role of aldosterone in expression of BK-α and <t>BK-β4.</t>
    Anti Bk β4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 88/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson anti integrin β4
    Modulation of miRNAs in ΔCK2β-MCF10A cells. ( A ) Log2 fold change of the main miRNAs modulated in CK2β-depleted versus parental MCF10A cells measured by miRNA array analysis; ( B ) Changes of miRNA expression between CK2β-depleted and Mock-MCF10A cells were confirmed by using the indicated TaqMan probes. The relative amount of each miRNAs was determined by cross-normalization to ΔCK2β samples using the comparative method and miR-720 as an internal reference; ( C ) Two targets of miR-200 and miR-30 families, respectively Zeb1 and <t>integrin</t> β3, were analyzed by Western blot and/or immunofluorescence in Mock- and CK2β-depleted cells. The ratio ΔCK2β/Mock of signal intensity in western blot was determined (3.5 and 2.3 for Zeb1 and integrin β3 respectively). Arrows indicate integrin β3 localization; ( D ) Integrin β1 and <t>β4,</t> targets of miR-21 were analyzed by western blot and/or immunofluorescence in Mock- and CK2β-depleted cells. The ratio ΔCK2β/Mock of signal intensity in western blot was 0.4 for integrin β1. F-actin in green, nuclei in blue, and integrin β in red. Scale bar, 10 μm.
    Anti Integrin β4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore thymosin β4
    Modulation of miRNAs in ΔCK2β-MCF10A cells. ( A ) Log2 fold change of the main miRNAs modulated in CK2β-depleted versus parental MCF10A cells measured by miRNA array analysis; ( B ) Changes of miRNA expression between CK2β-depleted and Mock-MCF10A cells were confirmed by using the indicated TaqMan probes. The relative amount of each miRNAs was determined by cross-normalization to ΔCK2β samples using the comparative method and miR-720 as an internal reference; ( C ) Two targets of miR-200 and miR-30 families, respectively Zeb1 and <t>integrin</t> β3, were analyzed by Western blot and/or immunofluorescence in Mock- and CK2β-depleted cells. The ratio ΔCK2β/Mock of signal intensity in western blot was determined (3.5 and 2.3 for Zeb1 and integrin β3 respectively). Arrows indicate integrin β3 localization; ( D ) Integrin β1 and <t>β4,</t> targets of miR-21 were analyzed by western blot and/or immunofluorescence in Mock- and CK2β-depleted cells. The ratio ΔCK2β/Mock of signal intensity in western blot was 0.4 for integrin β1. F-actin in green, nuclei in blue, and integrin β in red. Scale bar, 10 μm.
    Thymosin β4, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam anti β4 integrin
    Inhibiting <t>β4</t> <t>Integrin</t> decreases fission. Organoids grown in control media (ENR-Y; A) or in the presence of a β4 Integrin blocking antibody (B) were recorded by time-lapse imaging. The number of branches were measured every 6 h (C, S9 Data ). Branch number increases more slowly in organoids grown in the presence of the β4 Integrin antibody ( p
    Anti β4 Integrin, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti β4 integrin
    Inhibiting <t>β4</t> <t>Integrin</t> decreases fission. Organoids grown in control media (ENR-Y; A) or in the presence of a β4 Integrin blocking antibody (B) were recorded by time-lapse imaging. The number of branches were measured every 6 h (C, S9 Data ). Branch number increases more slowly in organoids grown in the presence of the β4 Integrin antibody ( p
    Anti β4 Integrin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc anti integrin β4
    A working model of metformin-induced cancer cell anoikis. Metformin upregulates ubiquitin E3 ligase WWP1, leading to destabilization of ΔNp63α and downregulation of proteins involved in cell-matrix adhesion including <t>integrin</t> <t>β4</t> and fibronectin 1, which in turn results in cell detachment [ 12 ]. On the other hand, metformin inhibits ΔNp63α expression resulting in upregulation of integrin β1, which in turn promotes cell–cell adhesion and cell aggregation. Both disruption of cell-matrix adhesion and enhanced cell–cell adhesion contribute to metformin-induced anoikis.
    Anti Integrin β4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology β4
    A working model of metformin-induced cancer cell anoikis. Metformin upregulates ubiquitin E3 ligase WWP1, leading to destabilization of ΔNp63α and downregulation of proteins involved in cell-matrix adhesion including <t>integrin</t> <t>β4</t> and fibronectin 1, which in turn results in cell detachment [ 12 ]. On the other hand, metformin inhibits ΔNp63α expression resulting in upregulation of integrin β1, which in turn promotes cell–cell adhesion and cell aggregation. Both disruption of cell-matrix adhesion and enhanced cell–cell adhesion contribute to metformin-induced anoikis.
    β4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti integrin beta 4 antibody epr17517
    A working model of metformin-induced cancer cell anoikis. Metformin upregulates ubiquitin E3 ligase WWP1, leading to destabilization of ΔNp63α and downregulation of proteins involved in cell-matrix adhesion including <t>integrin</t> <t>β4</t> and fibronectin 1, which in turn results in cell detachment [ 12 ]. On the other hand, metformin inhibits ΔNp63α expression resulting in upregulation of integrin β1, which in turn promotes cell–cell adhesion and cell aggregation. Both disruption of cell-matrix adhesion and enhanced cell–cell adhesion contribute to metformin-induced anoikis.
    Anti Integrin Beta 4 Antibody Epr17517, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti integrin β4
    A working model of metformin-induced cancer cell anoikis. Metformin upregulates ubiquitin E3 ligase WWP1, leading to destabilization of ΔNp63α and downregulation of proteins involved in cell-matrix adhesion including <t>integrin</t> <t>β4</t> and fibronectin 1, which in turn results in cell detachment [ 12 ]. On the other hand, metformin inhibits ΔNp63α expression resulting in upregulation of integrin β1, which in turn promotes cell–cell adhesion and cell aggregation. Both disruption of cell-matrix adhesion and enhanced cell–cell adhesion contribute to metformin-induced anoikis.
    Anti Integrin β4, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti β4
    A working model of metformin-induced cancer cell anoikis. Metformin upregulates ubiquitin E3 ligase WWP1, leading to destabilization of ΔNp63α and downregulation of proteins involved in cell-matrix adhesion including <t>integrin</t> <t>β4</t> and fibronectin 1, which in turn results in cell detachment [ 12 ]. On the other hand, metformin inhibits ΔNp63α expression resulting in upregulation of integrin β1, which in turn promotes cell–cell adhesion and cell aggregation. Both disruption of cell-matrix adhesion and enhanced cell–cell adhesion contribute to metformin-induced anoikis.
    Anti β4, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti plcβ4
    A working model of metformin-induced cancer cell anoikis. Metformin upregulates ubiquitin E3 ligase WWP1, leading to destabilization of ΔNp63α and downregulation of proteins involved in cell-matrix adhesion including <t>integrin</t> <t>β4</t> and fibronectin 1, which in turn results in cell detachment [ 12 ]. On the other hand, metformin inhibits ΔNp63α expression resulting in upregulation of integrin β1, which in turn promotes cell–cell adhesion and cell aggregation. Both disruption of cell-matrix adhesion and enhanced cell–cell adhesion contribute to metformin-induced anoikis.
    Anti Plcβ4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti integrin beta 4 antibody 346 11a
    A working model of metformin-induced cancer cell anoikis. Metformin upregulates ubiquitin E3 ligase WWP1, leading to destabilization of ΔNp63α and downregulation of proteins involved in cell-matrix adhesion including <t>integrin</t> <t>β4</t> and fibronectin 1, which in turn results in cell detachment [ 12 ]. On the other hand, metformin inhibits ΔNp63α expression resulting in upregulation of integrin β1, which in turn promotes cell–cell adhesion and cell aggregation. Both disruption of cell-matrix adhesion and enhanced cell–cell adhesion contribute to metformin-induced anoikis.
    Anti Integrin Beta 4 Antibody 346 11a, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    Gene Silencers generally consist of pools of three to five target specific 19 25 nucleotide sequences in length For independent verification of β4 Tubulin gene silencing results individual duplex components
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    N/A
    Source Thymosin b4 is a 43 amino acid peptide which is regarded as the main intracellular G actin sequestering peptide It has a molecular weight of 4963 55 Da and
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    N/A
    Target species human CRISPR Cas9 KO Plasmids consists of β4 Tubulin specific 20 nt guide RNA sequences derived from the GeCKO v2 library For CRISPR gene knockout gRNA sequences direct
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    N/A
    Thymosin β4 is an actin sequestering peptide that has wound healing and anti inflammatory activities It regulates cytoskeletal rearrangement by binding to actin Ks 0 7 and 2 1 µM
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    Image Search Results


    Integrin β4 promotes resistance of gastric cancer cell lines to gefitinib. a The expression of integrin β4 with integrin β4 siRNA or over-expressed vector was assayed by Western blot (n = 4) and real-time PCR (n = 4). b The proliferation of gastric cancer cell lines was assayed by MTT (n = 5). c Apoptosis rate was assayed by flow cytometry (n = 4). *P

    Journal: Cancer Cell International

    Article Title: A cross-talk between integrin β4 and epidermal growth factor receptor induces gefitinib chemoresistance to gastric cancer

    doi: 10.1186/s12935-018-0548-5

    Figure Lengend Snippet: Integrin β4 promotes resistance of gastric cancer cell lines to gefitinib. a The expression of integrin β4 with integrin β4 siRNA or over-expressed vector was assayed by Western blot (n = 4) and real-time PCR (n = 4). b The proliferation of gastric cancer cell lines was assayed by MTT (n = 5). c Apoptosis rate was assayed by flow cytometry (n = 4). *P

    Article Snippet: Integrin β4 siRNA or over-expressed vector The full-length cDNA of integrin β4 was amplified from SGC7901/gefitinib cells and cloned into the Xho l I/Bam H I sites of the vector pcDNA3 in order to generate integrin β4 high expressed vector. siRNA sequence targeting human integrin β4 was purchased from Santa Cruz biotechnology.

    Techniques: Expressing, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, MTT Assay, Flow Cytometry, Cytometry

    Integrin β4 was negatively correlated with p-EGFR activation (n = 50). a p-EGFR and integrin β4 protein expressions were determined by western blot. b Pearson correlation coefficients between p-EGFR and integrin β4. 1–4, gefitinib-sensitive gastric cancer samples; 5–7, gefitinib-resistant gastric cancer samples. *P

    Journal: Cancer Cell International

    Article Title: A cross-talk between integrin β4 and epidermal growth factor receptor induces gefitinib chemoresistance to gastric cancer

    doi: 10.1186/s12935-018-0548-5

    Figure Lengend Snippet: Integrin β4 was negatively correlated with p-EGFR activation (n = 50). a p-EGFR and integrin β4 protein expressions were determined by western blot. b Pearson correlation coefficients between p-EGFR and integrin β4. 1–4, gefitinib-sensitive gastric cancer samples; 5–7, gefitinib-resistant gastric cancer samples. *P

    Article Snippet: Integrin β4 siRNA or over-expressed vector The full-length cDNA of integrin β4 was amplified from SGC7901/gefitinib cells and cloned into the Xho l I/Bam H I sites of the vector pcDNA3 in order to generate integrin β4 high expressed vector. siRNA sequence targeting human integrin β4 was purchased from Santa Cruz biotechnology.

    Techniques: Activation Assay, Western Blot

    p-EGFR and intergrin β4 expression were assayed by IFA ( a ) and p-EGFR and p-FAK were assayed by western blot ( b ). Integrin β4 over expression induced rapid internalization of surface EGFR to the cytoplasm, while integrin β4 knockdown promoted the expression of EGFR. c The interaction between p-EGFR and intergrin β4 was assayed by immunocoprecipitation with anti-p-EGFR antibody. The first two samples were from SGC7901 cells and the second two samples were from clinic gefitinib-sensitive samples. *P

    Journal: Cancer Cell International

    Article Title: A cross-talk between integrin β4 and epidermal growth factor receptor induces gefitinib chemoresistance to gastric cancer

    doi: 10.1186/s12935-018-0548-5

    Figure Lengend Snippet: p-EGFR and intergrin β4 expression were assayed by IFA ( a ) and p-EGFR and p-FAK were assayed by western blot ( b ). Integrin β4 over expression induced rapid internalization of surface EGFR to the cytoplasm, while integrin β4 knockdown promoted the expression of EGFR. c The interaction between p-EGFR and intergrin β4 was assayed by immunocoprecipitation with anti-p-EGFR antibody. The first two samples were from SGC7901 cells and the second two samples were from clinic gefitinib-sensitive samples. *P

    Article Snippet: Integrin β4 siRNA or over-expressed vector The full-length cDNA of integrin β4 was amplified from SGC7901/gefitinib cells and cloned into the Xho l I/Bam H I sites of the vector pcDNA3 in order to generate integrin β4 high expressed vector. siRNA sequence targeting human integrin β4 was purchased from Santa Cruz biotechnology.

