Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, Venn diagram indicating differentially up-regulated genes (Nutlin-3A/DMSO, fold-change ≥2, adjusted p < 0.05) in MCF10A or SkFib cells. B, gene ontology analysis of differentially expressed genes from MCF10A and SkFib cells showing up-regulated genes for the top three significant biological processes. B–D, qRT-PCR validation of two representative common (C), MCF10A-specific (D), and SkFib-specific (E) Nutlin-3A–induced targets. Target gene expression is normalized to GAPDH (relative expression: Rel. Exp.), and data represent technical replicates from three independent biological replicates. Error bars represent S.E. with p values calculated by Student's t test, ****, p < 0.0001. F, intersection between SkFib (top) or MCF10A (bottom) p53 targets identified in this work compared with the meta-analysis of p53 targets identified in at least three independent experiments (32). G, immunoblotting for p53 expression at 6 h of DMSO (D) or Nutlin-3A (N) treatment in SkFib cells stably expressing shRNA against p53 (p53sh) or a nontargeting control shRNA (scr). H, qRT-PCR analysis of p53 expression at 6 h of DMSO (D) or Nutlin-3A (N) treatment in SkFib cells stably expressing shRNA against p53 or a nontargeting control shRNA. p53 expression is normalized to GAPDH expression (relative expression). Error bars represent S.E.; ****, p < 0.0001, and ***, p < 0.001, calculated by Student's t test. I, qRT-PCR analysis of SkFib-specific gene targets, GDNF and TRIM55, in SkFib cells stably expressing an shRNA to p53 or a nontargeting control shRNA. Gene expression is normalized to GAPDH expression. Error bars represent S.E.; ****, p < 0.0001; ***, p < 0.001; and **, p < 0.01, calculated by Student's t test. J, immunoblotting for p53 expression at 6 h of DMSO or Nutlin-3A treatment in response to p53 knockdown in MCF10A cells stably expressing shRNA against p53 or a nontargeting control shRNA. K, qRT-PCR analysis of p53 expression at 6 h of DMSO or Nutlin-3A treatment in response to p53 knockdown in MCF10A cells stably expressing shRNA against p53 or a nontargeting control shRNA. p53 RNA expression is normalized to GAPDH expression. Error bars represent S.E.; ****, p < 0.0001, calculated by Student's t test. L, qRT-PCR analysis of MCF10A-specific Nutlin-3A–induced genes, RIC3 and IL1B, in MCF10A cells stably expressing an shRNA to p53 (p53 sh) or a nontargeting control shRNA. Expression is normalized to GAPDH expression. Error bars represent S.E.; ****, p < 0.0001, calculated by Student's t test.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Targeted Gene Expression, Expressing, Western Blot, Stable Transfection, shRNA, Control, Gene Expression, Knockdown, RNA Expression

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, Venn diagram depicting overlap between significantly-enriched (p < 0. 01, MACS version 2) Nutlin-3A–induced p53 peaks in MCF10A or SkFib. B, boxplots depicting enrichment of p53 (input-normalized, log2) at common p53-binding sites found in both MCF10A (blue, left) and SkFib (green, right). C, fold-change ratio of Nutlin-3A/DMSO of common input-subtracted p53 enrichment for MCF10A or SkFib. D and E, boxplot analysis of the input-subtracted p53 enrichment for MCF10A (blue, left) or SkFib (green, right) for MCF10A-specific (D) or SkFib-specific p53-binding sites (right) (E) in response to DMSO (D) or Nutlin-3A (N) treatment. F–H, Chip-qPCR validation of the common (F), skin fibroblast–specific (G), and MCF10A-specific (H) p53 target CDKN1A/p21 at p53-binding and -nonbinding sites. Genome browser view of replicate ChIP-seq data for MCF10A (blue) and SkFib (green) with the location of qPCR primers for p53-binding site and negative region are shown above the qPCR data. qPCR data represent three biological replicates in MCF10A and SkFib cells with either control (scr, nontargeting) or p53-targeting shRNA after 6 h of Nutlin-3A or DMSO treatment. Statistical analysis was performed by using one-way ANOVA; ****, p < 0.0001; ***, p < 0.001; and **, p < 0.01.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

