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  • mcf7  (DSMZ)
    95
    DSMZ mcf7
    Spatial-Temporal Regulation of EGFR Phosphorylation by PTPN2 and PTPRG/J (A) Spatial-temporal maps (STMs) depicting EGFR-mTFP fluorescence (left) and pY 1068 phosphorylation (middle) in control cells (n ∼ 90 cells per time point for a total of ∼360 cells, N = 6 experiments) and following transfection with non-targeting siRNA pool (right, n ∼ 60, N = 4). (B) Columns 1–3: effect of PTPN2-mCitrine expression (column 1) on STMs of EGFR-mTFP localization (column 2) and phosphorylation fold change (1/PFC pY1068 -cDNA, column 3) (n ∼ 60, N = 3). Column 4: effect of siRNA-mediated PTPN2 knockdown on EGFR-mTFP phosphorylation fold change (PFC pY1068 -siRNA, n ∼ 45, N = 3). Column 5: STM of fraction of EGFR-mTFP interacting with PTPN2 C216S -mCitrine trapping mutant as determined by FLIM (α TM , n = 15–30, N = 2). (C) STMs of the same quantities as in (B) upon PTPRG-mCitrine expression/siRNA-mediated knockdown (n ∼ 60, N = 3; α TM PTPRG C1060S -mCitrine n = 15–30, N = 2). (D) STMs of the same quantities as in (B) upon PTPRJ-mCitrine expression/siRNA-mediated knockdown (n ∼ 40, N = 2; α TM PTPRJ D1205A -mCitrine, n ∼ 30, N = 2). In (A) to (D), cells were stimulated with 200 ng/mL 5P-EGF; transparent areas denote non-significant PFCs, p > 0.05. (E) Effect of siRNA-mediated knockdown of PTPRG, PTPN2, and PTPRJ on the fraction of phosphorylated EGFR (α) in single <t>MCF7</t> cells expressing EGFR-mCitrine (donor) and PTB-mCherry (acceptor). FLIM measurements were made prior to (gray) and 2 min after saturating 320 ng/mL EGF-Alexa647 stimulation (blue). α mean ± SD for control: n = 14 (gray), n = 17 (blue); PTPRG: n = 15 (gray), n = 11 (blue); PTPN2: n = 9 (gray), n = 8 (blue); PTPRJ: n = 6 (gray), n = 6 (blue). N = 1–2. ∗∗ p = 0.0018 and ∗∗∗ p
    Mcf7, supplied by DSMZ, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mcf 7  (DSMZ)
    86
    DSMZ mcf 7
    (a-c) <t>MCF-7</t> cells were labeled with 4sU for up to 8 h in two replicates. RNA was either biotinylated and employed in biochemical separation or subjected to chemical conversion using SLAM-, TLS- or TUC-seq protocols. ( a ) Quantification of dot blot analysis of biotinylated RNAs (input) and supernatant of the biochemical separation with Streptavidin-HRP. ( b ) RNA concentration of biotin-enriched fractions (eluate) determined by absorption measurement. ( c ) RT-qPCR analysis of MYC, PDLIM5 and GAPDH mRNA in eluate fractions of the biochemical separation, normalized to the respective input. In a, b and c, mean and SD of two replicates are shown. ( d ) Upper left panel: schematic representation of the restriction enzyme digestion assay probing the efficiency of chemical conversion employing the positive control substrate (C), containing a Not I cleavage site, the negative control substrate (U) or the chemical conversion substrate (4sU). All substrates were analyzed untreated and after chemical conversion using SLAM, TLS and TUC protocols. Lower left panel: analysis of the reaction products on 10% TBE gels. Right panel: quantification of the relative fraction of full length band and the two cleavage products derived from Not I digestion (+ Not I) in three independent experiments. untr. = untreated.
    Mcf 7, supplied by DSMZ, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mcf 7/product/DSMZ
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mcf 7 - by Bioz Stars, 2022-05
    86/100 stars
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    Image Search Results