    Techniques: Expressing, Immunofluorescence, Western Blot, Over Expression

    Integrin β4 associates with sensitivity of gefitinib of gastric cancer cell lines. a The sensitivity of the gastric cancer cell lines to gefitinib was assayed by MTT (n = 6). SGC7901R cells exhibited obvious resistance to gefitinib. b The proliferation of SGC7901R and SGC7901 was assayed by MTT (n = 6). The proliferation of SGC7901R was significantly up-regulated. c The resistant gene P-gp was assayed by real-time PCR. The expression of P-gp was increased significantly in SGC7901R cells. d The expression of integrin β4 was assayed by western blot (n = 4). The expression of integrin β4 was up-regulated in SGC7901R cells

    Journal: Cancer Cell International

    Article Title: A cross-talk between integrin β4 and epidermal growth factor receptor induces gefitinib chemoresistance to gastric cancer

    doi: 10.1186/s12935-018-0548-5

    Figure Lengend Snippet: Integrin β4 associates with sensitivity of gefitinib of gastric cancer cell lines. a The sensitivity of the gastric cancer cell lines to gefitinib was assayed by MTT (n = 6). SGC7901R cells exhibited obvious resistance to gefitinib. b The proliferation of SGC7901R and SGC7901 was assayed by MTT (n = 6). The proliferation of SGC7901R was significantly up-regulated. c The resistant gene P-gp was assayed by real-time PCR. The expression of P-gp was increased significantly in SGC7901R cells. d The expression of integrin β4 was assayed by western blot (n = 4). The expression of integrin β4 was up-regulated in SGC7901R cells

    Article Snippet: Integrin β4 siRNA or over-expressed vector The full-length cDNA of integrin β4 was amplified from SGC7901/gefitinib cells and cloned into the Xho l I/Bam H I sites of the vector pcDNA3 in order to generate integrin β4 high expressed vector. siRNA sequence targeting human integrin β4 was purchased from Santa Cruz biotechnology.

    Techniques: MTT Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot

    Determination of DHHC proteins responsible for β4 integrin palmitoylation. a MDA-MB-231 cells were treated with 10 mg/ml of brefeldin A (Golgi inhibitor), 2 h prior to metabolic [ 3 H]palmitate labeling. After cell lysis in 1% Brij96 buffer, β4

    Journal: Cellular and molecular life sciences : CMLS

    Article Title: Palmitoylation by DHHC3 is critical for the function, expression, and stability of integrin ?6?4

    doi: 10.1007/s00018-012-0924-6

    Figure Lengend Snippet: Determination of DHHC proteins responsible for β4 integrin palmitoylation. a MDA-MB-231 cells were treated with 10 mg/ml of brefeldin A (Golgi inhibitor), 2 h prior to metabolic [ 3 H]palmitate labeling. After cell lysis in 1% Brij96 buffer, β4

    Article Snippet: Monoclonal (mAb, MAB1964) and polyclonal (pAb, AB1922) antibodies to β4 integrin were from Chemicon.

    Techniques: Multiple Displacement Amplification, Labeling, Lysis

    DHHC3 palmitoylation specificity among integrin subunits. PC3 cells with vector (V) and DHHC3 (D3) stable knockdown were labeled with [ 3 H]palmitate for 2 h. Then α3, β1, α6, β4 integrin and CD9 proteins were immunoprecipitated

    Journal: Cellular and molecular life sciences : CMLS

    Article Title: Palmitoylation by DHHC3 is critical for the function, expression, and stability of integrin ?6?4

    doi: 10.1007/s00018-012-0924-6

    Figure Lengend Snippet: DHHC3 palmitoylation specificity among integrin subunits. PC3 cells with vector (V) and DHHC3 (D3) stable knockdown were labeled with [ 3 H]palmitate for 2 h. Then α3, β1, α6, β4 integrin and CD9 proteins were immunoprecipitated

    Article Snippet: Monoclonal (mAb, MAB1964) and polyclonal (pAb, AB1922) antibodies to β4 integrin were from Chemicon.

    Techniques: Plasmid Preparation, Labeling, Immunoprecipitation

    Pepstatin A restores β4 integrin expression after DHHC3 ablation. a PC3 cells with vector control (V) or DHHC3 stable knockdown (D3) were treated with the indicated protease inhibitors or mock-treated (M) for 24 h. After cell lysis, β4

    Journal: Cellular and molecular life sciences : CMLS

    Article Title: Palmitoylation by DHHC3 is critical for the function, expression, and stability of integrin ?6?4

    doi: 10.1007/s00018-012-0924-6

    Figure Lengend Snippet: Pepstatin A restores β4 integrin expression after DHHC3 ablation. a PC3 cells with vector control (V) or DHHC3 stable knockdown (D3) were treated with the indicated protease inhibitors or mock-treated (M) for 24 h. After cell lysis, β4

    Article Snippet: Monoclonal (mAb, MAB1964) and polyclonal (pAb, AB1922) antibodies to β4 integrin were from Chemicon.

    Techniques: Expressing, Plasmid Preparation, Lysis

    Decreased levels of β4 integrin in nerves of PMP22-deficient mice. A–F , Cryosections of sciatic nerves from +/+ ( A , C , E ) and PMP22−/− ( B , D , F ) mice were immunostained with monoclonal rat (rt) anti-β4 integrin ( A , B ), anti-β1 integrin ( C , D ), or polyclonal rabbit (rb) anti-laminin (Lam) ( E , F ) antibodies. In nerves of P10 +/+ mice, β4 and β1 integrin are detected at the abaxonal SC surface ( A , C , arrows). In comparison, abaxonal integrin-like staining is only discernable around a fraction of the fibers in the −/− samples ( B , D , arrows). In addition, when the images were collected at the same exposure times, the level of β4-like immunoreactivity was reduced (compare A , B ). Laminin was detected at the SC basal lamina in +/+ ( E , arrow) and −/− ( F ) nerves, with thickened basal lamina (arrowheads) and a tomaculum (open arrowhead) marked in the affected sample ( F ). Nonspecific rat ( A , B , inset) and rabbit ( E , F , inset) sera serve as controls for staining specificity. Scale bar, 5 μm. G , Sciatic nerve lysates (20 μg/lane) from P10 +/+, +/−, and −/− mice were analyzed with polyclonal rabbit anti-β4 integrin and anti-laminin and monoclonal rat anti-β1 integrin antibodies. The blots were reprobed with monoclonal mouse anti-GAPDH antibody as a protein loading control. Molecular mass is in kilodaltons.

    Journal: The Journal of Neuroscience

    Article Title: Peripheral Myelin Protein 22 Is in Complex with α6β4 Integrin, and Its Absence Alters the Schwann Cell Basal Lamina

    doi: 10.1523/JNEUROSCI.2618-05.2006

    Figure Lengend Snippet: Decreased levels of β4 integrin in nerves of PMP22-deficient mice. A–F , Cryosections of sciatic nerves from +/+ ( A , C , E ) and PMP22−/− ( B , D , F ) mice were immunostained with monoclonal rat (rt) anti-β4 integrin ( A , B ), anti-β1 integrin ( C , D ), or polyclonal rabbit (rb) anti-laminin (Lam) ( E , F ) antibodies. In nerves of P10 +/+ mice, β4 and β1 integrin are detected at the abaxonal SC surface ( A , C , arrows). In comparison, abaxonal integrin-like staining is only discernable around a fraction of the fibers in the −/− samples ( B , D , arrows). In addition, when the images were collected at the same exposure times, the level of β4-like immunoreactivity was reduced (compare A , B ). Laminin was detected at the SC basal lamina in +/+ ( E , arrow) and −/− ( F ) nerves, with thickened basal lamina (arrowheads) and a tomaculum (open arrowhead) marked in the affected sample ( F ). Nonspecific rat ( A , B , inset) and rabbit ( E , F , inset) sera serve as controls for staining specificity. Scale bar, 5 μm. G , Sciatic nerve lysates (20 μg/lane) from P10 +/+, +/−, and −/− mice were analyzed with polyclonal rabbit anti-β4 integrin and anti-laminin and monoclonal rat anti-β1 integrin antibodies. The blots were reprobed with monoclonal mouse anti-GAPDH antibody as a protein loading control. Molecular mass is in kilodaltons.

    Article Snippet: Polyclonal anti-PMP22 (1:2000); anti-β-galactosidase (β-gal; 1:1000; Molecular Probes); anti-laminin (1:10, 000; Sigma-Aldrich); anti-β4 integrin (1:1000) and monoclonal rat anti-β1 integrin (1:1000; both from Chemicon, Temecula, CA); and monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (a kind gift from Dr. G. Shaw, University of Florida, Gainesville, FL) were used.

    Techniques: Mouse Assay, Laser Capture Microdissection, Staining

    Coexpression of PMP22 and integrins during myelination. A , Sciatic nerve cryosections from 3-month-old +/+ mice were labeled with monoclonal rat anti-β4 and polyclonal rabbit anti-PMP22 antibodies and examined by confocal microscopy. The merged single plane image reveals the partial colocalization (merge, yellow) of β4 integrin (green) and PMP22 (red). Scale bar, 5 μm. Nonspecific rat (rt) and rabbit (rb) sera (bottom insets) serve as controls for staining specificity. B , Entire protein lysates of P1, P3, P8, and P21 sciatic nerves (10 μg/lane) from +/+ mice were analyzed with anti-PMP22 and anti-β4 integrin antibodies. The arrows indicate the migration of β4 integrin at ∼200 kDa (top) and of PMP22 at ∼22 kDa (bottom), whereas the arrowhead marks a nonspecific immunoreactive band. C , Cell-surface biotinylation of myelinating DRG–SC cocultures identifies β4 integrin and PMP22 in the avidin pull down (AP), from which actin is excluded. Total lysate (T) and agarose bead preclear (PC) fractions are also shown. Molecular mass is in kilodaltons. PMP, PMP22.

    Journal: The Journal of Neuroscience

    Article Title: Peripheral Myelin Protein 22 Is in Complex with α6β4 Integrin, and Its Absence Alters the Schwann Cell Basal Lamina

    doi: 10.1523/JNEUROSCI.2618-05.2006

    Figure Lengend Snippet: Coexpression of PMP22 and integrins during myelination. A , Sciatic nerve cryosections from 3-month-old +/+ mice were labeled with monoclonal rat anti-β4 and polyclonal rabbit anti-PMP22 antibodies and examined by confocal microscopy. The merged single plane image reveals the partial colocalization (merge, yellow) of β4 integrin (green) and PMP22 (red). Scale bar, 5 μm. Nonspecific rat (rt) and rabbit (rb) sera (bottom insets) serve as controls for staining specificity. B , Entire protein lysates of P1, P3, P8, and P21 sciatic nerves (10 μg/lane) from +/+ mice were analyzed with anti-PMP22 and anti-β4 integrin antibodies. The arrows indicate the migration of β4 integrin at ∼200 kDa (top) and of PMP22 at ∼22 kDa (bottom), whereas the arrowhead marks a nonspecific immunoreactive band. C , Cell-surface biotinylation of myelinating DRG–SC cocultures identifies β4 integrin and PMP22 in the avidin pull down (AP), from which actin is excluded. Total lysate (T) and agarose bead preclear (PC) fractions are also shown. Molecular mass is in kilodaltons. PMP, PMP22.

    Article Snippet: Polyclonal anti-PMP22 (1:2000); anti-β-galactosidase (β-gal; 1:1000; Molecular Probes); anti-laminin (1:10, 000; Sigma-Aldrich); anti-β4 integrin (1:1000) and monoclonal rat anti-β1 integrin (1:1000; both from Chemicon, Temecula, CA); and monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (a kind gift from Dr. G. Shaw, University of Florida, Gainesville, FL) were used.

    Techniques: Mouse Assay, Labeling, Confocal Microscopy, Staining, Migration, Avidin-Biotin Assay

    PMP22 and β4 integrin are coimmunoprecipitated from clone A cells. A , B , Epitope (myc)-tagged hPMP22 was expressed in clone A cells, and samples were double immunolabeled with anti-β4 ( A ) and anti-myc ( B ) antibodies. C , As the merged image reveals, hPMP22 is targeted to the β4 integrin-positive plasma membrane of the cells ( A–C , arrows). Scale bar, 10 μm. D , Vector control and hPMP-myc-expressing clone A cells were lysed and processed for IP with the indicated antibodies. The precipitates were subsequently probed for the marked proteins by Western blot. The arrows on the right indicate the overexpressed glycosylated PMP22 (∼22 kDa) and the endogenous β4 integrin (∼200 kDa). The arrowheads point at the position of the deglycosylated ∼18 and ∼190 kDa forms. Molecular mass is in kilodaltons. T, Total lysate; PC, preclear.

    Journal: The Journal of Neuroscience

    Article Title: Peripheral Myelin Protein 22 Is in Complex with α6β4 Integrin, and Its Absence Alters the Schwann Cell Basal Lamina

    doi: 10.1523/JNEUROSCI.2618-05.2006

    Figure Lengend Snippet: PMP22 and β4 integrin are coimmunoprecipitated from clone A cells. A , B , Epitope (myc)-tagged hPMP22 was expressed in clone A cells, and samples were double immunolabeled with anti-β4 ( A ) and anti-myc ( B ) antibodies. C , As the merged image reveals, hPMP22 is targeted to the β4 integrin-positive plasma membrane of the cells ( A–C , arrows). Scale bar, 10 μm. D , Vector control and hPMP-myc-expressing clone A cells were lysed and processed for IP with the indicated antibodies. The precipitates were subsequently probed for the marked proteins by Western blot. The arrows on the right indicate the overexpressed glycosylated PMP22 (∼22 kDa) and the endogenous β4 integrin (∼200 kDa). The arrowheads point at the position of the deglycosylated ∼18 and ∼190 kDa forms. Molecular mass is in kilodaltons. T, Total lysate; PC, preclear.

    Article Snippet: Polyclonal anti-PMP22 (1:2000); anti-β-galactosidase (β-gal; 1:1000; Molecular Probes); anti-laminin (1:10, 000; Sigma-Aldrich); anti-β4 integrin (1:1000) and monoclonal rat anti-β1 integrin (1:1000; both from Chemicon, Temecula, CA); and monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (a kind gift from Dr. G. Shaw, University of Florida, Gainesville, FL) were used.