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

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, representative UCSC Genome Browser track view of GDNF locus, a fibroblast-specific p53 target, in response to DMSO (D) or Nutlin-3A (N) treatment. p53-bound, putative enhancers (H3K27ac+, H3K4me2+, and H3K4me3−) illustrated by a dashed box for GDNF and TRIM55, and the putative GDNF promoter (H3K27ac/H3K4me2/H3K4me3+) is represented by a solid box. MCF10A-N-1 is biological replicate of MCF10A-N-2, and SkFib-N-1 is a biological replicate of SkFib-N-2. The y axis is scaled to the maximum intensity between MCF10A or SkFib for each feature. B, representative UCSC Genome Browser track view at the RIC3 locus, illustrating p53 binding to an MCF10A-specific enhancer signature (H3K27ac/H3K4me2+ and H3K4me3−) in response to Nutlin-3A treatment (dashed box). MCF10A-N-1 is a biological replicate of MCF10A-N-2, and SkFib-N-1 is a biological replicate of SkFib-N-2. The y axis is scaled to the maximum intensity between MCF10A or SkFib for each feature.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, p53 ChIP-seq enrichment from either MCF10A (blue) or SkFib (green) at MCF10A-specific p53-binding sites. (−/+ 2000 bp from the p53 motif; −2 indicates 2000 bp downstream from the motif; 0 indicates the center of the p53 DNA motif, and +2 indicates 2000 bp upstream of the motif.) Two biological replicates for each dataset are shown. H3K4me2 (B) and H3K27ac (C) enrichment in DMSO or Nutlin-3A–treated MCF10A or SkFib within a 4000-bp window (−/+ 2000 bp from the p53 motif; 2 indicates 2000 bp downstream from the motif, 0 indicates the motif center, and +2 indicates 2000 bp upstream from the motif). D–F, percentage of intersecting H3K27ac peaks (input-normalized, MACS version 2, p < 0.01) with p53 peaks (input-normalized, MACS version 2, p < 0.01) that are common to MCF10A and SkFib (D), MCF10A-specific (E), or SkFib-specific (F) in response to DMSO (D) or Nutlin-3A (N) treatment. Adjacent boxplots depict H3K27ac enrichment over a 500-bp window from the p53-binding site.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: ChIP-sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, Homer-derived transcription factor motif enrichment found in MCF10A-specific (top) or SkFib-specific (bottom) enhancers (H3K4me2+/H3K27ac+/H3K4me3−). Full list of transcription factor enrichment and facet-specific enhancers are found in Table S4. B, qRT-PCR analysis of p53, p63, or p73 of cell lysates from MCF10A, SkFib, HUVEC, and HaCaT cells at 6 h after DMSO (D) or Nutlin-3A (N) treatment. Expressions detected by qRT-PCR are normalized to GAPDH expression. HaCaT cells were used as positive control for p63 and p73 expression. Statistical analysis was performed by using one-way ANOVA; ****, p < 0.0001. C, qRT-PCR analysis of TA–p63 and ΔN-p63 of cell lysates from MCF10A, SkFib, and HCT116-TA–p63 cells. Transiently transfected HCT116 cells with TA–p63 were used as a positive control for TA–p63 expression. Statistical analysis was performed by using one-way ANOVA; ****, p < 0.0001. D, immunoblotting for p63, TA–p63, and ΔN–p63 of cell lysates from HCT116, HCT116-TAp63, and HCT116-ΔNp63 transiently transfected cells to confirm antibody specificity for different isoforms. E, immunoblotting for p63, TA–p63, or ΔN-p63 in cell lysates from MCF10A cells. HCT116 cell lysate is used as negative control. F, representative UCSC Genome Browser track view of the IL1B locus, illustrating three MCF10A-specific putative enhancers bound by p53 and p63 in response to Nutlin-3A treatment (H3K27ac+, H3K4me2+, H3K4me3−; dashed box). The y axis is scaled to the maximum intensity for each data set. MCF10A-D-1 is a biological replicate of MCF10A-D-2; MCF10A-N-1 is a biological replicate of MCF10A-N-2; and SkFib-N-1 is a biological replicate of SkFib-N-2. G, representative UCSC Genome Browser track view of the RNASE7 locus, illustrating a MCF10A-specific putative enhancer bound by p53 and p63 in response to DMSO (D) or Nutlin-3A (N) treatment (H3K27Ac+, H3K4me2+, H3K4me3−; dashed box). The y axis is scaled to the maximum intensity for each data set. MCF10A-D-1 is a biological replicate of MCF10A-D-2; MCF10A-N-1 is a biological replicate of MCF10A-N-2; and SkFib-N-1 is a biological replicate of SkFib-N-2. H, Venn diagram representation of overlapping p53 and p63 ChIP-seq peaks (input-normalized, p53/p63 motif-positive, MACS version 2, p < 0.01) in MCF10A when treated with Nutlin-3A. I, heatmap plots of p53 and p63 enrichment at shared binding sites in replicate MCF10A cells within a 2000-bp window (−/+ 1000 bp from the peak center) in response to Nutlin-3A treatment. J, percentage of intersecting H3K27ac/H3K4me2+ peaks (input-normalized, MACS version 2, p < 0.01) with p53 only, p63 only, or p53/p63 peaks observed in MCF10A cells (input-normalized, MACS version 2, p < 0.01). K, percentage of p63-binding sites observed in MCF10A cells at varying distances to the nearest TSS of all RefSeq genes (white) or Nutlin-3A–induced genes (red).