    Spatial-Temporal Regulation of EGFR Phosphorylation by PTPN2 and PTPRG/J (A) Spatial-temporal maps (STMs) depicting EGFR-mTFP fluorescence (left) and pY 1068 phosphorylation (middle) in control cells (n ∼ 90 cells per time point for a total of ∼360 cells, N = 6 experiments) and following transfection with non-targeting siRNA pool (right, n ∼ 60, N = 4). (B) Columns 1–3: effect of PTPN2-mCitrine expression (column 1) on STMs of EGFR-mTFP localization (column 2) and phosphorylation fold change (1/PFC pY1068 -cDNA, column 3) (n ∼ 60, N = 3). Column 4: effect of siRNA-mediated PTPN2 knockdown on EGFR-mTFP phosphorylation fold change (PFC pY1068 -siRNA, n ∼ 45, N = 3). Column 5: STM of fraction of EGFR-mTFP interacting with PTPN2 C216S -mCitrine trapping mutant as determined by FLIM (α TM , n = 15–30, N = 2). (C) STMs of the same quantities as in (B) upon PTPRG-mCitrine expression/siRNA-mediated knockdown (n ∼ 60, N = 3; α TM PTPRG C1060S -mCitrine n = 15–30, N = 2). (D) STMs of the same quantities as in (B) upon PTPRJ-mCitrine expression/siRNA-mediated knockdown (n ∼ 40, N = 2; α TM PTPRJ D1205A -mCitrine, n ∼ 30, N = 2). In (A) to (D), cells were stimulated with 200 ng/mL 5P-EGF; transparent areas denote non-significant PFCs, p > 0.05. (E) Effect of siRNA-mediated knockdown of PTPRG, PTPN2, and PTPRJ on the fraction of phosphorylated EGFR (α) in single MCF7 cells expressing EGFR-mCitrine (donor) and PTB-mCherry (acceptor). FLIM measurements were made prior to (gray) and 2 min after saturating 320 ng/mL EGF-Alexa647 stimulation (blue). α mean ± SD for control: n = 14 (gray), n = 17 (blue); PTPRG: n = 15 (gray), n = 11 (blue); PTPN2: n = 9 (gray), n = 8 (blue); PTPRJ: n = 6 (gray), n = 6 (blue). N = 1–2. ∗∗ p = 0.0018 and ∗∗∗ p

    Journal: Cell Systems

    Article Title: Interdependence between EGFR and Phosphatases Spatially Established by Vesicular Dynamics Generates a Growth Factor Sensing and Responding Network

    doi: 10.1016/j.cels.2018.06.006

    Figure Lengend Snippet: Spatial-Temporal Regulation of EGFR Phosphorylation by PTPN2 and PTPRG/J (A) Spatial-temporal maps (STMs) depicting EGFR-mTFP fluorescence (left) and pY 1068 phosphorylation (middle) in control cells (n ∼ 90 cells per time point for a total of ∼360 cells, N = 6 experiments) and following transfection with non-targeting siRNA pool (right, n ∼ 60, N = 4). (B) Columns 1–3: effect of PTPN2-mCitrine expression (column 1) on STMs of EGFR-mTFP localization (column 2) and phosphorylation fold change (1/PFC pY1068 -cDNA, column 3) (n ∼ 60, N = 3). Column 4: effect of siRNA-mediated PTPN2 knockdown on EGFR-mTFP phosphorylation fold change (PFC pY1068 -siRNA, n ∼ 45, N = 3). Column 5: STM of fraction of EGFR-mTFP interacting with PTPN2 C216S -mCitrine trapping mutant as determined by FLIM (α TM , n = 15–30, N = 2). (C) STMs of the same quantities as in (B) upon PTPRG-mCitrine expression/siRNA-mediated knockdown (n ∼ 60, N = 3; α TM PTPRG C1060S -mCitrine n = 15–30, N = 2). (D) STMs of the same quantities as in (B) upon PTPRJ-mCitrine expression/siRNA-mediated knockdown (n ∼ 40, N = 2; α TM PTPRJ D1205A -mCitrine, n ∼ 30, N = 2). In (A) to (D), cells were stimulated with 200 ng/mL 5P-EGF; transparent areas denote non-significant PFCs, p > 0.05. (E) Effect of siRNA-mediated knockdown of PTPRG, PTPN2, and PTPRJ on the fraction of phosphorylated EGFR (α) in single MCF7 cells expressing EGFR-mCitrine (donor) and PTB-mCherry (acceptor). FLIM measurements were made prior to (gray) and 2 min after saturating 320 ng/mL EGF-Alexa647 stimulation (blue). α mean ± SD for control: n = 14 (gray), n = 17 (blue); PTPRG: n = 15 (gray), n = 11 (blue); PTPN2: n = 9 (gray), n = 8 (blue); PTPRJ: n = 6 (gray), n = 6 (blue). N = 1–2. ∗∗ p = 0.0018 and ∗∗∗ p

    Article Snippet: MCF7 and MCF10A cells were authenticated by Short Tandem Repeat (STR) analysis and did not contain DNA sequences from mouse, rat and hamster (Leibniz-Institut DSMZ).