    Techniques: Immunolabeling, Plasmid Preparation, Expressing, Western Blot

    PMP22 is in a complex with α6β4 integrin and laminin. Sciatic nerve lysates (T lanes) from P21 +/+ mice were processed for IP, after preclearing (PC lanes) with nonspecific Igs of the appropriate isotype. Lysates were incubated with polyclonal rabbit anti-β4 integrin ( A , left, C ), anti-PMP22 ( A , right), monoclonal rat anti-α6 integrin ( B , left), or polyclonal rabbit anti-laminin ( B , right) antibodies, and captured immunoprecipitates were probed for the indicated proteins ( A–C ) as designated at the right of each blot. On reprobes (marked with asterisks), after stripping the membranes, the anti-β4 integrin and anti-laminin antibodies did not work efficiently when 1 μg/lane total (T) nerve protein was analyzed. IP with anti-β4 integrin on nerve lysates of homozygous PMP22-deficient mice is shown as a negative control ( C ). Molecular mass is in kilodaltons. PMP, PMP22; Lam, laminin; βDys, β-dystroglycan.

    Journal: The Journal of Neuroscience

    Article Title: Peripheral Myelin Protein 22 Is in Complex with α6β4 Integrin, and Its Absence Alters the Schwann Cell Basal Lamina

    doi: 10.1523/JNEUROSCI.2618-05.2006

    Figure Lengend Snippet: PMP22 is in a complex with α6β4 integrin and laminin. Sciatic nerve lysates (T lanes) from P21 +/+ mice were processed for IP, after preclearing (PC lanes) with nonspecific Igs of the appropriate isotype. Lysates were incubated with polyclonal rabbit anti-β4 integrin ( A , left, C ), anti-PMP22 ( A , right), monoclonal rat anti-α6 integrin ( B , left), or polyclonal rabbit anti-laminin ( B , right) antibodies, and captured immunoprecipitates were probed for the indicated proteins ( A–C ) as designated at the right of each blot. On reprobes (marked with asterisks), after stripping the membranes, the anti-β4 integrin and anti-laminin antibodies did not work efficiently when 1 μg/lane total (T) nerve protein was analyzed. IP with anti-β4 integrin on nerve lysates of homozygous PMP22-deficient mice is shown as a negative control ( C ). Molecular mass is in kilodaltons. PMP, PMP22; Lam, laminin; βDys, β-dystroglycan.

    Article Snippet: Polyclonal anti-PMP22 (1:2000); anti-β-galactosidase (β-gal; 1:1000; Molecular Probes); anti-laminin (1:10, 000; Sigma-Aldrich); anti-β4 integrin (1:1000) and monoclonal rat anti-β1 integrin (1:1000; both from Chemicon, Temecula, CA); and monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (a kind gift from Dr. G. Shaw, University of Florida, Gainesville, FL) were used.

    Techniques: Mouse Assay, Incubation, Stripping Membranes, Negative Control, Laser Capture Microdissection

    Cell surface biomarker expression. a – d The MCF10A cell-formed acini in TMG and Matrigel were IF stained with either β1 or β4 integrin (red), E-cadherin (green), and Hoechst (blue). Scale bars, 100 μm. e – g The expression of CD44 in MM231 cells cultured on the three types of hydrogels were examined using IF staining. CD44, red; Col I, green; nucleus/Hoechst, blue. Scale bars, 10 μm

    Journal: Breast Cancer Research : BCR

    Article Title: Distinct phenotypes of cancer cells on tissue matrix gel

    doi: 10.1186/s13058-020-01321-7

    Figure Lengend Snippet: Cell surface biomarker expression. a – d The MCF10A cell-formed acini in TMG and Matrigel were IF stained with either β1 or β4 integrin (red), E-cadherin (green), and Hoechst (blue). Scale bars, 100 μm. e – g The expression of CD44 in MM231 cells cultured on the three types of hydrogels were examined using IF staining. CD44, red; Col I, green; nucleus/Hoechst, blue. Scale bars, 10 μm

    Article Snippet: The cells were probed with primary antibodies against β1 or β4 integrin, and E-cadherin (Santa Cruz Biotechnology), followed by fluorophore-conjugated secondary antibody and Hoechst staining and imaged for acini structures under a fluorescence microscope.

    Techniques: Biomarker Assay, Expressing, Staining, Cell Culture

    Matrix metalloproteinases (MMP)-10 and integrin α3 protein levels in the corneas of rats fed normal chow (lane 1) or 50% galactose in the absence (lane 2) or presence (lane 3) of aldose reductase inhibitor were analyzed by immunoblotting. The protein expression level of MMP-10 was enhanced by exposure to 50% galactose, while integrin α3 expression decreased. These changes were normalized with the treatment of aldose reductase inhibitor. No significant change was found in the expression levels of integrin α6 or β4. The bar graphs illustrate the intensities of the bands relative to β-actin. The values are mean±standard error of the mean; *p

    Journal: Molecular Vision

    Article Title: Aldose reductase inhibitor counteracts the enhanced expression of matrix metalloproteinase-10 and improves corneal wound healing in galactose-fed rats

    doi:

    Figure Lengend Snippet: Matrix metalloproteinases (MMP)-10 and integrin α3 protein levels in the corneas of rats fed normal chow (lane 1) or 50% galactose in the absence (lane 2) or presence (lane 3) of aldose reductase inhibitor were analyzed by immunoblotting. The protein expression level of MMP-10 was enhanced by exposure to 50% galactose, while integrin α3 expression decreased. These changes were normalized with the treatment of aldose reductase inhibitor. No significant change was found in the expression levels of integrin α6 or β4. The bar graphs illustrate the intensities of the bands relative to β-actin. The values are mean±standard error of the mean; *p

    Article Snippet: The membranes were then stripped and reprobed with anti-integrin α3, anti-integrin α6, anti-integrin β4 or anti-β-actin antibodies (Santa Cruz Biotechnology).

    Techniques: Expressing

    Matrix-deprived cells are susceptible to ferroptosis in the absence of the α6β4-integrin. (A) Control and β4-depleted cells were detached for 24 h in the presence of either DMSO or 2 µM ferrostatin-1, and the number of viable cells was quantified. (B) Control and β4-depleted Hs578t cells were detached for 24 h in the presence of either DMSO or 2 µM ferrostatin-1, and the number of viable cells was quantified. (C) Control and β4-depleted SUM-159 cells were detached for 24 h in the presence of either DMSO or 2 µM liproxstatin-1, and the number of viable cells was quantified. (D) Control and β4-depleted MCF-10A and SUM-159 cells were detached for 24 h in the presence of either DMSO, 100 µM DFO, 100 µM α-tocopherol, or 500 µM Trolox, and the number of viable cells was quantified. (E) MCF10-A or SUM-159 vector control and β4-depleted cells were assayed for LDH after 6 h of detachment with either DMSO or 2 µM ferrostatin-1. (F) Vector and β4-depleted cells were incubated for the times indicated with either DMSO or 2 µM ferrostatin-1, and viable cells were quantified. (G) Vector and β4-depleted cells were detached for 24 h in the presence of either DMSO, 2 µM ferrostatin-1, 25 µM Z-VAD-FMK, or ferrostatin-1 and Z-VAD-FMK, and the number of viable cells was quantified. All experiments were performed independently three times and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: The α6β4 integrin promotes resistance to ferroptosis

    doi: 10.1083/jcb.201701136

    Figure Lengend Snippet: Matrix-deprived cells are susceptible to ferroptosis in the absence of the α6β4-integrin. (A) Control and β4-depleted cells were detached for 24 h in the presence of either DMSO or 2 µM ferrostatin-1, and the number of viable cells was quantified. (B) Control and β4-depleted Hs578t cells were detached for 24 h in the presence of either DMSO or 2 µM ferrostatin-1, and the number of viable cells was quantified. (C) Control and β4-depleted SUM-159 cells were detached for 24 h in the presence of either DMSO or 2 µM liproxstatin-1, and the number of viable cells was quantified. (D) Control and β4-depleted MCF-10A and SUM-159 cells were detached for 24 h in the presence of either DMSO, 100 µM DFO, 100 µM α-tocopherol, or 500 µM Trolox, and the number of viable cells was quantified. (E) MCF10-A or SUM-159 vector control and β4-depleted cells were assayed for LDH after 6 h of detachment with either DMSO or 2 µM ferrostatin-1. (F) Vector and β4-depleted cells were incubated for the times indicated with either DMSO or 2 µM ferrostatin-1, and viable cells were quantified. (G) Vector and β4-depleted cells were detached for 24 h in the presence of either DMSO, 2 µM ferrostatin-1, 25 µM Z-VAD-FMK, or ferrostatin-1 and Z-VAD-FMK, and the number of viable cells was quantified. All experiments were performed independently three times and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Article Snippet: The following antibodies were used: GPX4 (Abcam), actin (Sigma-Aldrich), integrin β4 (505 [ ] and 439-9b [Abcam]), α6 (GoH3; MilliporeSigma), and integrin β1 (BD), phospho-Src Y418 (R & D Systems), total Src (Santa Cruz Biotechnology, Inc.), ACSL4 (Santa Cruz Biotechnology, Inc.), phospho-STAT3 Y705 (Cell Signaling Technology), and total STAT3 (Cell Signaling Technology).

    Techniques: Plasmid Preparation, Incubation

    Matrix-deprived cells exhibit increased lipid peroxidation and an inability to sustain GPX4 expression in the absence of the α6β4-integrin. (A) GPX4 mRNA expression was quantified by qPCR in control and β4-depleted MCF-10A and SUM-159 cells under adherent or 2-h matrix-deprived conditions. (B) Expression of GPX4 was assessed by immunoblotting in vector control, β4-depleted, and β4-rescued cells after 2 h of detachment. Relative densitometry values are shown. (C) Lipid peroxidation was quantified using the MDA assay in control and β4-depleted, MCF-10A and SUM-159 cells under adherent or 4-h matrix-deprived conditions. (D) GPX enzyme activity was assayed in control and β4-depleted, MCF-10A and SUM-159 cells under adherent or 4-h matrix-deprived conditions. (E) Control and β4-depleted SUM-159 cells were transfected with either a vector control or a GPX4 expression vector. GPX4 mRNA expression was quantified by qPCR. (F) Control and β4-depleted MCF10-A and SUM-159 cells that had been transfected with either a vector control or a GPX4 expression vector were detached for 24 h, and the number of viable cells was quantified. (G) Control and β4-depleted cells that had been transfected with either a vector control or a GPX4 expression vector were detached for 24 h with in the presence of either DMSO or 10 µM erastin, and the number of viable cells was quantified. Experiments were performed independently three times and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: The α6β4 integrin promotes resistance to ferroptosis

    doi: 10.1083/jcb.201701136

    Figure Lengend Snippet: Matrix-deprived cells exhibit increased lipid peroxidation and an inability to sustain GPX4 expression in the absence of the α6β4-integrin. (A) GPX4 mRNA expression was quantified by qPCR in control and β4-depleted MCF-10A and SUM-159 cells under adherent or 2-h matrix-deprived conditions. (B) Expression of GPX4 was assessed by immunoblotting in vector control, β4-depleted, and β4-rescued cells after 2 h of detachment. Relative densitometry values are shown. (C) Lipid peroxidation was quantified using the MDA assay in control and β4-depleted, MCF-10A and SUM-159 cells under adherent or 4-h matrix-deprived conditions. (D) GPX enzyme activity was assayed in control and β4-depleted, MCF-10A and SUM-159 cells under adherent or 4-h matrix-deprived conditions. (E) Control and β4-depleted SUM-159 cells were transfected with either a vector control or a GPX4 expression vector. GPX4 mRNA expression was quantified by qPCR. (F) Control and β4-depleted MCF10-A and SUM-159 cells that had been transfected with either a vector control or a GPX4 expression vector were detached for 24 h, and the number of viable cells was quantified. (G) Control and β4-depleted cells that had been transfected with either a vector control or a GPX4 expression vector were detached for 24 h with in the presence of either DMSO or 10 µM erastin, and the number of viable cells was quantified. Experiments were performed independently three times and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Article Snippet: The following antibodies were used: GPX4 (Abcam), actin (Sigma-Aldrich), integrin β4 (505 [ ] and 439-9b [Abcam]), α6 (GoH3; MilliporeSigma), and integrin β1 (BD), phospho-Src Y418 (R & D Systems), total Src (Santa Cruz Biotechnology, Inc.), ACSL4 (Santa Cruz Biotechnology, Inc.), phospho-STAT3 Y705 (Cell Signaling Technology), and total STAT3 (Cell Signaling Technology).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Multiple Displacement Amplification, Activity Assay, Transfection