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Positive Control, Transfection, Western Blot, Negative Control, ChIP-sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, qRT-PCR analysis of p53 and p63 in response to p63 knockdown in MCF10A cells stably expressing shRNA to p63 or a nontargeting control shRNA (scr) after 6 h of DMSO (D) or Nutlin-3A (N) treatment. Target gene expression is normalized to GAPDH for qRT-PCR analysis. Statistical analysis was performed by using one-way ANOVA; ****, p < 0.0001, and ***, p < 0.001. B, immunoblotting for p53, p63, and GAPDH in MCF10A cells stably expressing shRNA to p53, p63, or a nontargeting control shRNA after 6 h of DMSO or Nutlin-3A treatment. This immunoblot image is an uncropped version of Fig. 1J. C, RNA-seq analysis of MCF10A-specific, Nutlin-3A–induced genes in MCF10A cells expressing shRNA targeting either a nontargeting control, p53, or p63. Data are graphed as fold-change (Nutlin-3A/DMSO, log2, median in black). Statistical analysis was performed by using one-way ANOVA. ****, p < 0.0001; ***, p < 0.001; and *, p < 0.05. D, qRT-PCR analysis of MCF10A-specific Nutlin-3A–induced genes, RIC3, IL1A, and IL1B. Expression is normalized to GAPDH expression. Error bars represent S.E.; ****, p < 0.0001, and **, p < 0.01, calculated by Student's t test. E, percent of p53, p63, and p53/p63 co-bound sites relative to the three classes of p63-regulated p53 gene targets (p63-dependent, p63-independent, and p63-inhibited) in binned distance regions (under 25 kb and over 25 kb). Statistics represent χ2 test using the distance of each binding site class to nonregulated genes.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Quantitative RT-PCR, Knockdown, Stable Transfection, Expressing, shRNA, Control, Targeted Gene Expression, Western Blot, RNA Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A, H3K4me2 enrichment (input-subtracted H3K4me2, −/+ 250 bp from p53 motif center) at p53-binding sites in MCF10A cells expressing control (scr), p53, or p63-targeted shRNA in response to DMSO or Nutlin-3A treatment. Statistical analysis was performed by using one-way ANOVA. ****, p < 0.0001. B, H3K27ac enrichment at p53-binding sites in MCF10A cells expressing the represented shRNA molecules after 6 h of DMSO (D) or 5 μm Nutlin-3A (N) treatment. Statistical analysis was performed by using one-way ANOVA. ****, p < 0.0001, and ***, p < 0.001. C, bar graph displaying the number of p53-binding sites (left) or total cellular complement of H3K27ac+/H3K4me2+/H3K4me3− enhancers with more than 2-fold change in H3K27ac enrichment after Nutlin-3A treatment of MCF10 cells expressing the indicated shRNA. D, H3K4me2 enrichment at p53-binding sites (25) in HCT116 TP53+/+ or −/− cells in response to DMSO or Nutlin-3A treatment. Statistical analysis was performed by using one-way ANOVA. ****, p < 0.0001, and **, p < 0.01. E, H3K4me1 and H3K4me2 enrichment at p53-binding sites (85) in Trp53+/+ or Trp53−/− mouse embryonic fibroblasts.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Binding Assay, Expressing, Control, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Control of p53-dependent transcription and enhancer activity by the p53 family member p63