    Techniques: Fluorescence, Transfection, Expressing, Mutagenesis

    Differential Regulation of EGFR Responsiveness by PTPN2 and PTPRs (A) Averaged single-cell dose-response measurements following PTP X knockdown. PTPRG knockdown results in EGFR phosphorylation in absence of stimulus (top, blue dots on the y axis as in Figure 3 E). Dose response of EGFR-mCitrine phosphorylation (red, n = 21, N = 6) is significantly altered upon siRNA-mediated PTPRJ knockdown (bottom, blue line, p = 0.004; n = 11, N = 3) and less upon PTPN2 knockdown (middle, blue line, p = 0.17; n = 14, N = 6). Shaded bounds as in Figure 1 D. Solid lines: model-based fits to the phosphorylated EGFR fraction ( STAR Methods and Figure S4 A). (B) Dose response of EGFR-mTFP phosphorylation (red) is significantly altered upon PTPRG-mCitrine co-expression (blue lines, n = 28, N = 14, p = 0.027; top), PTPN2-mCitrine (blue lines, n = 34, N = 13, p = 0.001; middle), or PTPRJ-mCitrine co-expression (n = 16, N = 7, p = 4 × 10 −4 ; bottom). Solid lines: model-based fits to the phosphorylated EGFR fraction ( STAR Methods and Figure S4 A). Best fits are with the model shown in the inset. (C) NOX inhibition by DPI (10 μM, 30 min pre-incubation) significantly flattens dose response of EGFR phosphorylation upon ectopic PTPRG-mCitrine (top, yellow lines, p = 0.06; n = 26, N = 10), but has no effect upon PTPN2-mCitrine (middle, p = 0.19; n = 45, N = 12) or PTPRJ-mCitrine expression (bottom, p = 0.162; n = 10, N = 5). (D) Quantification of PTPRG-mCitrine (top), PTPN2-mCitrine (middle), and PTPRJ-mCitrine (bottom) catalytic cysteine oxidation for different EGF-Alexa647 doses (blue bars, means ± SEM, N = 4–7; Figure S4 G) and with 10 μM DPI pre-incubation (yellow bars, means ± SEM, N = 5; Figure S4 G). p values given as numbers above the bars are calculated with respect to the unstimulated case. (E) Quantification of PTPRG-mCitrine catalytic cysteine oxidation in control (left) and upon knockdown of NOX component p22 phox in MCF7 cells treated with 80 ng/mL EGF-Alexa647 with or without 10 μM DPI 20-min pre-incubation, or 4 mM H 2 O 2 (mean ± SEM, N = 4, Figures S4 H and S4I).

    Journal: Cell Systems

    Article Title: Interdependence between EGFR and Phosphatases Spatially Established by Vesicular Dynamics Generates a Growth Factor Sensing and Responding Network