    The integrin α6β4 represses ACSL4 to inhibit ferroptosis. Expression of ACSL4 was assessed by qPCR (A) and immunoblotting (B) in vector control and β4-depleted cells after 2 h of ECM detachment. (C) Expression of ACSL4 was quantified by qPCR in vector control MCF10-A or SUM-159 cells under either adherent or detached conditions after 2 h of treatment with DMSO or 10 µM PP2. (D) Expression of ACSL4 was quantified in adherent vector control, β4-depleted, and β4-rescued cells by qPCR. (E) Vector control and β4-depleted cells were assessed for viability after 24 h of detachment in the presence of either DMSO or the ACSL4 inhibitor 5 µM rosiglitazone. (F) SUM-159 vector control and β4-depleted adherent cells that expressed a doxycycline-inducible, constitutively active Src were incubated for 24 h with 2 µg/ml doxycycline, and the expression of ACSL4 was quantified by qPCR. (G) Expression of ITGB4 and ACSL4 was correlated using a published gene expression database (cBioportal) comprising 70 human breast tumors. The correlation coefficient ( r ) was calculated using Pearson’s correlation. Experiments were performed independently three times, and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: The α6β4 integrin promotes resistance to ferroptosis

    doi: 10.1083/jcb.201701136

    Figure Lengend Snippet: The integrin α6β4 represses ACSL4 to inhibit ferroptosis. Expression of ACSL4 was assessed by qPCR (A) and immunoblotting (B) in vector control and β4-depleted cells after 2 h of ECM detachment. (C) Expression of ACSL4 was quantified by qPCR in vector control MCF10-A or SUM-159 cells under either adherent or detached conditions after 2 h of treatment with DMSO or 10 µM PP2. (D) Expression of ACSL4 was quantified in adherent vector control, β4-depleted, and β4-rescued cells by qPCR. (E) Vector control and β4-depleted cells were assessed for viability after 24 h of detachment in the presence of either DMSO or the ACSL4 inhibitor 5 µM rosiglitazone. (F) SUM-159 vector control and β4-depleted adherent cells that expressed a doxycycline-inducible, constitutively active Src were incubated for 24 h with 2 µg/ml doxycycline, and the expression of ACSL4 was quantified by qPCR. (G) Expression of ITGB4 and ACSL4 was correlated using a published gene expression database (cBioportal) comprising 70 human breast tumors. The correlation coefficient ( r ) was calculated using Pearson’s correlation. Experiments were performed independently three times, and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Article Snippet: The following antibodies were used: GPX4 (Abcam), actin (Sigma-Aldrich), integrin β4 (505 [ ] and 439-9b [Abcam]), α6 (GoH3; MilliporeSigma), and integrin β1 (BD), phospho-Src Y418 (R & D Systems), total Src (Santa Cruz Biotechnology, Inc.), ACSL4 (Santa Cruz Biotechnology, Inc.), phospho-STAT3 Y705 (Cell Signaling Technology), and total STAT3 (Cell Signaling Technology).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Incubation

    Evasion of ferroptosis is specific to the α6β4-integrin. (A) β4-depleted SUM-159 cells were transfected with either a control plasmid (vector) or a β4 expression construct in which the PAM sequences targeted by the guide RNAs were mutated. Rescue of β4 expression was confirmed by immunoblotting and qPCR. Vector control cells, β4-depleted cells, vector control cells transfected with β4 containing PAM mutations, and β4-depleted cells transfected with β4 containing PAM mutations (β4-rescued cells) were detached for 24 h, and the number of viable cells was quantified. (B) MCF10-A and SUM-159 α3-depleted cells were detached and compared with control cells for their viability after 24 h. (C and D) PDXs of triple-negative breast tumors were isolated, dissociated, and lineage-depleted, and the tumor cells were sorted based on the level of β4 surface expression into β4 high (red) and β4 low (blue) populations. Those two populations were assessed for viability in detached conditions, either in the presence or absence of ferrostatin-1. All experiments were performed independently three times, and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: The α6β4 integrin promotes resistance to ferroptosis

    doi: 10.1083/jcb.201701136

    Figure Lengend Snippet: Evasion of ferroptosis is specific to the α6β4-integrin. (A) β4-depleted SUM-159 cells were transfected with either a control plasmid (vector) or a β4 expression construct in which the PAM sequences targeted by the guide RNAs were mutated. Rescue of β4 expression was confirmed by immunoblotting and qPCR. Vector control cells, β4-depleted cells, vector control cells transfected with β4 containing PAM mutations, and β4-depleted cells transfected with β4 containing PAM mutations (β4-rescued cells) were detached for 24 h, and the number of viable cells was quantified. (B) MCF10-A and SUM-159 α3-depleted cells were detached and compared with control cells for their viability after 24 h. (C and D) PDXs of triple-negative breast tumors were isolated, dissociated, and lineage-depleted, and the tumor cells were sorted based on the level of β4 surface expression into β4 high (red) and β4 low (blue) populations. Those two populations were assessed for viability in detached conditions, either in the presence or absence of ferrostatin-1. All experiments were performed independently three times, and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Article Snippet: The following antibodies were used: GPX4 (Abcam), actin (Sigma-Aldrich), integrin β4 (505 [ ] and 439-9b [Abcam]), α6 (GoH3; MilliporeSigma), and integrin β1 (BD), phospho-Src Y418 (R & D Systems), total Src (Santa Cruz Biotechnology, Inc.), ACSL4 (Santa Cruz Biotechnology, Inc.), phospho-STAT3 Y705 (Cell Signaling Technology), and total STAT3 (Cell Signaling Technology).

    Techniques: Transfection, Plasmid Preparation, Expressing, Construct, Real-time Polymerase Chain Reaction, Isolation

    The integrin α6β4 activates Src to inhibit ferroptosis. (A) Vector control and β4-depleted cells were assessed for phosphorylated (Y418) Src by immunoblotting 3 h after ECM detachment. Relative phosphorylated Src was quantified by densitometry. (B) Vector control and β4-depleted cells were assessed for viability after 24 h of detachment in the presence of either DMSO, 10 µM PP2, PP2 and 2 µM ferrostatin-1, PP2 and 500 µM α-tocopherol, or PP2 and 2 µM liproxstatin-1. (C) Vector control and β4-depleted cells were assessed for phosphorylated (Y418) Src by immunoblotting after 3 h of incubation with 10 µM erastin. Relative phosphorylated Src was quantified by densitometry. (D) SUM-159 vector control and β4-depleted cells that expressed a doxycycline-inducible, constitutively active Src were incubated for 24 h with 2 µg/ml doxycycline and then assessed for viability after 24 h of detachment. All experiments were performed independently three times, and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: The α6β4 integrin promotes resistance to ferroptosis

    doi: 10.1083/jcb.201701136

    Figure Lengend Snippet: The integrin α6β4 activates Src to inhibit ferroptosis. (A) Vector control and β4-depleted cells were assessed for phosphorylated (Y418) Src by immunoblotting 3 h after ECM detachment. Relative phosphorylated Src was quantified by densitometry. (B) Vector control and β4-depleted cells were assessed for viability after 24 h of detachment in the presence of either DMSO, 10 µM PP2, PP2 and 2 µM ferrostatin-1, PP2 and 500 µM α-tocopherol, or PP2 and 2 µM liproxstatin-1. (C) Vector control and β4-depleted cells were assessed for phosphorylated (Y418) Src by immunoblotting after 3 h of incubation with 10 µM erastin. Relative phosphorylated Src was quantified by densitometry. (D) SUM-159 vector control and β4-depleted cells that expressed a doxycycline-inducible, constitutively active Src were incubated for 24 h with 2 µg/ml doxycycline and then assessed for viability after 24 h of detachment. All experiments were performed independently three times, and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Article Snippet: The following antibodies were used: GPX4 (Abcam), actin (Sigma-Aldrich), integrin β4 (505 [ ] and 439-9b [Abcam]), α6 (GoH3; MilliporeSigma), and integrin β1 (BD), phospho-Src Y418 (R & D Systems), total Src (Santa Cruz Biotechnology, Inc.), ACSL4 (Santa Cruz Biotechnology, Inc.), phospho-STAT3 Y705 (Cell Signaling Technology), and total STAT3 (Cell Signaling Technology).

    Techniques: Plasmid Preparation, Incubation

    The α6β4 integrin promotes evasion of ferroptosis induced by erastin. (A) The β4-integrin subunit was depleted in MCF10-A and SUM-159 cells by CRISPR/CAS9 using two independent guide RNAs (β4-3 and β4-4). Depletion of β4 expression was verified by immunoblotting. (B) Extracts of β4-depleted cells were immunoprecipitated with an α6 antibody and immunoblotted with a β1 antibody to verify that these cells express α6β1 (left blot). Vector control and β4-depleted cells were assessed for surface expression of the α6-integrin by flow cytometry. (C and D) Control and β4-depleted MCF10-A or SUM-159 cells (5 × 10 2 ) were plated in 60-mm dishes in the presence of either DMSO, 10 µM erastin, erastin and 2 µM ferrostatin-1, or erastin and 500 µM α-tocopherol, and survival was quantified after 7 d by either DMSO extraction of crystal violet-stained cells or colony counting, respectively. (E and F) MCF10-A or SUM-159 vector control and β4-depleted cells were assayed for LDH after 6 h of treatment with either DMSO control or 10 µM erastin. (G) shRNA-mediated depletion of the α3-integrin subunit in MCF10-A and SUM-159 cells was confirmed by qPCR after 7 d of puromycin selection. (H) MCF10-A and SUM-159 α3-depleted cells were plated at clonal density, and survival was quantified as in C and E. All experiments were performed independently three times, and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Journal: The Journal of Cell Biology

    Article Title: The α6β4 integrin promotes resistance to ferroptosis

    doi: 10.1083/jcb.201701136

    Figure Lengend Snippet: The α6β4 integrin promotes evasion of ferroptosis induced by erastin. (A) The β4-integrin subunit was depleted in MCF10-A and SUM-159 cells by CRISPR/CAS9 using two independent guide RNAs (β4-3 and β4-4). Depletion of β4 expression was verified by immunoblotting. (B) Extracts of β4-depleted cells were immunoprecipitated with an α6 antibody and immunoblotted with a β1 antibody to verify that these cells express α6β1 (left blot). Vector control and β4-depleted cells were assessed for surface expression of the α6-integrin by flow cytometry. (C and D) Control and β4-depleted MCF10-A or SUM-159 cells (5 × 10 2 ) were plated in 60-mm dishes in the presence of either DMSO, 10 µM erastin, erastin and 2 µM ferrostatin-1, or erastin and 500 µM α-tocopherol, and survival was quantified after 7 d by either DMSO extraction of crystal violet-stained cells or colony counting, respectively. (E and F) MCF10-A or SUM-159 vector control and β4-depleted cells were assayed for LDH after 6 h of treatment with either DMSO control or 10 µM erastin. (G) shRNA-mediated depletion of the α3-integrin subunit in MCF10-A and SUM-159 cells was confirmed by qPCR after 7 d of puromycin selection. (H) MCF10-A and SUM-159 α3-depleted cells were plated at clonal density, and survival was quantified as in C and E. All experiments were performed independently three times, and a representative experiment is shown. The bars in graphs represent means ± SD. *, P

    Article Snippet: The following antibodies were used: GPX4 (Abcam), actin (Sigma-Aldrich), integrin β4 (505 [ ] and 439-9b [Abcam]), α6 (GoH3; MilliporeSigma), and integrin β1 (BD), phospho-Src Y418 (R & D Systems), total Src (Santa Cruz Biotechnology, Inc.), ACSL4 (Santa Cruz Biotechnology, Inc.), phospho-STAT3 Y705 (Cell Signaling Technology), and total STAT3 (Cell Signaling Technology).

    Techniques: CRISPR, Expressing, Immunoprecipitation, Plasmid Preparation, Flow Cytometry, Cytometry, Staining, shRNA, Real-time Polymerase Chain Reaction, Selection

    STAT3 represses transcription of ACSL4 downstream of the integrin α6β4. (A) Analysis of MCF10-A cells using the ENCODE database showed binding of STAT3 to the promoter region. The phosphorylation of STAT3 was assessed by immunoblotting in vector control and β4-depleted MCF-10A cells after 3 h of detachment (B) or after incubation with erastin (D) and was also quantified by densitometry (C and E). (F) Expression of ACSL4 was quantified by qPCR in vector control MCF10-A and SUM-159 adherent cells after 2 h of incubation with the STAT3 inhibitor DPP. Experiments were performed independently three times, and a representative experiment is shown. The bars in graphs represent means ± SD. **, P

    Journal: The Journal of Cell Biology

    Article Title: The α6β4 integrin promotes resistance to ferroptosis

    doi: 10.1083/jcb.201701136

    Figure Lengend Snippet: STAT3 represses transcription of ACSL4 downstream of the integrin α6β4. (A) Analysis of MCF10-A cells using the ENCODE database showed binding of STAT3 to the promoter region. The phosphorylation of STAT3 was assessed by immunoblotting in vector control and β4-depleted MCF-10A cells after 3 h of detachment (B) or after incubation with erastin (D) and was also quantified by densitometry (C and E). (F) Expression of ACSL4 was quantified by qPCR in vector control MCF10-A and SUM-159 adherent cells after 2 h of incubation with the STAT3 inhibitor DPP. Experiments were performed independently three times, and a representative experiment is shown. The bars in graphs represent means ± SD. **, P

    Article Snippet: The following antibodies were used: GPX4 (Abcam), actin (Sigma-Aldrich), integrin β4 (505 [ ] and 439-9b [Abcam]), α6 (GoH3; MilliporeSigma), and integrin β1 (BD), phospho-Src Y418 (R & D Systems), total Src (Santa Cruz Biotechnology, Inc.), ACSL4 (Santa Cruz Biotechnology, Inc.), phospho-STAT3 Y705 (Cell Signaling Technology), and total STAT3 (Cell Signaling Technology).