doi: 10.1074/jbc.RA119.007965

Figure Lengend Snippet: A and B, input-subtracted H327ac (A) or H3K4me2 (B) enrichment at p63-binding sites (left, −/+ 250 bp from p63 peak center) or at all remaining enhancers (H3K27ac+, H3K4me2+, H3K4me3−, right) in MCF10A cells expressing nontargeting (Scr) or p63 shRNA. C, representative UCSC Genome Browser track view of the RRM1 locus, illustrating three MCF10A-specific putative enhancers bound by p63 that are lost in response to p63 depletion (H3K27ac+, H3K4me2+, H3K4me3−; dashed box). D, representative UCSC Genome Browser track view of the EDN2 locus, illustrating three MCF10A-specific putative enhancers bound by p63 that are lost in response to p63 depletion (H3K27ac+, H3K4me2+, H3K4me3−; three separate dashed boxes). The y axis is scaled to the maximum intensity for each data set. E, bar graphs depicting the percent of p63 peaks that show 2-fold gains or losses of H3K27ac (left) or H3K4me2 (right) in response to either p53 or p63 depletion relative to nontargeting control shRNA. F, number of p63-sensitive, H3K4me2-marked enhancers (out of 1496 total) overlapping DHS across epithelial and nonepithelial cell types analyzed by the ENCODE project. Error bars represent the median and 95% confidence interval. G, Jitter plot depicting the fraction of p63-dependent or -independent enhancers overlapping regions of DHS across both epithelial and nonepithelial cell lines as assayed by the ENCODE project. H, heatmap of k-means (k = 3) clustered Bonferroni-corrected p values (q-values) for motif enrichment found at p63-dependent or -independent enhancers. Blue represents adjusted p values less than 0.05, and white represents adjusted p values greater than 0.05. A full list of motifs and their enrichment statistics can be found in Table S7.

Article Snippet: MCF10A mammalian epithelial cells and foreskin fibroblast cells (AG22153, Coriell Institute) were cultured at 37 °C in 5% CO 2 in HuMEC Complete media (Gibco, catalog no. 12752010) and Dulbecco's modified Eagle's media (with 10% fetal bovine serum and 1% penicillin/streptomycin, catalog no. VWR-0101-0500), respectively.