    doi: 10.1016/j.cels.2018.06.006

    Figure Lengend Snippet: Differential Regulation of EGFR Responsiveness by PTPN2 and PTPRs (A) Averaged single-cell dose-response measurements following PTP X knockdown. PTPRG knockdown results in EGFR phosphorylation in absence of stimulus (top, blue dots on the y axis as in Figure 3 E). Dose response of EGFR-mCitrine phosphorylation (red, n = 21, N = 6) is significantly altered upon siRNA-mediated PTPRJ knockdown (bottom, blue line, p = 0.004; n = 11, N = 3) and less upon PTPN2 knockdown (middle, blue line, p = 0.17; n = 14, N = 6). Shaded bounds as in Figure 1 D. Solid lines: model-based fits to the phosphorylated EGFR fraction ( STAR Methods and Figure S4 A). (B) Dose response of EGFR-mTFP phosphorylation (red) is significantly altered upon PTPRG-mCitrine co-expression (blue lines, n = 28, N = 14, p = 0.027; top), PTPN2-mCitrine (blue lines, n = 34, N = 13, p = 0.001; middle), or PTPRJ-mCitrine co-expression (n = 16, N = 7, p = 4 × 10 −4 ; bottom). Solid lines: model-based fits to the phosphorylated EGFR fraction ( STAR Methods and Figure S4 A). Best fits are with the model shown in the inset. (C) NOX inhibition by DPI (10 μM, 30 min pre-incubation) significantly flattens dose response of EGFR phosphorylation upon ectopic PTPRG-mCitrine (top, yellow lines, p = 0.06; n = 26, N = 10), but has no effect upon PTPN2-mCitrine (middle, p = 0.19; n = 45, N = 12) or PTPRJ-mCitrine expression (bottom, p = 0.162; n = 10, N = 5). (D) Quantification of PTPRG-mCitrine (top), PTPN2-mCitrine (middle), and PTPRJ-mCitrine (bottom) catalytic cysteine oxidation for different EGF-Alexa647 doses (blue bars, means ± SEM, N = 4–7; Figure S4 G) and with 10 μM DPI pre-incubation (yellow bars, means ± SEM, N = 5; Figure S4 G). p values given as numbers above the bars are calculated with respect to the unstimulated case. (E) Quantification of PTPRG-mCitrine catalytic cysteine oxidation in control (left) and upon knockdown of NOX component p22 phox in MCF7 cells treated with 80 ng/mL EGF-Alexa647 with or without 10 μM DPI 20-min pre-incubation, or 4 mM H 2 O 2 (mean ± SEM, N = 4, Figures S4 H and S4I).

    Article Snippet: MCF7 and MCF10A cells were authenticated by Short Tandem Repeat (STR) analysis and did not contain DNA sequences from mouse, rat and hamster (Leibniz-Institut DSMZ).

    Techniques: Expressing, Inhibition, Incubation

    Dynamics of the Spatially Distributed EGFR-PTP Network (A) Scheme of the EGFR-PTP interaction network established through EGFR trafficking dynamics. EGFR interacts with PTPRG/PTPRJ at the PM and PTPN2 in the cytoplasm. All notations as in Figure 1 L. (B) Causality diagram that corresponds to (A). Red/blue lines: causal interactions; green arrow: ligand binding. (C) 3D-bifurcation diagram for double-negative EGFR-PTPRG feedback network topology at the plasma membrane, showing the dependence of monomeric EGFR phosphorylation (EGFRp) on PTPRG/EGFR expression ratio and fraction of liganded receptors. Forward (green) and backward (red) dose-response trajectories are shown for PTPRG/EGFR = 1.9, with corresponding orthographic projections on the right profile plane. (D) 3D-bifurcation diagram as in (C), for the combined toggle-switch/negative regulation/negative-feedback network topology established by ligandless EGFR vesicular recycling. Projections are the same as in (C). (E) Simulated temporal profiles of the fractions of liganded (dark) and phosphorylated receptors (light) in response to a train of pulses (gray), when the system is organized in the bistable regime (left), close to the bistability region (middle), and in the monostable regime (right) for the complete EGFR/PTP network as in (D). (F) Temporal traces of the fraction of ligand-bound (EGF-Alexa647/EGFR-mCitrine, dark color) and phosphorylated EGFR estimated by PTB-mCherry translocation to the plasma membrane (PTB-mCherry/EGFR-mCitrine, light color) in live MCF7 cells expressing non-targeting siRNA (middle, n = 4, N = 1), following siRNA-mediated knockdown of PTPRG (left, n = 5, N = 2), and ectopic Rab11 S25N expression (right, n = 16, N = 2). Data were acqui red at 1-min intervals following 20 ng/mL 5P-EGF every 30 min. Means ± SD are shown. Lower boxes depict the normalized differences between the fraction of phosphorylated and liganded EGFR.