    Techniques: Binding Assay, Plasmid Preparation, Incubation, Expressing, Real-time Polymerase Chain Reaction

    Immunostaining of basal and luminal markers after LC ablation and antibody permeability a – f , Confocal imaging of immunostaining for E-cadherin, K5 and tdTomato ( a – c ) or E-cadherin, K14 and tdTomato ( d – f ) in CTR ( a , d ) or K5CreER/tdTomato/K8rtTA/TetO-DTA mice after IDI of DOX and chased for 1 week ( b , e ) or 2 weeks ( c , f ) ( n = 3 mice per condition). g , Quantification of the proportion of hybrid K8 + K14 + tdTomato + cells and K8 + tdTomato + cells on the total K8 + tdTomato + cells at 1 week ( n = 7 mice), 2 weeks ( n = 4 mice), 4 weeks ( n = 3 mice) and 7 weeks ( n = 3 mice) after DOX treatment. The P value between 1 week and 7 weeks, derived from ANOVA followed by two-sided Dunnett’s test, is shown. h , i , Confocal imaging of immunostaining for K14, p63 and K8 ( h ) and quantification of p63 + cells in K8 + K14 + cells ( i ) in CTR or K5CreER/tdTomato/K8rtTA/TetO-DTA mice after DOX IDI and chased for 1 week. n = 3 mice per condition. j , k , Confocal imaging of immunostaining for K14, Foxa1 and K8 ( j ) and quantification of Foxa1 + cells in K8 + K14 + cells ( k ) in CTR or K5CreER/tdTomato/K8rtTA/TetO-DTA mice after DOX IDI and chased for 1 week. n = 3 mice per condition. l , Confocal imaging of immunostaining for K14 and anti-rat Alexa Fluor 488 (green) in CTR MG not injected or MG after IDI of rat anti-β4 integrin (CD104) and chased for 2 days. n = 3 mice per condition. Data are mean ± s.e.m. For the immunofluorescence data, Hoechst nuclear staining is shown in blue. Scale bars, 5 μm.

    Journal: Nature

    Article Title: Heterotypic cell–cell communication regulates glandular stem cell multipotency

    doi: 10.1038/s41586-020-2632-y

    Figure Lengend Snippet: Immunostaining of basal and luminal markers after LC ablation and antibody permeability a – f , Confocal imaging of immunostaining for E-cadherin, K5 and tdTomato ( a – c ) or E-cadherin, K14 and tdTomato ( d – f ) in CTR ( a , d ) or K5CreER/tdTomato/K8rtTA/TetO-DTA mice after IDI of DOX and chased for 1 week ( b , e ) or 2 weeks ( c , f ) ( n = 3 mice per condition). g , Quantification of the proportion of hybrid K8 + K14 + tdTomato + cells and K8 + tdTomato + cells on the total K8 + tdTomato + cells at 1 week ( n = 7 mice), 2 weeks ( n = 4 mice), 4 weeks ( n = 3 mice) and 7 weeks ( n = 3 mice) after DOX treatment. The P value between 1 week and 7 weeks, derived from ANOVA followed by two-sided Dunnett’s test, is shown. h , i , Confocal imaging of immunostaining for K14, p63 and K8 ( h ) and quantification of p63 + cells in K8 + K14 + cells ( i ) in CTR or K5CreER/tdTomato/K8rtTA/TetO-DTA mice after DOX IDI and chased for 1 week. n = 3 mice per condition. j , k , Confocal imaging of immunostaining for K14, Foxa1 and K8 ( j ) and quantification of Foxa1 + cells in K8 + K14 + cells ( k ) in CTR or K5CreER/tdTomato/K8rtTA/TetO-DTA mice after DOX IDI and chased for 1 week. n = 3 mice per condition. l , Confocal imaging of immunostaining for K14 and anti-rat Alexa Fluor 488 (green) in CTR MG not injected or MG after IDI of rat anti-β4 integrin (CD104) and chased for 2 days. n = 3 mice per condition. Data are mean ± s.e.m. For the immunofluorescence data, Hoechst nuclear staining is shown in blue. Scale bars, 5 μm.

    Article Snippet: To check the antibody penetration into the basal layer, the fourth gland of CD1 mice was injected intraductally with 15 μl of rat anti-β4 integrin (clone 346-11A, 553745, BD Biosciences), and after 2 days the gland was stained with anti-rat conjugated to Alexa Fluor 488 secondary antibody.

    Techniques: Immunostaining, Permeability, Imaging, Mouse Assay, Derivative Assay, Injection, Immunofluorescence, Staining

    p53 deletion in CPs leads to BCC formation. (a) Immunostaining of β4-integrin/SmoM2 in ear and tail skin of K14 and Inv-CreER/Rosa-SmoM2/p53fl/fl mice 24w after Tamoxifen administration (b) Whole mount immunostaining of K31/SmoM2 in tail epidermis overtime. (c) Quantification of normal, hyperplastic, dysplastic and BCC clones in the interscale region. Description of number of counted clones is found in the method section. (d) Distribution of basal clone sizes in K14 and Inv-CreER/Rosa-SmoM2/p53fl/fl mice. The number of clones analysed is indicated. Clone merger events were observed after 12w following oncogenic activation in K14Cre-ER/Rosa-SmoM2/p53fl/fl preventing the accurate quantification of clonal persistence and clone size at longer times. (e) Quantification of the proportion of apoptotic cells in different clones (K14 n= 82 clones and Inv n=90 clones from 3 independent experiments). (f) Percentage of double-labelled EdU/BrdU SmoM2-expressing cells after 6 days of continuous BrdU administration following a 24h pulse of EdU at 12w post-induction. *P≤0,05, **P≤0,01. (i) Mean basal clone size in Inv-CreER/Rosa-SmoM2/p53fl/fl and K14-CreER/Rosa-SmoM2/p53fl/fl clones. The prediction of the model is indicated by the blue and red lines. Histograms and error bars represent the mean and s.e.m. Shaded areas represent 95% confidence intervals for the model prediction in i. Scale bars, 100μm.

    Journal: Nature

    Article Title: Defining the clonal dynamics leading to mouse skin tumour initiation

    doi: 10.1038/nature19069

    Figure Lengend Snippet: p53 deletion in CPs leads to BCC formation. (a) Immunostaining of β4-integrin/SmoM2 in ear and tail skin of K14 and Inv-CreER/Rosa-SmoM2/p53fl/fl mice 24w after Tamoxifen administration (b) Whole mount immunostaining of K31/SmoM2 in tail epidermis overtime. (c) Quantification of normal, hyperplastic, dysplastic and BCC clones in the interscale region. Description of number of counted clones is found in the method section. (d) Distribution of basal clone sizes in K14 and Inv-CreER/Rosa-SmoM2/p53fl/fl mice. The number of clones analysed is indicated. Clone merger events were observed after 12w following oncogenic activation in K14Cre-ER/Rosa-SmoM2/p53fl/fl preventing the accurate quantification of clonal persistence and clone size at longer times. (e) Quantification of the proportion of apoptotic cells in different clones (K14 n= 82 clones and Inv n=90 clones from 3 independent experiments). (f) Percentage of double-labelled EdU/BrdU SmoM2-expressing cells after 6 days of continuous BrdU administration following a 24h pulse of EdU at 12w post-induction. *P≤0,05, **P≤0,01. (i) Mean basal clone size in Inv-CreER/Rosa-SmoM2/p53fl/fl and K14-CreER/Rosa-SmoM2/p53fl/fl clones. The prediction of the model is indicated by the blue and red lines. Histograms and error bars represent the mean and s.e.m. Shaded areas represent 95% confidence intervals for the model prediction in i. Scale bars, 100μm.

    Article Snippet: Primary antibodies used were the following: anti-GFP (Rabbit, 1/100, BD, ref. A11122), anti-GFP (Goat, 1:800,Abcam, ref. Ab6673), anti-active-caspase3 (Rabbit, 1/600,R & D, ref. AF835), anti-β4-integrin(Rat, 1:200, BD, ref. 553745) and anti-K31 (Guinea Pig, 1:200, Progen, ref. GP-hHa1).

    Techniques: Immunostaining, Mouse Assay, Clone Assay, Activation Assay, Expressing

    SmoM2 expression in CPs induces clonal expansion that does not progress into BCC. (a) Immunostaining for β4-integrin, YFP and SmoM2 in Inv-CreER/Rosa-YFP and Inv-CreER/Rosa-SmoM2 epidermis at different time points.(b) Distribution of Inv-CreER/Rosa-YFP and Inv-CreER/Rosa-SmoM2 basal clone sizes. The number of clones analysed in Inv-CreER/Rosa-SmoM2 is indicated for each time point and for Inv-CreER/Rosa-YFP indicated in Fig. 2b . (c) Quantification of EdU/BrdU double-labelled cells during continuous BrdU administration, at different time points post clonal induction. The lines represent the model fit ( Supplementary Theory ). (d) Quantification of the proportion of apoptotic cells in dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones 8w post-induction (n= 73 clones analysed from 4 independent experiments). (e) Division rate determined from EdU/BrdU double-labelling experiments (data in black, fit in blue dashed line). (f) Mean basal and suprabasal clone size in the interscale. The lines represent the model fit from which we inferred the cell fate probabilities displayed in g. (g) Cell fate probabilities of the tumour progenitor expressing SmoM2. (h) Basal clone size distribution of Inv-CreER/Rosa-SmoM2 clones (black). Consistent with the hypothesis of a single equipotent progenitor pool, all distributions are well-fit by single exponential. Blue lines represent the model prediction using only the parameters extracted from g. Shaded areas represent 95% confidence intervals for the model prediction. D: dysplasia; H: hyperplasia; N: normal differentiation. Hoechst nuclear staining in blue; scale bars, 10μm. Histograms and error bars represent the mean and the s.e.m. (b–f) and the s.d. (h).

    Journal: Nature

    Article Title: Defining the clonal dynamics leading to mouse skin tumour initiation

    doi: 10.1038/nature19069

    Figure Lengend Snippet: SmoM2 expression in CPs induces clonal expansion that does not progress into BCC. (a) Immunostaining for β4-integrin, YFP and SmoM2 in Inv-CreER/Rosa-YFP and Inv-CreER/Rosa-SmoM2 epidermis at different time points.(b) Distribution of Inv-CreER/Rosa-YFP and Inv-CreER/Rosa-SmoM2 basal clone sizes. The number of clones analysed in Inv-CreER/Rosa-SmoM2 is indicated for each time point and for Inv-CreER/Rosa-YFP indicated in Fig. 2b . (c) Quantification of EdU/BrdU double-labelled cells during continuous BrdU administration, at different time points post clonal induction. The lines represent the model fit ( Supplementary Theory ). (d) Quantification of the proportion of apoptotic cells in dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones 8w post-induction (n= 73 clones analysed from 4 independent experiments). (e) Division rate determined from EdU/BrdU double-labelling experiments (data in black, fit in blue dashed line). (f) Mean basal and suprabasal clone size in the interscale. The lines represent the model fit from which we inferred the cell fate probabilities displayed in g. (g) Cell fate probabilities of the tumour progenitor expressing SmoM2. (h) Basal clone size distribution of Inv-CreER/Rosa-SmoM2 clones (black). Consistent with the hypothesis of a single equipotent progenitor pool, all distributions are well-fit by single exponential. Blue lines represent the model prediction using only the parameters extracted from g. Shaded areas represent 95% confidence intervals for the model prediction. D: dysplasia; H: hyperplasia; N: normal differentiation. Hoechst nuclear staining in blue; scale bars, 10μm. Histograms and error bars represent the mean and the s.e.m. (b–f) and the s.d. (h).

    Article Snippet: Primary antibodies used were the following: anti-GFP (Rabbit, 1/100, BD, ref. A11122), anti-GFP (Goat, 1:800,Abcam, ref. Ab6673), anti-active-caspase3 (Rabbit, 1/600,R & D, ref. AF835), anti-β4-integrin(Rat, 1:200, BD, ref. 553745) and anti-K31 (Guinea Pig, 1:200, Progen, ref. GP-hHa1).

    Techniques: Expressing, Immunostaining, Clone Assay, Staining

    Clonal dynamics of interscale K14-CreER/Rosa-SmoM2 clones is consistent with two populations (a) Distribution of K14-CreER/Rosa-YFP (black) and K14-CreER/Rosa-SmoM2 (red) clone sizes as measured by total cell content, imaged by confocal microscopy on whole mount tail epidermis from 1w to 24w post-induction. The number of clones analyzed for K14-CreER/Rosa-SmoM2 is indicated in Fig 4b; the number of clones counted in K14-CreER/Rosa-YFP is as indicated in Fig. 2a. (b) Evolution of the clonal persistence and (c) labelled cell fraction for K14-CreER/Rosa-SmoM2 clones in the interscale. (d) Fraction of EdU/BrdU double-labelled cells as a function of basal clone size at 8w for K14-CreER/Rosa-SmoM2 clones, for 2 (left), 4 (centre) and 6 (right) days of continuous BrdU incorporation. (e) Immunostaining for β4-integrin, SmoM2 and active-caspase-3 in K14-CreER/Rosa-SmoM2 clones 8w after SmoM2 activation. (f) Percentage of BCC, dysplastic, hyperplastic and normally differentiating clones presenting at least one active-caspase-3 positive cell at 8w post-induction (n=117 clones analysed from 4 independent experiments). (g) Quantification of the number (%) of basal and suprabasal apoptotic cells in dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones 8w after SmoM2-activation. (h) Percentage of dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones presenting basal and suprabasal apoptosis 8w after oncogenic activation. (i) Cumulative distribution of the fraction of basal apoptosis as a function of basal cell number in a K14-CreER/Rosa-SmoM2 clone at 8w (data in red). The green line is the expected theoretical distribution of apoptotic fraction if apoptosis occurred randomly (following a Poisson process), in any clone with the same probability. The data is statistically different from the random theory, showing that apoptosis clusters in certain clones at a given time point. (j) Quantification of EdU/BrdU double-labelled cells as a function of the period of continuous BrdU incorporation for large K14 clones at 4w (black), 8w (orange) and 12w (red) post clonal induction. The dashed lines represent the model fit ( Supplementary Theory ). (k) Whisker plot of the suprabasal clone size in the interscale. The boxes delineate the first and third quartiles of the data, while the whiskers delineate the first and last decile of the data at a given time point. The thick continuous line is the best fit from the model from which we extract the probability of fate choices in tumor SC and progenitors, displayed in Fig.4g . The thin lines represent the mean clone sizes of stem cells (top curve) and progenitors (bottom curve) if they were alone. (l) Short-term fate outcome of progenitors in K14-CreER/Rosa-SmoM2 clones at 8w, as assessed by using EdU as a clonal marker. We count only cell doublets and classify them as either basal-basal, basal-suprabasal, or suprabasal-suprabasal. (n=49 clones from 3 independent experiments) Immunostaining for β4-integrin, EdU and SmoM2 in K14-CreER/Rosa-SmoM2 hyperplastic/dysplastic clones (top) and in BCC (bottom panel). BCC: basal cell carcinoma; D: dysplasia; H: hyperplasia; N: normal differentiation; SB: suprabasal. Hoechst nuclear staining is represented in blue; Scale bars, 10μm. Error bars represent the s.e.m.