Techniques: Binding Assay, Expressing, shRNA, Control

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Fibronectin Fibrils Regulate TGF-β1-induced Epithelial-Mesenchymal Transition

doi: 10.1016/j.matbio.2017.01.001

Figure Lengend Snippet: FN fibril inhibition blocks the TGF-β pathway, inhibits cell size, cell number, and decreases migration in MCF10As. (A) Representative immunofluorescence images of Smad2 in MCF10As after 48 hours of treatment with TGF-β1 and FUD. (B) Immunofluorescence images of nuclei from part A (C) Quantification of nuclear colocalization of Smad2. N ≥ 16 for each condition. *p < 0.01 significantly different from TGF-β1, Student's t-test. (D) Quantification of the average cell size per condition. N ≥ 16 for each condition. *p < 0.01 significantly different from TGF-β1, Student's t-test. (E) Quantification of cell density. N ≥ 16 for each condition. *p < 0.01 significantly different from TGF-β1, Student's t-test. (F-G) Migration kinetics of MCF10A cells by continuous monitoring of live cell migration for approximately 48 hours. Direct comparison of FN fibril inhibitor &/or TGF-β1 effects on cell migration (F) after 24 hours, and (G) after 48 hours. Control samples served as the baseline cell index levels for comparison with wells containing a combination of FUD &/or TGF-β1. Cell index values from 4 wells per condition ± SD correspond to impedance from microelectrode sensor. **p < 0.05 significantly different from control or TGF-β1, Student's t-test. Scale bar is 50 μm.

Article Snippet: Human MCF10A mammary epithelial cells were obtained from the National Cancer Institute Physical Sciences in Oncology Bioresource Core Facility, in conjunction with American Type Culture Collection (Manassas, VA).

Techniques: Inhibition, Migration, Immunofluorescence, Comparison, Control

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Fibronectin Fibrils Regulate TGF-β1-induced Epithelial-Mesenchymal Transition

doi: 10.1016/j.matbio.2017.01.001

Figure Lengend Snippet: Blocking the LTBP-1/FN binding site inhibits TGF-β1–induced EMT in MCF10A cells. (A) Representative immunofluorescence images of MCF10A cells cultured for 48 hours with TGF-β1 and/or monoclonal ab. Ab staining for F-actin (orange) FN (green), and monoclonal ab (red). Composite displays colocalization of FN and monoclonal ab in yellow. mRNA levels for (B) FN (C) LTBP-1, and (D) EMT markers αSMA and vimentin, were determined by means of RT-qPCR. N = 3 for each condition. *p ≤ 0.01, **p ≤ 0.05, and ***p ≤ 0.1 significantly different from TGF-β1, Student's t-test. Scale bar is 10 μm.

Article Snippet: Human MCF10A mammary epithelial cells were obtained from the National Cancer Institute Physical Sciences in Oncology Bioresource Core Facility, in conjunction with American Type Culture Collection (Manassas, VA).

Techniques: Blocking Assay, Binding Assay, Immunofluorescence, Cell Culture, Staining, Quantitative RT-PCR

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Fibronectin Fibrils Regulate TGF-β1-induced Epithelial-Mesenchymal Transition

doi: 10.1016/j.matbio.2017.01.001

Figure Lengend Snippet: FN lacking the growth factor binding domains III 11-14 inhibits TGF-β1–induced EMT in MCF10A cells. (A) Representative immunofluorescence images of MCF10A cells cultured for 24 hours with TGF-β1 and/or FN/Δ11-14. Ab staining for F-actin (orange), FN (green), and LTBP-1 (red). Composite displays colocalization of FN and LTBP-1 in yellow. mRNA levels for (B) EMT markers twist and vimentin, were determined by means of RT-qPCR. N = 3 for each condition. *p < 0.005, and **p < 0.05 significantly different from TGF-β1, Student's t-test. Scale bar is 10 μm.

Article Snippet: Human MCF10A mammary epithelial cells were obtained from the National Cancer Institute Physical Sciences in Oncology Bioresource Core Facility, in conjunction with American Type Culture Collection (Manassas, VA).