    Journal: Cell Systems

    Article Title: Interdependence between EGFR and Phosphatases Spatially Established by Vesicular Dynamics Generates a Growth Factor Sensing and Responding Network

    doi: 10.1016/j.cels.2018.06.006

    Figure Lengend Snippet: Dynamics of the Spatially Distributed EGFR-PTP Network (A) Scheme of the EGFR-PTP interaction network established through EGFR trafficking dynamics. EGFR interacts with PTPRG/PTPRJ at the PM and PTPN2 in the cytoplasm. All notations as in Figure 1 L. (B) Causality diagram that corresponds to (A). Red/blue lines: causal interactions; green arrow: ligand binding. (C) 3D-bifurcation diagram for double-negative EGFR-PTPRG feedback network topology at the plasma membrane, showing the dependence of monomeric EGFR phosphorylation (EGFRp) on PTPRG/EGFR expression ratio and fraction of liganded receptors. Forward (green) and backward (red) dose-response trajectories are shown for PTPRG/EGFR = 1.9, with corresponding orthographic projections on the right profile plane. (D) 3D-bifurcation diagram as in (C), for the combined toggle-switch/negative regulation/negative-feedback network topology established by ligandless EGFR vesicular recycling. Projections are the same as in (C). (E) Simulated temporal profiles of the fractions of liganded (dark) and phosphorylated receptors (light) in response to a train of pulses (gray), when the system is organized in the bistable regime (left), close to the bistability region (middle), and in the monostable regime (right) for the complete EGFR/PTP network as in (D). (F) Temporal traces of the fraction of ligand-bound (EGF-Alexa647/EGFR-mCitrine, dark color) and phosphorylated EGFR estimated by PTB-mCherry translocation to the plasma membrane (PTB-mCherry/EGFR-mCitrine, light color) in live MCF7 cells expressing non-targeting siRNA (middle, n = 4, N = 1), following siRNA-mediated knockdown of PTPRG (left, n = 5, N = 2), and ectopic Rab11 S25N expression (right, n = 16, N = 2). Data were acqui red at 1-min intervals following 20 ng/mL 5P-EGF every 30 min. Means ± SD are shown. Lower boxes depict the normalized differences between the fraction of phosphorylated and liganded EGFR.

    Article Snippet: MCF7 and MCF10A cells were authenticated by Short Tandem Repeat (STR) analysis and did not contain DNA sequences from mouse, rat and hamster (Leibniz-Institut DSMZ).

    Techniques: Ligand Binding Assay, Expressing, Translocation Assay

    EGFR Phosphorylation and Vesicular Dynamics (A) Quantifying ectopic EGFR-mTFP expression in MCF7 cells. Average EGF-Alexa647 versus EGFR-mTFP fluorescence in single MCF7 (green) or MCF10A cells without EGFR-mTFP (black). Histograms (left) reflect that levels of EGF-Alexa647 binding to MCF7 with ectopic EGFR-mTFP expression (green) and MCF10A with endogenous EGFR (black) are similar. (B) EGFR Y 1068 phosphorylation (left) and Akt phosphorylation (right) in MCF7 cells ectopically expressing EGFR-mTFP (solid lines) and for endogenous EGFR in MCF10A cells (dashed lines), following 5-min pulsed (5P-EGF, 200 ng/mL, blue) or sustained EGF stimulation (S-EGF, 200 ng/mL, red), determined by in-cell western assay. Data are normalized to the maximum response in each respective condition (means ± SEM, N = 3). (C) Representative fluorescence image series of EGF-Alexa647, EGFR-mTFP, PTB-mCherry, and PTB-mCherry (magenta)/EGFR-mTFP (green) overlay from single-cell dose-response experiment. Cells were stimulated every ∼1.5 min with increasing EGF-Alexa647 doses (2.5–600 ng/mL). Scale bar, 20 μm. (D) Fraction of phosphorylated versus ligand-bound EGFR-mTFP (n = 21, N = 10; Figures S1 B–S1D). Dashed lines: moving averages from single cells; shaded bounds: SDs; dash-dotted lines: estimated contribution of ligandless to the fraction of phosphorylated EGFR. (E) Live cell fluorescence anisotropy microscopy measurements of EGFR-QG-mCitrine dimerization state as a function of the fraction of ligand-bound receptor (mean ± SEM, n = 30, N = 3, Figures S1 F and S1G). (F–H) Average spatial-temporal maps of the estimated fraction of ligand-bound EGFR (F, EGF-Alexa647/EGFR-mCitrine), ligandless EGFR (G, 1 − [EGF-Alexa647/EGFR-mCitrine]), and the fraction of phosphorylated EGFR-mCitrine estimated by PTB-mCherry translocation (H, PTB-mCherry/EGFR-mCitrine). Data were acquired at 1-min intervals in live MCF7 cells following 200 ng/mL S-EGF (top, n = 16, N = 3; Figures S1 I and S1J) or 5P-EGF (n = 14, N = 2; Figures S1 I and S1J) stimulation. White dotted lines: trajectories representing the change in distribution of ligand-bound (F) and ligandless (G) EGFR. PM, plasma membrane; NM, nuclear membrane. (I) The respective plasma membrane fractions of ligand-bound (EGF-Alexa647/EGFR-mCitrine, red) and phosphorylated EGFR (PTB-mCherry/EGFR-mCitrine, blue) derived from (F) and (H) (median ± AMD). Extracellular EGF-Alexa647 fluorescence is shown in gray. (J) Dimerization state measured by anisotropy (black) and the fraction of ligand-bound EGFR-QG-mCitrine (red) at the plasma membrane for live cells following 200 ng/mL S-EGF (top, n = 5, N = 3) or 5P-EGF (bottom, n = 5, N = 3) stimulation (means ± SEM). (K) The dose response of EGFR-mTFP phosphorylation (red, control) is significantly altered upon ectopic Rab11 S25N expression (green; p = 0.02; n = 12, N = 4). Lines are the same as in (D). (L) EGFR trafficking dynamics: ligandless EGFR recycles via early (EE) and recycling endosomes (RE) to the plasma membrane (red arrows) whereas upon EGF binding (thin green arrow), ubiquitinated EGF-EGFR Ub unidirectionally traffics via the early to the late endosomes (LE, green arrow) to be degraded in lysosomes (∅). Causal links are denoted by solid black lines.