    Journal: Nature

    Article Title: Defining the clonal dynamics leading to mouse skin tumour initiation

    doi: 10.1038/nature19069

    Figure Lengend Snippet: Clonal dynamics of interscale K14-CreER/Rosa-SmoM2 clones is consistent with two populations (a) Distribution of K14-CreER/Rosa-YFP (black) and K14-CreER/Rosa-SmoM2 (red) clone sizes as measured by total cell content, imaged by confocal microscopy on whole mount tail epidermis from 1w to 24w post-induction. The number of clones analyzed for K14-CreER/Rosa-SmoM2 is indicated in Fig 4b; the number of clones counted in K14-CreER/Rosa-YFP is as indicated in Fig. 2a. (b) Evolution of the clonal persistence and (c) labelled cell fraction for K14-CreER/Rosa-SmoM2 clones in the interscale. (d) Fraction of EdU/BrdU double-labelled cells as a function of basal clone size at 8w for K14-CreER/Rosa-SmoM2 clones, for 2 (left), 4 (centre) and 6 (right) days of continuous BrdU incorporation. (e) Immunostaining for β4-integrin, SmoM2 and active-caspase-3 in K14-CreER/Rosa-SmoM2 clones 8w after SmoM2 activation. (f) Percentage of BCC, dysplastic, hyperplastic and normally differentiating clones presenting at least one active-caspase-3 positive cell at 8w post-induction (n=117 clones analysed from 4 independent experiments). (g) Quantification of the number (%) of basal and suprabasal apoptotic cells in dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones 8w after SmoM2-activation. (h) Percentage of dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones presenting basal and suprabasal apoptosis 8w after oncogenic activation. (i) Cumulative distribution of the fraction of basal apoptosis as a function of basal cell number in a K14-CreER/Rosa-SmoM2 clone at 8w (data in red). The green line is the expected theoretical distribution of apoptotic fraction if apoptosis occurred randomly (following a Poisson process), in any clone with the same probability. The data is statistically different from the random theory, showing that apoptosis clusters in certain clones at a given time point. (j) Quantification of EdU/BrdU double-labelled cells as a function of the period of continuous BrdU incorporation for large K14 clones at 4w (black), 8w (orange) and 12w (red) post clonal induction. The dashed lines represent the model fit ( Supplementary Theory ). (k) Whisker plot of the suprabasal clone size in the interscale. The boxes delineate the first and third quartiles of the data, while the whiskers delineate the first and last decile of the data at a given time point. The thick continuous line is the best fit from the model from which we extract the probability of fate choices in tumor SC and progenitors, displayed in Fig.4g . The thin lines represent the mean clone sizes of stem cells (top curve) and progenitors (bottom curve) if they were alone. (l) Short-term fate outcome of progenitors in K14-CreER/Rosa-SmoM2 clones at 8w, as assessed by using EdU as a clonal marker. We count only cell doublets and classify them as either basal-basal, basal-suprabasal, or suprabasal-suprabasal. (n=49 clones from 3 independent experiments) Immunostaining for β4-integrin, EdU and SmoM2 in K14-CreER/Rosa-SmoM2 hyperplastic/dysplastic clones (top) and in BCC (bottom panel). BCC: basal cell carcinoma; D: dysplasia; H: hyperplasia; N: normal differentiation; SB: suprabasal. Hoechst nuclear staining is represented in blue; Scale bars, 10μm. Error bars represent the s.e.m.

    Article Snippet: Primary antibodies used were the following: anti-GFP (Rabbit, 1/100, BD, ref. A11122), anti-GFP (Goat, 1:800,Abcam, ref. Ab6673), anti-active-caspase3 (Rabbit, 1/600,R & D, ref. AF835), anti-β4-integrin(Rat, 1:200, BD, ref. 553745) and anti-K31 (Guinea Pig, 1:200, Progen, ref. GP-hHa1).

    Techniques: Clone Assay, Confocal Microscopy, BrdU Incorporation Assay, Immunostaining, Activation Assay, Whisker Assay, Marker, Staining

    Clonal dynamics of interscale InvSmoM2 clones is consistent with a single imbalanced population of progenitors slowing down in time (a) Distribution of Inv-CreER/Rosa-YFP (black) and Inv-CreER/Rosa-SmoM2 (red) clone sizes as measured by total cell content, imaged by confocal microscopy on whole mount tail epidermis from 1w to 24w following Tamoxifen administration. The number of clones analysed in Inv-CreER/Rosa-SmoM2 is indicated in Fig 3b . The number of clones counted in Inv-CreER/Rosa-YFP is as indicated in Fig. 2b . (b) Evolution of the clonal persistence for interscale Inv-CreER/Rosa-SmoM2 clones. (c) Labelled cell fraction for interscale Inv-CreER/Rosa-SmoM2 clones. (d) Fraction of EdU/BrdU double-labelled cells as a function of basal clone size at 8w for Inv-CreER/Rosa-SmoM2 clones, for 2 (left), 4 (centre) and 6 (right) days of continuous BrdU incorporation. (e) Immunostaining for β4-integrin, SmoM2 and active-caspase-3 in Inv-CreER/Rosa-SmoM2 clones at 8w post-induction. (f) Percentage of dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones presenting at least one active-caspase positive cell within the clone at 8w post-induction (n= 73 clones analysed from 4 independent experiments). (g) Quantification of the number (%) of basal and suprabasal apoptotic cells in dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones 8w after SmoM2-activation. (h) Percentage of dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones presenting apoptosis in basal and suprabasal compartments 8w after oncogenic activation. (i) Cumulative distribution of the fraction of basal apoptosis as a function of basal cell number in an Inv-CreER/Rosa-SmoM2 clone at 8w (data in blue). The green line is the expected theoretical distribution of apoptotic fraction if apoptosis occurred randomly (following a Poisson process), in any clone with the same probability. The data is statistically different from the random theory, showing that apoptosis clusters in certain clones at a given time point. (j) Short-term fate outcome of progenitors in Inv-CreER/Rosa-SmoM2 clones at 8w, as assessed by using EdU as a clonal marker. We count only cell doublets and classify them as either basal-basal, basal-suprabasal, or suprabasal-suprabasal. (n=47 clones from 3 independent experiments). Immunostaining for β4-integrin, EdU and SmoM2 showing the different type of cell fate outcomes found in Inv-CreER/Rosa-SmoM2 clones. D: dysplasia; H: hyperplasia; N: normal differentiation. Hoechst nuclear staining is represented in blue; Scale bars, 10μm. Histograms and error bars represent the mean and the s.e.m.

    Journal: Nature

    Article Title: Defining the clonal dynamics leading to mouse skin tumour initiation

    doi: 10.1038/nature19069

    Figure Lengend Snippet: Clonal dynamics of interscale InvSmoM2 clones is consistent with a single imbalanced population of progenitors slowing down in time (a) Distribution of Inv-CreER/Rosa-YFP (black) and Inv-CreER/Rosa-SmoM2 (red) clone sizes as measured by total cell content, imaged by confocal microscopy on whole mount tail epidermis from 1w to 24w following Tamoxifen administration. The number of clones analysed in Inv-CreER/Rosa-SmoM2 is indicated in Fig 3b . The number of clones counted in Inv-CreER/Rosa-YFP is as indicated in Fig. 2b . (b) Evolution of the clonal persistence for interscale Inv-CreER/Rosa-SmoM2 clones. (c) Labelled cell fraction for interscale Inv-CreER/Rosa-SmoM2 clones. (d) Fraction of EdU/BrdU double-labelled cells as a function of basal clone size at 8w for Inv-CreER/Rosa-SmoM2 clones, for 2 (left), 4 (centre) and 6 (right) days of continuous BrdU incorporation. (e) Immunostaining for β4-integrin, SmoM2 and active-caspase-3 in Inv-CreER/Rosa-SmoM2 clones at 8w post-induction. (f) Percentage of dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones presenting at least one active-caspase positive cell within the clone at 8w post-induction (n= 73 clones analysed from 4 independent experiments). (g) Quantification of the number (%) of basal and suprabasal apoptotic cells in dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones 8w after SmoM2-activation. (h) Percentage of dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones presenting apoptosis in basal and suprabasal compartments 8w after oncogenic activation. (i) Cumulative distribution of the fraction of basal apoptosis as a function of basal cell number in an Inv-CreER/Rosa-SmoM2 clone at 8w (data in blue). The green line is the expected theoretical distribution of apoptotic fraction if apoptosis occurred randomly (following a Poisson process), in any clone with the same probability. The data is statistically different from the random theory, showing that apoptosis clusters in certain clones at a given time point. (j) Short-term fate outcome of progenitors in Inv-CreER/Rosa-SmoM2 clones at 8w, as assessed by using EdU as a clonal marker. We count only cell doublets and classify them as either basal-basal, basal-suprabasal, or suprabasal-suprabasal. (n=47 clones from 3 independent experiments). Immunostaining for β4-integrin, EdU and SmoM2 showing the different type of cell fate outcomes found in Inv-CreER/Rosa-SmoM2 clones. D: dysplasia; H: hyperplasia; N: normal differentiation. Hoechst nuclear staining is represented in blue; Scale bars, 10μm. Histograms and error bars represent the mean and the s.e.m.

    Article Snippet: Primary antibodies used were the following: anti-GFP (Rabbit, 1/100, BD, ref. A11122), anti-GFP (Goat, 1:800,Abcam, ref. Ab6673), anti-active-caspase3 (Rabbit, 1/600,R & D, ref. AF835), anti-β4-integrin(Rat, 1:200, BD, ref. 553745) and anti-K31 (Guinea Pig, 1:200, Progen, ref. GP-hHa1).

    Techniques: Clone Assay, Confocal Microscopy, BrdU Incorporation Assay, Immunostaining, Activation Assay, Marker, Staining

    The fate of oncogene targeted clones is determined by the initial targeted cell (SC or CP) and their location in scale or interscale regions (a) Orthogonal view used to quantify the number of clones, cells stained with β4-integrin/SmoM2. (left). Quantification of the number of clones induced 1 week after Tamoxifen administration in scale and interscale regions in K14-CreER/Rosa-SmoM2 (n=4 animals, 0.1mg Tamoxifen) and Inv-CreER/ Rosa-SmoM2 (n=3 animals, 2.5 mg Tamoxifen) (right). (b) Immunostaining for β4-integrin and SmoM2 in K14-CreER/Rosa-SmoM2 and Inv-CreER/Rosa-SmoM2 clones located in the scale and interscale regions, 8w after oncogene activation. (c) Immunostaining for the differentiation marker keratin-10, K10, and SmoM2 in K14-CreER/Rosa-SmoM2 and Inv-CreER/Rosa-SmoM2 clones 8w after oncogene activation, showing absence of differentiated cells in K14-CreER/Rosa-SmoM2 clones and alteration of the differentiation in Inv-CreER/Rosa-SmoM2 clones. Hoechst nuclear staining is represented in blue; Scale bars, 10μm.

    Journal: Nature

    Article Title: Defining the clonal dynamics leading to mouse skin tumour initiation

    doi: 10.1038/nature19069

    Figure Lengend Snippet: The fate of oncogene targeted clones is determined by the initial targeted cell (SC or CP) and their location in scale or interscale regions (a) Orthogonal view used to quantify the number of clones, cells stained with β4-integrin/SmoM2. (left). Quantification of the number of clones induced 1 week after Tamoxifen administration in scale and interscale regions in K14-CreER/Rosa-SmoM2 (n=4 animals, 0.1mg Tamoxifen) and Inv-CreER/ Rosa-SmoM2 (n=3 animals, 2.5 mg Tamoxifen) (right). (b) Immunostaining for β4-integrin and SmoM2 in K14-CreER/Rosa-SmoM2 and Inv-CreER/Rosa-SmoM2 clones located in the scale and interscale regions, 8w after oncogene activation. (c) Immunostaining for the differentiation marker keratin-10, K10, and SmoM2 in K14-CreER/Rosa-SmoM2 and Inv-CreER/Rosa-SmoM2 clones 8w after oncogene activation, showing absence of differentiated cells in K14-CreER/Rosa-SmoM2 clones and alteration of the differentiation in Inv-CreER/Rosa-SmoM2 clones. Hoechst nuclear staining is represented in blue; Scale bars, 10μm.

    Article Snippet: Primary antibodies used were the following: anti-GFP (Rabbit, 1/100, BD, ref. A11122), anti-GFP (Goat, 1:800,Abcam, ref. Ab6673), anti-active-caspase3 (Rabbit, 1/600,R & D, ref. AF835), anti-β4-integrin(Rat, 1:200, BD, ref. 553745) and anti-K31 (Guinea Pig, 1:200, Progen, ref. GP-hHa1).