Techniques: Binding Assay, Immunofluorescence, Cell Culture, Staining, Quantitative RT-PCR

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Fibronectin Fibrils Regulate TGF-β1-induced Epithelial-Mesenchymal Transition

doi: 10.1016/j.matbio.2017.01.001

Figure Lengend Snippet: MCF10As on cell-extracted ECM. (A) Representative immunofluorescence images of the resulting MCF10A matrix after treatment with or without TGF-β1 for 48 hours. (B) Representative immunofluorescence images of MCF10As cultured on pre-assembled ECM from cells treated with or without TGF-β1 for 48 hours and cultured for an additional 24 hours. Ab staining for F-actin (red), E-cadherin (green), and FN (white). Scale bar is 20 μm.

Article Snippet: Human MCF10A mammary epithelial cells were obtained from the National Cancer Institute Physical Sciences in Oncology Bioresource Core Facility, in conjunction with American Type Culture Collection (Manassas, VA).

Techniques: Immunofluorescence, Cell Culture, Staining

Journal: Nature Communications

Article Title: Spatial cycles mediated by UNC119 solubilisation maintain Src family kinases plasma membrane localisation

doi: 10.1038/s41467-017-00116-3

Figure Lengend Snippet: UNC119-solubilising activity maintains SFK PM localisation. Confocal micrographs of MCF10a cells expressing Src-mCit ( a ) or Fyn-mCit ( b ) and the ER marker, CalR-mCer transfected with UNC119 targeting siRNA ( lower rows ) or non-targeting siRNA control nucleotides ( upper rows ). c Representative western blot showing UNC119 levels of transfected cells and the GAPDH-loading control. Bar graph depicts the quantification of UNC119 kD in which UNC119 levels were normalised to the tubulin-loading control and the non-targeting siRNA control ( n = 4, data are mean ± SD; significance determined by Student’s t -test). d Quantification of co-localisation with Mander’s coefficient for SFK-mCit with the ER CalR-mCer marker in MCF10a cells ( n > 30 cells per condition from two independent experiments; data are mean ± SD. * P < 0.05; *** P < 0.001; Student’s t -test). Confocal micrographs of HeLa cells expressing Src-mCit e or Fyn-mCit f and the ER marker; CalR-mCer e or stained with anti-Calnexin antibodies f , transfected with UNC119 targeting siRNA ( lower rows ) or non-targeting siRNA control nucleotides ( upper rows ). g Quantification of co-localisation with Mander’s coefficient for SFK-mCit with the ER markers for HeLa cells ( n > 15 cells per condition from two independent experiments; data are mean ± SD. * P < 0.05; *** P < 0.001; Student’s t -test). h Quantification of UNC119 KD in HeLa cells by western blot using UNC119 and tubulin antibodies. UNC119 levels were normalised to the tubulin-loading control and the non-targeting siRNA control ( n = 5, data are mean ± SD; ** P < 0.001; Student’s t -test). i Confocal micrographs of MCF10a cells transfected with UNC119A and UNC119B targeting siRNA or non-targeting siRNA control nucleotides and then stained with an antibody against SFKs ( green ) and with DAPI ( blue ). Traces below show the vertical intensity profile plots of SFK for the yellow dashed area . j The mean SFK intensity profile plots for multiple cells ( n = 20 cells per condition, mean intensity ± SEM). Scale bars , 10 μm

Article Snippet: MCF10a cells (CRL-10317; LGC Genomics GmbH, Berlin, Germany) were cultured in a DMEM/F12 culture medium (PAN-Biotech GmbH) supplemented with glutamine, 5% horse serum (PAN-Biotech GmbH), 20 ng/ml epidermal growth factor (Sigma-Aldrich Chemie GmbH, Munich, Germany), 0.5 mg/ml hydrocortisone (Sigma-Aldrich Chemie GmbH), 100 ng/ml cholera toxin (Sigma-Aldrich Chemie GmbH) and 10 µg/ml insulin (Sigma-Aldrich Chemie GmbH).

Techniques: Activity Assay, Expressing, Marker, Transfection, Western Blot, Staining