    Journal: Cell Systems

    Article Title: Interdependence between EGFR and Phosphatases Spatially Established by Vesicular Dynamics Generates a Growth Factor Sensing and Responding Network

    doi: 10.1016/j.cels.2018.06.006

    Figure Lengend Snippet: EGFR Phosphorylation and Vesicular Dynamics (A) Quantifying ectopic EGFR-mTFP expression in MCF7 cells. Average EGF-Alexa647 versus EGFR-mTFP fluorescence in single MCF7 (green) or MCF10A cells without EGFR-mTFP (black). Histograms (left) reflect that levels of EGF-Alexa647 binding to MCF7 with ectopic EGFR-mTFP expression (green) and MCF10A with endogenous EGFR (black) are similar. (B) EGFR Y 1068 phosphorylation (left) and Akt phosphorylation (right) in MCF7 cells ectopically expressing EGFR-mTFP (solid lines) and for endogenous EGFR in MCF10A cells (dashed lines), following 5-min pulsed (5P-EGF, 200 ng/mL, blue) or sustained EGF stimulation (S-EGF, 200 ng/mL, red), determined by in-cell western assay. Data are normalized to the maximum response in each respective condition (means ± SEM, N = 3). (C) Representative fluorescence image series of EGF-Alexa647, EGFR-mTFP, PTB-mCherry, and PTB-mCherry (magenta)/EGFR-mTFP (green) overlay from single-cell dose-response experiment. Cells were stimulated every ∼1.5 min with increasing EGF-Alexa647 doses (2.5–600 ng/mL). Scale bar, 20 μm. (D) Fraction of phosphorylated versus ligand-bound EGFR-mTFP (n = 21, N = 10; Figures S1 B–S1D). Dashed lines: moving averages from single cells; shaded bounds: SDs; dash-dotted lines: estimated contribution of ligandless to the fraction of phosphorylated EGFR. (E) Live cell fluorescence anisotropy microscopy measurements of EGFR-QG-mCitrine dimerization state as a function of the fraction of ligand-bound receptor (mean ± SEM, n = 30, N = 3, Figures S1 F and S1G). (F–H) Average spatial-temporal maps of the estimated fraction of ligand-bound EGFR (F, EGF-Alexa647/EGFR-mCitrine), ligandless EGFR (G, 1 − [EGF-Alexa647/EGFR-mCitrine]), and the fraction of phosphorylated EGFR-mCitrine estimated by PTB-mCherry translocation (H, PTB-mCherry/EGFR-mCitrine). Data were acquired at 1-min intervals in live MCF7 cells following 200 ng/mL S-EGF (top, n = 16, N = 3; Figures S1 I and S1J) or 5P-EGF (n = 14, N = 2; Figures S1 I and S1J) stimulation. White dotted lines: trajectories representing the change in distribution of ligand-bound (F) and ligandless (G) EGFR. PM, plasma membrane; NM, nuclear membrane. (I) The respective plasma membrane fractions of ligand-bound (EGF-Alexa647/EGFR-mCitrine, red) and phosphorylated EGFR (PTB-mCherry/EGFR-mCitrine, blue) derived from (F) and (H) (median ± AMD). Extracellular EGF-Alexa647 fluorescence is shown in gray. (J) Dimerization state measured by anisotropy (black) and the fraction of ligand-bound EGFR-QG-mCitrine (red) at the plasma membrane for live cells following 200 ng/mL S-EGF (top, n = 5, N = 3) or 5P-EGF (bottom, n = 5, N = 3) stimulation (means ± SEM). (K) The dose response of EGFR-mTFP phosphorylation (red, control) is significantly altered upon ectopic Rab11 S25N expression (green; p = 0.02; n = 12, N = 4). Lines are the same as in (D). (L) EGFR trafficking dynamics: ligandless EGFR recycles via early (EE) and recycling endosomes (RE) to the plasma membrane (red arrows) whereas upon EGF binding (thin green arrow), ubiquitinated EGF-EGFR Ub unidirectionally traffics via the early to the late endosomes (LE, green arrow) to be degraded in lysosomes (∅). Causal links are denoted by solid black lines.