    Techniques: Clone Assay, Staining, Immunostaining, Activation Assay, Marker

    SCs but not CPs are competent to initiate BCC formation upon HH activation (a) Genetic strategy to activate SmoM2 expression in SCs and CPs. (b) Immunostaining of β4-integrin/SmoM2 in ear and tail skin 24w after SmoM2 activation. (c) Immunostaining of β4-integrin/K14 in ventral skin 24w after Ptch1 deletion. (d) Quantification of tumour burden (total tumour area divided by length of epidermis) following Ptch1 deletion. Quantification of BCC number per length (mm) following Ptch1 deletion. (n=4 Inv-CreER/Ptch1KO animals and n=3 K14CreER/Ptch1KO animals (e) Immunostaining of K31/ SmoM2 in whole mount tail skin. (f) Quantification of the morphology of SmoM2-expressing clones. Description of number of counted clones is found in the method section. Hoechst nuclear staining in blue; scale bars, 100μm. *P≤0,05, **P≤0,01, ***P≤0,001. Histograms and error bars represent the mean and the standard error of the mean (s.e.m).

    Journal: Nature

    Article Title: Defining the clonal dynamics leading to mouse skin tumour initiation

    doi: 10.1038/nature19069

    Figure Lengend Snippet: SCs but not CPs are competent to initiate BCC formation upon HH activation (a) Genetic strategy to activate SmoM2 expression in SCs and CPs. (b) Immunostaining of β4-integrin/SmoM2 in ear and tail skin 24w after SmoM2 activation. (c) Immunostaining of β4-integrin/K14 in ventral skin 24w after Ptch1 deletion. (d) Quantification of tumour burden (total tumour area divided by length of epidermis) following Ptch1 deletion. Quantification of BCC number per length (mm) following Ptch1 deletion. (n=4 Inv-CreER/Ptch1KO animals and n=3 K14CreER/Ptch1KO animals (e) Immunostaining of K31/ SmoM2 in whole mount tail skin. (f) Quantification of the morphology of SmoM2-expressing clones. Description of number of counted clones is found in the method section. Hoechst nuclear staining in blue; scale bars, 100μm. *P≤0,05, **P≤0,01, ***P≤0,001. Histograms and error bars represent the mean and the standard error of the mean (s.e.m).

    Article Snippet: Primary antibodies used were the following: anti-GFP (Rabbit, 1/100, BD, ref. A11122), anti-GFP (Goat, 1:800,Abcam, ref. Ab6673), anti-active-caspase3 (Rabbit, 1/600,R & D, ref. AF835), anti-β4-integrin(Rat, 1:200, BD, ref. 553745) and anti-K31 (Guinea Pig, 1:200, Progen, ref. GP-hHa1).

    Techniques: Activation Assay, Expressing, Immunostaining, Clone Assay, Staining

    SmoM2 expression in SCs induces tumour SCs that lead to BCC formation. (a) Immunostaining for β4-integrin, YFP and SmoM2 in K14-CreER/Rosa-YFP and K14-CreER/Rosa-SmoM2 epidermis at different time points. (b) Distribution of K14-CreER/Rosa-YFP and K14-CreER/Rosa-SmoM2 basal clone sizes. The number of clones analysed for K14-CreER/Rosa-SmoM2 is indicated and for K14-CreER/Rosa-YFP indicated in Fig. 2a . (c) Quantification of EdU/BrdU double-labelled cells following continuous BrdU administration, at 8w post clonal induction for small K14-CreER clones, Inv-CreER clones, and large K14-CreER clones. (d) Quantification of the number of apoptotic cells in BCC, dysplastic, hyperplastic and normally differentiating K14-CreER/Rosa-SmoM2 clones 8w post-induction (n= 117 clones analysed from 4 independent experiments). (e) Division rate in large K14 clones determined from double-labelling experiments (data in black, fit in red dashed line). (f) Whisker plot of the mean basal clone size in the interscale. The boxes delineate the first and third quartiles of the data, while the whiskers delineate the first and last decile of the data. The thick continuous line is the best fit from the model from which we extract the probability of fate choices in tumour SCs and progenitors displayed in g. The thin dashed lines represent the predicted mean clone sizes of SCs- (top thin curve) and progenitors- (bottom thin curve) derived clones alone. (g) Cell fate probabilities of the tumour SC upon SmoM2 activation. (h) Basal clone size distribution of K14CreER/SmoM2 clones (black). Red lines are the model prediction using only the parameters extracted from g. Shaded areas represent 95% confidence intervals for the model prediction. BCC: basal cell carcinoma; D: dysplasia; H: hyperplasia; N: normal differentiation. Hoechst nuclear staining in blue; scale bars, 10μm. Histograms and error bars represent the mean and the s.e.m. (b–f) and the s.d. (h).

    Journal: Nature

    Article Title: Defining the clonal dynamics leading to mouse skin tumour initiation

    doi: 10.1038/nature19069

    Figure Lengend Snippet: SmoM2 expression in SCs induces tumour SCs that lead to BCC formation. (a) Immunostaining for β4-integrin, YFP and SmoM2 in K14-CreER/Rosa-YFP and K14-CreER/Rosa-SmoM2 epidermis at different time points. (b) Distribution of K14-CreER/Rosa-YFP and K14-CreER/Rosa-SmoM2 basal clone sizes. The number of clones analysed for K14-CreER/Rosa-SmoM2 is indicated and for K14-CreER/Rosa-YFP indicated in Fig. 2a . (c) Quantification of EdU/BrdU double-labelled cells following continuous BrdU administration, at 8w post clonal induction for small K14-CreER clones, Inv-CreER clones, and large K14-CreER clones. (d) Quantification of the number of apoptotic cells in BCC, dysplastic, hyperplastic and normally differentiating K14-CreER/Rosa-SmoM2 clones 8w post-induction (n= 117 clones analysed from 4 independent experiments). (e) Division rate in large K14 clones determined from double-labelling experiments (data in black, fit in red dashed line). (f) Whisker plot of the mean basal clone size in the interscale. The boxes delineate the first and third quartiles of the data, while the whiskers delineate the first and last decile of the data. The thick continuous line is the best fit from the model from which we extract the probability of fate choices in tumour SCs and progenitors displayed in g. The thin dashed lines represent the predicted mean clone sizes of SCs- (top thin curve) and progenitors- (bottom thin curve) derived clones alone. (g) Cell fate probabilities of the tumour SC upon SmoM2 activation. (h) Basal clone size distribution of K14CreER/SmoM2 clones (black). Red lines are the model prediction using only the parameters extracted from g. Shaded areas represent 95% confidence intervals for the model prediction. BCC: basal cell carcinoma; D: dysplasia; H: hyperplasia; N: normal differentiation. Hoechst nuclear staining in blue; scale bars, 10μm. Histograms and error bars represent the mean and the s.e.m. (b–f) and the s.d. (h).

    Article Snippet: Primary antibodies used were the following: anti-GFP (Rabbit, 1/100, BD, ref. A11122), anti-GFP (Goat, 1:800,Abcam, ref. Ab6673), anti-active-caspase3 (Rabbit, 1/600,R & D, ref. AF835), anti-β4-integrin(Rat, 1:200, BD, ref. 553745) and anti-K31 (Guinea Pig, 1:200, Progen, ref. GP-hHa1).

    Techniques: Expressing, Immunostaining, Clone Assay, Whisker Assay, Derivative Assay, Activation Assay, Staining

    Role of aldosterone in expression of BK-α and BK-β4.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Regulation of BK-α expression in the distal nephron by aldosterone and urine pH

    doi: 10.1152/ajprenal.00171.2013

    Figure Lengend Snippet: Role of aldosterone in expression of BK-α and BK-β4.

    Article Snippet: Primary antibodies included anti-BK-α (mouse monoclonal, diluted 1:200; Neuromab), anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories), anti-cadherin (goat polyclonal, diluted 1:500; Santa Cruz Biotechnology), anti-GAPDH (goat polyclonal, diluted 1:500, Santa Cruz Biotechnology), and anti-β-actin (mouse monoclonal, diluted 1:500; Santa Cruz Biotechnology) with goat anti-rabbit IgG, donkey anti-mouse IgG, or donkey anti-goat IgG-conjugated horseradish peroxidase (diluted 1:10,000–1:20,000; Santa Cruz Biotechnology).

    Techniques: Expressing

    Western blot analysis of renal BK-α and BK- β4 expressions in mice with acid, neutral or alkaline drinking water. Representative immunoblots of total cellular BK-α expression ( A ) and summary bar plots of total BK-β4 expression

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Regulation of BK-α expression in the distal nephron by aldosterone and urine pH

    doi: 10.1152/ajprenal.00171.2013

    Figure Lengend Snippet: Western blot analysis of renal BK-α and BK- β4 expressions in mice with acid, neutral or alkaline drinking water. Representative immunoblots of total cellular BK-α expression ( A ) and summary bar plots of total BK-β4 expression

    Article Snippet: Primary antibodies included anti-BK-α (mouse monoclonal, diluted 1:200; Neuromab), anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories), anti-cadherin (goat polyclonal, diluted 1:500; Santa Cruz Biotechnology), anti-GAPDH (goat polyclonal, diluted 1:500, Santa Cruz Biotechnology), and anti-β-actin (mouse monoclonal, diluted 1:500; Santa Cruz Biotechnology) with goat anti-rabbit IgG, donkey anti-mouse IgG, or donkey anti-goat IgG-conjugated horseradish peroxidase (diluted 1:10,000–1:20,000; Santa Cruz Biotechnology).

    Techniques: Western Blot, Mouse Assay, Expressing

    Role of acid-base consumption on expression of BK-α and BK-β4.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Regulation of BK-α expression in the distal nephron by aldosterone and urine pH

    doi: 10.1152/ajprenal.00171.2013

    Figure Lengend Snippet: Role of acid-base consumption on expression of BK-α and BK-β4.

    Article Snippet: Primary antibodies included anti-BK-α (mouse monoclonal, diluted 1:200; Neuromab), anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories), anti-cadherin (goat polyclonal, diluted 1:500; Santa Cruz Biotechnology), anti-GAPDH (goat polyclonal, diluted 1:500, Santa Cruz Biotechnology), and anti-β-actin (mouse monoclonal, diluted 1:500; Santa Cruz Biotechnology) with goat anti-rabbit IgG, donkey anti-mouse IgG, or donkey anti-goat IgG-conjugated horseradish peroxidase (diluted 1:10,000–1:20,000; Santa Cruz Biotechnology).

    Techniques: Expressing

    Western blot analysis of renal BK-α and BK-β4 expression in WT on HK-alk diets treated with vehicle or spironolactone. Representative immunoblots ( A ) and summary bar plots of relative densitometries of total cell lysate BK-β4 (

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Regulation of BK-α expression in the distal nephron by aldosterone and urine pH

    doi: 10.1152/ajprenal.00171.2013

    Figure Lengend Snippet: Western blot analysis of renal BK-α and BK-β4 expression in WT on HK-alk diets treated with vehicle or spironolactone. Representative immunoblots ( A ) and summary bar plots of relative densitometries of total cell lysate BK-β4 (

    Article Snippet: Primary antibodies included anti-BK-α (mouse monoclonal, diluted 1:200; Neuromab), anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories), anti-cadherin (goat polyclonal, diluted 1:500; Santa Cruz Biotechnology), anti-GAPDH (goat polyclonal, diluted 1:500, Santa Cruz Biotechnology), and anti-β-actin (mouse monoclonal, diluted 1:500; Santa Cruz Biotechnology) with goat anti-rabbit IgG, donkey anti-mouse IgG, or donkey anti-goat IgG-conjugated horseradish peroxidase (diluted 1:10,000–1:20,000; Santa Cruz Biotechnology).

    Techniques: Western Blot, Expressing

    Western blot analysis of BK-β4 and PM BK-α expression in MDCK-C11 cells. Immunoblots ( A ) and summary bar plots ( B ) demonstrating the relative densitometries of cadherin/GAPDH in the PM, OM, and cytosolic (C) fractions of MDCK-C11 cells.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Regulation of BK-α expression in the distal nephron by aldosterone and urine pH

    doi: 10.1152/ajprenal.00171.2013

    Figure Lengend Snippet: Western blot analysis of BK-β4 and PM BK-α expression in MDCK-C11 cells. Immunoblots ( A ) and summary bar plots ( B ) demonstrating the relative densitometries of cadherin/GAPDH in the PM, OM, and cytosolic (C) fractions of MDCK-C11 cells.

    Article Snippet: Primary antibodies included anti-BK-α (mouse monoclonal, diluted 1:200; Neuromab), anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories), anti-cadherin (goat polyclonal, diluted 1:500; Santa Cruz Biotechnology), anti-GAPDH (goat polyclonal, diluted 1:500, Santa Cruz Biotechnology), and anti-β-actin (mouse monoclonal, diluted 1:500; Santa Cruz Biotechnology) with goat anti-rabbit IgG, donkey anti-mouse IgG, or donkey anti-goat IgG-conjugated horseradish peroxidase (diluted 1:10,000–1:20,000; Santa Cruz Biotechnology).

    Techniques: Western Blot, Expressing

    Effects of high-K diets and BK-β4 on BK-α expression.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Regulation of BK-α expression in the distal nephron by aldosterone and urine pH

    doi: 10.1152/ajprenal.00171.2013

    Figure Lengend Snippet: Effects of high-K diets and BK-β4 on BK-α expression.

    Article Snippet: Primary antibodies included anti-BK-α (mouse monoclonal, diluted 1:200; Neuromab), anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories), anti-cadherin (goat polyclonal, diluted 1:500; Santa Cruz Biotechnology), anti-GAPDH (goat polyclonal, diluted 1:500, Santa Cruz Biotechnology), and anti-β-actin (mouse monoclonal, diluted 1:500; Santa Cruz Biotechnology) with goat anti-rabbit IgG, donkey anti-mouse IgG, or donkey anti-goat IgG-conjugated horseradish peroxidase (diluted 1:10,000–1:20,000; Santa Cruz Biotechnology).