    Article Snippet: MCF7 and MCF10A cells were authenticated by Short Tandem Repeat (STR) analysis and did not contain DNA sequences from mouse, rat and hamster (Leibniz-Institut DSMZ).

    Techniques: Expressing, Fluorescence, Binding Assay, In-Cell ELISA, Microscopy, Translocation Assay, Derivative Assay

    Measuring proteome and genome changes in cancer versus normal cells. For proteomic analysis lysates of each of the non-labeled cells (HMEC, HCC1143 and HCC2218) were mixed with lysate of SILAC-labeled MCF7 cells. Proteins were trypsin-digested and analyzed by LC-MS using high resolution mass spectrometry. For genomic analysis, genomic DNA was isolated from HMEC, HCC1143 and HCC2218 cells and hybridized with a SNP arrays.

    Journal: PLoS Genetics

    Article Title: Proteomic Changes Resulting from Gene Copy Number Variations in Cancer Cells

    doi: 10.1371/journal.pgen.1001090

    Figure Lengend Snippet: Measuring proteome and genome changes in cancer versus normal cells. For proteomic analysis lysates of each of the non-labeled cells (HMEC, HCC1143 and HCC2218) were mixed with lysate of SILAC-labeled MCF7 cells. Proteins were trypsin-digested and analyzed by LC-MS using high resolution mass spectrometry. For genomic analysis, genomic DNA was isolated from HMEC, HCC1143 and HCC2218 cells and hybridized with a SNP arrays.

    Article Snippet: MCF7 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

    Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation

    (a-c) MCF-7 cells were labeled with 4sU for up to 8 h in two replicates. RNA was either biotinylated and employed in biochemical separation or subjected to chemical conversion using SLAM-, TLS- or TUC-seq protocols. ( a ) Quantification of dot blot analysis of biotinylated RNAs (input) and supernatant of the biochemical separation with Streptavidin-HRP. ( b ) RNA concentration of biotin-enriched fractions (eluate) determined by absorption measurement. ( c ) RT-qPCR analysis of MYC, PDLIM5 and GAPDH mRNA in eluate fractions of the biochemical separation, normalized to the respective input. In a, b and c, mean and SD of two replicates are shown. ( d ) Upper left panel: schematic representation of the restriction enzyme digestion assay probing the efficiency of chemical conversion employing the positive control substrate (C), containing a Not I cleavage site, the negative control substrate (U) or the chemical conversion substrate (4sU). All substrates were analyzed untreated and after chemical conversion using SLAM, TLS and TUC protocols. Lower left panel: analysis of the reaction products on 10% TBE gels. Right panel: quantification of the relative fraction of full length band and the two cleavage products derived from Not I digestion (+ Not I) in three independent experiments. untr. = untreated.

    Journal: Briefings in Bioinformatics

    Article Title: A comparison of metabolic labeling and statistical methods to infer genome-wide dynamics of RNA turnover

    doi: 10.1093/bib/bbab219

    Figure Lengend Snippet: (a-c) MCF-7 cells were labeled with 4sU for up to 8 h in two replicates. RNA was either biotinylated and employed in biochemical separation or subjected to chemical conversion using SLAM-, TLS- or TUC-seq protocols. ( a ) Quantification of dot blot analysis of biotinylated RNAs (input) and supernatant of the biochemical separation with Streptavidin-HRP. ( b ) RNA concentration of biotin-enriched fractions (eluate) determined by absorption measurement. ( c ) RT-qPCR analysis of MYC, PDLIM5 and GAPDH mRNA in eluate fractions of the biochemical separation, normalized to the respective input. In a, b and c, mean and SD of two replicates are shown. ( d ) Upper left panel: schematic representation of the restriction enzyme digestion assay probing the efficiency of chemical conversion employing the positive control substrate (C), containing a Not I cleavage site, the negative control substrate (U) or the chemical conversion substrate (4sU). All substrates were analyzed untreated and after chemical conversion using SLAM, TLS and TUC protocols. Lower left panel: analysis of the reaction products on 10% TBE gels. Right panel: quantification of the relative fraction of full length band and the two cleavage products derived from Not I digestion (+ Not I) in three independent experiments. untr. = untreated.

    Article Snippet: MCF-7 cells (ACC-115) were obtained from the Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures.

    Techniques: Labeling, Dot Blot, Concentration Assay, Quantitative RT-PCR, Positive Control, Negative Control, Derivative Assay

    4sU-tagging overview. ( a ) MCF-7 cells were pulse-labeled, and total RNA was used for four different labeling protocols: biochemical enrichment by BSA purification (similar to standard 4sU-seq), SLAM-seq, TimeLapse-seq (TLS-seq) and TUC-seq. SLAM-seq, TLS-seq and TUC-seq methods induce T to C substitutions, which are used to separate pre-existing and newly transcribed RNAs in the sequencing data. Theoretically, the fraction ratio is reflected by the read counts, but due to non-negligible other sources of T to C mismatches, appropriate bioinformatics tools must be employed. In the BSA purification protocol, ERCC RNA spike-in controls are added to normalize the fraction ratio in the libraries. ( b ) pulseR and GRAND-SLAM were used to estimate decay rates from all 4sU-tagging methods. pulseR is an RNA-seq count-based parameter estimation framework. GRAND-SLAM is a statistical software package to estimate NTR ‘out-of-the-box’. While both methods handle nucleotide conversion labeling experiments, only pulseR provides estimates for the BSA purification protocol.

    Journal: Briefings in Bioinformatics

    Article Title: A comparison of metabolic labeling and statistical methods to infer genome-wide dynamics of RNA turnover

    doi: 10.1093/bib/bbab219

    Figure Lengend Snippet: 4sU-tagging overview. ( a ) MCF-7 cells were pulse-labeled, and total RNA was used for four different labeling protocols: biochemical enrichment by BSA purification (similar to standard 4sU-seq), SLAM-seq, TimeLapse-seq (TLS-seq) and TUC-seq. SLAM-seq, TLS-seq and TUC-seq methods induce T to C substitutions, which are used to separate pre-existing and newly transcribed RNAs in the sequencing data. Theoretically, the fraction ratio is reflected by the read counts, but due to non-negligible other sources of T to C mismatches, appropriate bioinformatics tools must be employed. In the BSA purification protocol, ERCC RNA spike-in controls are added to normalize the fraction ratio in the libraries. ( b ) pulseR and GRAND-SLAM were used to estimate decay rates from all 4sU-tagging methods. pulseR is an RNA-seq count-based parameter estimation framework. GRAND-SLAM is a statistical software package to estimate NTR ‘out-of-the-box’. While both methods handle nucleotide conversion labeling experiments, only pulseR provides estimates for the BSA purification protocol.

    Article Snippet: MCF-7 cells (ACC-115) were obtained from the Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures.

    Techniques: Labeling, Purification, Sequencing, RNA Sequencing Assay, Software