    Techniques: Expressing

    Modulation of miRNAs in ΔCK2β-MCF10A cells. ( A ) Log2 fold change of the main miRNAs modulated in CK2β-depleted versus parental MCF10A cells measured by miRNA array analysis; ( B ) Changes of miRNA expression between CK2β-depleted and Mock-MCF10A cells were confirmed by using the indicated TaqMan probes. The relative amount of each miRNAs was determined by cross-normalization to ΔCK2β samples using the comparative method and miR-720 as an internal reference; ( C ) Two targets of miR-200 and miR-30 families, respectively Zeb1 and integrin β3, were analyzed by Western blot and/or immunofluorescence in Mock- and CK2β-depleted cells. The ratio ΔCK2β/Mock of signal intensity in western blot was determined (3.5 and 2.3 for Zeb1 and integrin β3 respectively). Arrows indicate integrin β3 localization; ( D ) Integrin β1 and β4, targets of miR-21 were analyzed by western blot and/or immunofluorescence in Mock- and CK2β-depleted cells. The ratio ΔCK2β/Mock of signal intensity in western blot was 0.4 for integrin β1. F-actin in green, nuclei in blue, and integrin β in red. Scale bar, 10 μm.

    Journal: Cancers

    Article Title: Stem Cell-Like Properties of CK2β-down Regulated Mammary Cells

    doi: 10.3390/cancers9090114

    Figure Lengend Snippet: Modulation of miRNAs in ΔCK2β-MCF10A cells. ( A ) Log2 fold change of the main miRNAs modulated in CK2β-depleted versus parental MCF10A cells measured by miRNA array analysis; ( B ) Changes of miRNA expression between CK2β-depleted and Mock-MCF10A cells were confirmed by using the indicated TaqMan probes. The relative amount of each miRNAs was determined by cross-normalization to ΔCK2β samples using the comparative method and miR-720 as an internal reference; ( C ) Two targets of miR-200 and miR-30 families, respectively Zeb1 and integrin β3, were analyzed by Western blot and/or immunofluorescence in Mock- and CK2β-depleted cells. The ratio ΔCK2β/Mock of signal intensity in western blot was determined (3.5 and 2.3 for Zeb1 and integrin β3 respectively). Arrows indicate integrin β3 localization; ( D ) Integrin β1 and β4, targets of miR-21 were analyzed by western blot and/or immunofluorescence in Mock- and CK2β-depleted cells. The ratio ΔCK2β/Mock of signal intensity in western blot was 0.4 for integrin β1. F-actin in green, nuclei in blue, and integrin β in red. Scale bar, 10 μm.

    Article Snippet: Western Blot Analysis Primary antibodies were anti-actin (Abcam, Cambridge, UK, ab8226), anti-Zeb (Santa Cruz Biotechnology, Heidelberg, Germany, sc-25388), anti-integrin β1, anti- integrin β4 (BD Biosciences, 610467, 611232, respectively), anti- Integrin β3 (Abcam, Ab7167), and anti-PARP (Thermo Scientific, Courtaboeuf, France, 9542).

    Techniques: Expressing, Western Blot, Immunofluorescence

    Inhibiting β4 Integrin decreases fission. Organoids grown in control media (ENR-Y; A) or in the presence of a β4 Integrin blocking antibody (B) were recorded by time-lapse imaging. The number of branches were measured every 6 h (C, S9 Data ). Branch number increases more slowly in organoids grown in the presence of the β4 Integrin antibody ( p

    Journal: PLoS Biology

    Article Title: Paneth Cell-Rich Regions Separated by a Cluster of Lgr5+ Cells Initiate Crypt Fission in the Intestinal Stem Cell Niche

    doi: 10.1371/journal.pbio.1002491

    Figure Lengend Snippet: Inhibiting β4 Integrin decreases fission. Organoids grown in control media (ENR-Y; A) or in the presence of a β4 Integrin blocking antibody (B) were recorded by time-lapse imaging. The number of branches were measured every 6 h (C, S9 Data ). Branch number increases more slowly in organoids grown in the presence of the β4 Integrin antibody ( p

    Article Snippet: Anti-β4 Integrin (Abcam) was added at 5 μg/ml.

    Techniques: Blocking Assay, Imaging

    Mislocalising Paneth cells leads to more asymmetric fission. Shown are 3D projections of organoids stained to visualise F-actin (Phalloidin, red), Paneth cells (Lysozyme, white), and β4 Integrin (green), grown in control (ENR) media (A) or after the addition of an inhibitory Eph fragment (B). Note the bud formation between three Paneth cells located away from the base of the crypt (arrow in B). (C) The frequency of Paneth cells along the crypt axis was recorded relative to the crypt base and is shown in a histogram (bin every 10 μm), revealing their displacement away from the crypt base after addition of the Eph fragment ( n = 304 Paneth cells from 48 crypts for ENR and 423 Paneth cells from 45 crypts for Eph, S6 Data ). The crypt base was defined as the tip of an organoid branch. (D) Symmetry ratio (length of short daughter ÷ length of long daughter) in organoids grown in ENR with or without Eph fragments shows that organoids grown in the Eph fragment have fewer symmetrical fissions ( p

    Journal: PLoS Biology

    Article Title: Paneth Cell-Rich Regions Separated by a Cluster of Lgr5+ Cells Initiate Crypt Fission in the Intestinal Stem Cell Niche

    doi: 10.1371/journal.pbio.1002491

    Figure Lengend Snippet: Mislocalising Paneth cells leads to more asymmetric fission. Shown are 3D projections of organoids stained to visualise F-actin (Phalloidin, red), Paneth cells (Lysozyme, white), and β4 Integrin (green), grown in control (ENR) media (A) or after the addition of an inhibitory Eph fragment (B). Note the bud formation between three Paneth cells located away from the base of the crypt (arrow in B). (C) The frequency of Paneth cells along the crypt axis was recorded relative to the crypt base and is shown in a histogram (bin every 10 μm), revealing their displacement away from the crypt base after addition of the Eph fragment ( n = 304 Paneth cells from 48 crypts for ENR and 423 Paneth cells from 45 crypts for Eph, S6 Data ). The crypt base was defined as the tip of an organoid branch. (D) Symmetry ratio (length of short daughter ÷ length of long daughter) in organoids grown in ENR with or without Eph fragments shows that organoids grown in the Eph fragment have fewer symmetrical fissions ( p

    Article Snippet: Anti-β4 Integrin (Abcam) was added at 5 μg/ml.

    Techniques: Staining

    Fission in Organoids Is Similar to Fission in Tissue. Crypts in organoids were stained to visualise F-actin (Phalloidin, white), Lgr5-GFP (green), and Paneth cells (Lysozyme, red) (A–C) and corresponding Imaris-rendered surfaces of the same images prepared (crypt lumen, blue; crypt wall, transparent yellow; Lgr5-GFP+ cells, green; and Paneth cells, red A′–C′). (A) Single crypts in organoids have a straight lumen and alternating Lgr5+ and Paneth cells at the crypt base. (B, C) Fission of organoid crypts can initiate at the crypt base so that two prospective daughter crypts are formed on either side of a bifurcation. (B) Early fission is identifiable by two short prospective daughter crypts at the base of the crypt. Paneth cells are located on either side of the bifurcation, whilst Lgr5+ cells are located between the two Paneth-cell-rich regions. (C) Late fission of organoid crypts is identifiable by two longer daughter crypts sharing a common lumen above the bifurcation point. At this time, both Lgr5+ and Paneth cells are mainly restricted to the two crypt bases. (D, D′) Time lapse of Life-ActGFP expressing organoids shows a crypt base widening and two daughter crypts forming and growing in opposing directions. (E) Extended focus optical sections of an organoid stained against F-actin (Phalloidin, green), β4 Integrin (red), Lysozyme (white), and nuclei (Hoechst, blue). Arrows indicate basal membrane of Paneth cells. (F) Quantifying β4 Integrin on the basal surface of Paneth cells (PC) and their neighbouring cells (CBC) reveals β4 Integrin signal intensity is higher on Paneth cells (±SEM, p

    Journal: PLoS Biology

    Article Title: Paneth Cell-Rich Regions Separated by a Cluster of Lgr5+ Cells Initiate Crypt Fission in the Intestinal Stem Cell Niche

    doi: 10.1371/journal.pbio.1002491

    Figure Lengend Snippet: Fission in Organoids Is Similar to Fission in Tissue. Crypts in organoids were stained to visualise F-actin (Phalloidin, white), Lgr5-GFP (green), and Paneth cells (Lysozyme, red) (A–C) and corresponding Imaris-rendered surfaces of the same images prepared (crypt lumen, blue; crypt wall, transparent yellow; Lgr5-GFP+ cells, green; and Paneth cells, red A′–C′). (A) Single crypts in organoids have a straight lumen and alternating Lgr5+ and Paneth cells at the crypt base. (B, C) Fission of organoid crypts can initiate at the crypt base so that two prospective daughter crypts are formed on either side of a bifurcation. (B) Early fission is identifiable by two short prospective daughter crypts at the base of the crypt. Paneth cells are located on either side of the bifurcation, whilst Lgr5+ cells are located between the two Paneth-cell-rich regions. (C) Late fission of organoid crypts is identifiable by two longer daughter crypts sharing a common lumen above the bifurcation point. At this time, both Lgr5+ and Paneth cells are mainly restricted to the two crypt bases. (D, D′) Time lapse of Life-ActGFP expressing organoids shows a crypt base widening and two daughter crypts forming and growing in opposing directions. (E) Extended focus optical sections of an organoid stained against F-actin (Phalloidin, green), β4 Integrin (red), Lysozyme (white), and nuclei (Hoechst, blue). Arrows indicate basal membrane of Paneth cells. (F) Quantifying β4 Integrin on the basal surface of Paneth cells (PC) and their neighbouring cells (CBC) reveals β4 Integrin signal intensity is higher on Paneth cells (±SEM, p

    Article Snippet: Anti-β4 Integrin (Abcam) was added at 5 μg/ml.

    Techniques: Staining, Expressing

    Paneth cells are more adherent to laminin than other crypt cells. (A, B) Immunofluorescence images of sectioned tissue show nuclei (Hoechst, blue), F-actin (Phalloidin, green), and β4 Integrin (red): (A) is an optical section; (B) is a 3D projection (from Imaris) of the same crypt. In the left panel, the basal surface of crypt base columnar (CBC) cells are pseudo-coloured yellow, and the basal surface of Paneth cells are pseudo-coloured purple. Paneth cells are identifiable as the large cells with round, basally placed nuclei; CBC cells are narrow and have compressed nuclei. White lines (left and right panels) indicate the boundaries between Paneth cells and their neighbours. (C) Average β4 Integrin intensity on the basal surface of Paneth cells (PC) was normalised to the average value for neighbouring cells (CBC). Left panels in (A) and (B) indicate the Imaris-rendered surfaces that were used to measure β4 Integrin signal intensity. ± SEM, p

    Journal: PLoS Biology

    Article Title: Paneth Cell-Rich Regions Separated by a Cluster of Lgr5+ Cells Initiate Crypt Fission in the Intestinal Stem Cell Niche

    doi: 10.1371/journal.pbio.1002491

    Figure Lengend Snippet: Paneth cells are more adherent to laminin than other crypt cells. (A, B) Immunofluorescence images of sectioned tissue show nuclei (Hoechst, blue), F-actin (Phalloidin, green), and β4 Integrin (red): (A) is an optical section; (B) is a 3D projection (from Imaris) of the same crypt. In the left panel, the basal surface of crypt base columnar (CBC) cells are pseudo-coloured yellow, and the basal surface of Paneth cells are pseudo-coloured purple. Paneth cells are identifiable as the large cells with round, basally placed nuclei; CBC cells are narrow and have compressed nuclei. White lines (left and right panels) indicate the boundaries between Paneth cells and their neighbours. (C) Average β4 Integrin intensity on the basal surface of Paneth cells (PC) was normalised to the average value for neighbouring cells (CBC). Left panels in (A) and (B) indicate the Imaris-rendered surfaces that were used to measure β4 Integrin signal intensity. ± SEM, p

    Article Snippet: Anti-β4 Integrin (Abcam) was added at 5 μg/ml.

    Techniques: Immunofluorescence

    A working model of metformin-induced cancer cell anoikis. Metformin upregulates ubiquitin E3 ligase WWP1, leading to destabilization of ΔNp63α and downregulation of proteins involved in cell-matrix adhesion including integrin β4 and fibronectin 1, which in turn results in cell detachment [ 12 ]. On the other hand, metformin inhibits ΔNp63α expression resulting in upregulation of integrin β1, which in turn promotes cell–cell adhesion and cell aggregation. Both disruption of cell-matrix adhesion and enhanced cell–cell adhesion contribute to metformin-induced anoikis.

    Journal: International Journal of Molecular Sciences

    Article Title: Integrin β1-Mediated Cell–Cell Adhesion Augments Metformin-Induced Anoikis

    doi: 10.3390/ijms20051161

    Figure Lengend Snippet: A working model of metformin-induced cancer cell anoikis. Metformin upregulates ubiquitin E3 ligase WWP1, leading to destabilization of ΔNp63α and downregulation of proteins involved in cell-matrix adhesion including integrin β4 and fibronectin 1, which in turn results in cell detachment [ 12 ]. On the other hand, metformin inhibits ΔNp63α expression resulting in upregulation of integrin β1, which in turn promotes cell–cell adhesion and cell aggregation. Both disruption of cell-matrix adhesion and enhanced cell–cell adhesion contribute to metformin-induced anoikis.

    Article Snippet: Antibodies for N-cadherin (14215, 1:1000) or Integrin β4 (4704, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing