Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Schematic of an ssDNA molecule (purple) tethered between two optically trapped micrometer‐sized polystyrene beads (grey) with hRAD51 complexes (green) bound to the ssDNA molecule. By controlling the position of the beads, the extension of the DNA molecule can be controlled while the tension in the molecule is monitored. At the same time, the proteins can be directly visualized with single‐fluorophore resolution using wide‐field fluorescence microscopy. The experiments are generally performed using a microfluidic flow system with four laminar channels. A typical experiment is comprised of the following steps: (1) capture of two beads; (2) tethering of a single dsDNA molecule between these beads; (3) probing the mechanical properties of the tethered dsDNA molecule, to ensure that it is a single molecule with the expected mechanical properties; (4) the tension on the dsDNA molecule is increased to generate an ssDNA molecule by force‐induced melting; (5) the ssDNA is incubated in the protein channel; and (6) the hRAD51‐ssDNA complex is brought into the buffer for imaging. Typical fluorescence intensity snapshots of hRAD51‐ssDNA complexes (buffer composition: 20 mM Tris pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 1 mM ATP, 10 mM DTT) at indicated time intervals. Scale bars: 2 μm. Fluorescence kymograph of the same hRAD51‐ssDNA complex as in (C). Scale bars: 2 μm (horizontal) and 5 min (vertical). Integrated fluorescence intensity along the DNA of the same hRAD51‐ssDNA complex as in (C and D) over time (red dataset), showing an exponential decay at a rate of (33 ± 1) 10 −4 s −1 , obtained from an exponential fit to the data (blue curve). After correcting for photobleaching, this gives, for this particular example, a hRAD51 disassembly rate of (17 ± 1) 10 −4 s −1 . Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence, Microscopy, Incubation, Imaging

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Fluorescence images and kymographs of hRAD51 disassembling from ssDNA at indicated ssDNA tensions (buffer composition: 20 mM Tris pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 1 mM ATP, 10 mM DTT). Images are typical examples of 16 (at 5 pN), 9 (at 20 pN) and 4 (at 50 pN) identical experiments. Scale bars: 2 μm (horizontal) and 5 min (vertical). Integrated fluorescence intensity along the DNA of the same complexes as in (A) over time and corresponding an exponential fit. Fits are normalized using the amplitude and offset of the exponential fit. Coloured edges in (A) show colour of corresponding force curve. Average disassembly rate as a function of tension. Error bars originate from the exponential fits on the individual datasets (number of datasets as in A). Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Fluorescence images and kymographs of hRAD51 disassembling from ssDNA for filaments assembled in 20 mM Tris pH 7.5, 40 mM KCl, 10 mM Mg(OAc) 2 , 2 mM ATP and 10 mM DTT and disassembled in 20 mM Tris pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 1 mM ATP and 10 mM DTT (right panel) or filaments assembled in 20 mM Tris pH 7.5, 40 mM KCl, 10 mM Mg(OAc) 2 , 2 mM ADP and 10 mM DTT and disassembled in 20 mM Tris pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 1 mM ADP and 10 mM DTT (right panel). Images are typical examples of 16 (for the ATP condition) and 11 (for the ADP condition). Scale bars: 2 μm (horizontal) and 5 s (vertical). Normalized integrated fluorescence intensity of the images shown in (A) over time. Exponential fits to these traces give disassembly rates of (6.7 ± 0.3) 10 −4 s −1 (black dataset; in the presence of ATP) and (17 ± 4) 10 −4 s −1 (red dataset; in the presence of ADP). Coloured edges in (A) show colour of corresponding force curve. Disassembly rate is correlated with the initial coverage of the DNA molecule. Red closed circles: measured in ATP at 5 pN; red open circles: measured in ATP at 20 pN; red closed triangles: measured in ATP at 50 pN; red open triangles: measured in ATP at 75 pN; black closed circles; measured in ADP in 100 mM KCl and 1 mM MgCl 2 ; black open circles; measured in ADP in 100 mM KCl and 10 mM MgCl 2 ; black triangles: measured in 1 mM MgCl 2 . Blue line: linear fit with a slope of (−21 ± 3) 10 −10 s −1 /AU (Pearson's correlation coefficient of the fit: −0.84) that was used to correct observed disassembly rates for differences in initial coverage. Average disassembly rates in ATP or ADP conditions after correcting for differences in initial coverage. Since the error bars of the black and red datasets overlap, there is no significant difference between the disassembly rate in the presence of ATP or ADP. All coverages were scaled to 400,000 using a linear approximation based on the fit in (C). Data information: Error bars: SEM, based on 11 molecules (black dataset) and 16 molecules (red dataset). Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Fluorescence images and kymographs of hRAD51 disassembling from ssDNA of filaments assembled in 20 mM Tris pH 7.5, 40 mM KCl, 10 mM Mg(OAc) 2 , 2 mM ADP and 10 mM DTT and disassembled in 20 mM Tris pH 7.5, 1 mM MgCl 2 , 1 mM ADP and 10 mM DTT (left panel); filaments assembled in 20 mM Tris pH 7.5, 40 mM KCl, 10 mM Mg(OAc) 2 , 2 mM ADP and 10 mM DTT and disassembled in 20 mM Tris pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 1 mM ADP and 10 mM DTT (middle panel); and filaments assembled in 20 mM Tris pH 7.5, 40 mM KCl, 10 mM Mg(OAc) 2 , 2 mM ADP and 10 mM DTT and disassembled in 20 mM Tris pH 7.5, 100 mM KCl, 10 mM MgCl 2 , 1 mM ADP and 10 mM DTT (right panel). Images are typical examples of 5, 11 and 3 identical experiments, respectively. Scale bars: 2 μm (horizontal) and 5 s (vertical). Normalized integrated fluorescence intensity of the images shown in (A) over time. Exponential fits to these traces give disassembly rates of (8 ± 1) 10 −4 s −1 (black dataset), (6.7 ± 0.3) 10 −4 s −1 (red dataset) and (6.7 ± 0.2) 10 −4 s −1 (blue dataset). Coloured edges in (A) show colour of corresponding force curve. Curves are typical examples of 5, 11 and 3 identical experiments, respectively. Average disassembly rates at indicated ionic strengths do not vary significantly. Error bars: SEM, based on 5, 11 and 3 identical experiments, respectively. Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: A Force‐extension/force‐relaxation cycles of a hRAD51‐coated ssDNA molecule in ATP and Ca 2+ (buffer: 20 mM Tris pH 7.5, 2 mM CaCl 2 , 2 mM ATP, 1 mM DTT). Time difference between cycles as indicated. Note that extension and relaxation curves of ssDNA (grey dataset) completely overlap. Arrows: direction in which the curves were recorded. Grey: ssDNA. Inset: fluorescence images with edges coloured as force curves. Representative example of two identical experiments. Scale bars: 2 μm. B Normalized integrated fluorescence intensity of the same construct over time. The amount of DNA‐bound hRAD51 remains constant; that is, photobleaching is negligible during these experiments. Representative example of two identical experiments. C The hysteresis area, defined as the area between extension and relaxation curve, remains roughly constant over time under these conditions. Representative example of two identical experiments. D Successive extension–relaxation cycles measured in the presence of Ca 2+ , Mg 2+ and ATP, showing reversible curves up to around 10 pN (first blue region), as all ATP‐bound NPFs are in ATP‐compact state. At higher forces, ATP‐bound NPFs transition to the ATP‐extended state and the curves show hysteresis (red region). Above 50 pN, all NPFs are in the ATP‐extended conformation (second blue region). Representative example of two identical experiments. E The relaxation curve is reversible. After converting all ATP‐bound NPFs into the ATP‐extended conformation and relaxing the molecule to 24 μm (black), the DNA is extended (red) before complete relaxation (blue). Red and blue curves overlap, showing reversibility of the relaxation curve. Representative example of two identical experiments. F Example of an eWLC fit (green) to a relaxation and extension curve (black) and corresponding contour length–extension curve (red) measured in the presence of ATP and Ca 2+ . Representative examples out of 38 experiments. eWLC fit parameters: persistence length 2.501 ± 0.009 nm, stretch modulus 1,599 ± 8 pN and contour length 29.01 ± 0.01 μm (error bars originate from individual eWLC fits). Contour length–extension curves were calculated under the assumption that upon conversion from ATP‐compact to ATP‐extended conformation, persistence length and stretch modulus remain constant. G–I Same as (A–C) but in presence of ADP, Ca 2+ and Mg 2+ (buffer: 20 mM Tris pH 7.5, 10 mM Mg(OAc) 2 , 2 mM CaCl 2 , 2 mM ADP, 1 mM DTT). Here, there is no ATP hydrolysis but hRAD51 can disassemble from the ssDNA. We see no hysteresis (G) and (I), while the integrated fluorescence intensity decreases (H) exponentially, yielding a disassembly rate of (4.2 ± 0.3) 10 −4 s −1 . Representative example of two identical experiments. Scale bars: 2 μm. Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence, Construct

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Force‐extension and force‐relaxation curve measured in a buffer containing ATP and Mg 2+ . Under these conditions, ATP hydrolysis and NPF disassembly can occur. We observe a slight hysteresis between extension and relaxation curves. However, the disassembly rate under these conditions is relatively high, such that the assumption that the amount of hRAD51 bound remains constant during one extension–relaxation cycle is no longer valid. Therefore, under these conditions, no quantitative analysis (such as shown for other conditions in Fig ) of the hysteresis and the structural transitions can be performed. Scale bar: 2 μm. Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques:

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: A–C Same as Fig A–C but recorded in the presence of ATP, Ca 2+ and Mg 2+ (buffer composition: 20 mM Tris pH 7.5, 10 mM Mg(OAc) 2 , 2 mM CaCl 2 , 2 mM ATP, 1 mM DTT). Under these conditions, ATP hydrolysis can occur, and thus, hRAD51 can disassemble from the ssDNA. Therefore, the difference between the extension and relaxation curves (A), the total fluorescence intensity (B) and hysteresis area (C) decrease over time. In (A), the blue curve is indistinguishable from the grey curve of bare ssDNA. The disassembly rate can be determined either by an exponential fit to the fluorescence data (B), yielding, after correcting for photobleaching, a rate of (3.5 ± 0.3) 10 −4 s −1 , or by an exponential fit to the hysteresis data (C), yielding a rate of (4 ± 1) 10 −4 s −1 . Data shown is a representative example of six identical experiments. D–F Same experiments as in (A–C) in the presence of Ca 2+ and Mg 2+ (20 mM Tris pH 7.5, 2 mM CaCl 2 , 10 mM Mg(OAc) 2 , 1 mM DTT). NPFs were formed in the presence of ATP, but there was no ATP or ADP in the observation channel. Under these conditions, ATP hydrolysis can occur, and thus, hRAD51 can disassemble from the ssDNA, but reloading of ATP to the NPF after ATP hydrolysis and ADP release is impossible. The differences between the extension and relaxation curves (D), the total fluorescence intensity (E) and hysteresis area (F) decrease over time. In (D), blue curve is indistinguishable from grey curve of bare ssDNA. The disassembly rate can be determined by an exponential fit to the data in (E), giving a rate of (4 ± 1) 10 −4 s −1 , or by an exponential fit to the data in (F), giving a rate of (4 ± 1) 10 −4 s −1 . Source data are available online for this figure.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Fluorescence

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: A, B Side (A) and top (B) views of the asymmetric unit content of the crystal, which consists of 14 ATP‐bound copies of hRAD51 arranged as two heptameric right‐handed filaments. The two heptameric oligomers form continuous helical filaments running through the crystal. The hRAD51 molecules are drawn as ribbons, colour‐coded in blue or green in the two heptamers, with cylinders marking the position of each alpha helix. The ATP molecule is drawn as spacefill model. The positions of the N‐terminal and ATPase domains of RAD51 are indicated in (A). The RAD51 chains in the two heptameric assemblies are labelled A to G and H to N in (B).

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques:

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Structural superposition of the 12 dimers present in the asymmetric unit of the hRAD51‐ATP filament. Each hRAD51 dimer is coloured in light blue or yellow, according to the interface type between its two protomers. The protein chains of the dimers are drawn as a C α backbone, with one ATP moiety shown as stick model. The illustration underneath the superposition reports the distribution of interface types between protomers in the two hRAD51‐ATP heptamers, with RAD51 chains labelled A to G and H to N, as in Fig B. Each interface is marked by a coloured bar, colour‐coded blue or yellow as in the structural superimposition. The relative displacement in the position of hRAD51 residues between the two dimer types is illustrated by drawing the hRAD51 C α backbone as tube of varying radius, in direct proportion to the rmsd value for each amino acid (a larger radius corresponds to a higher rmsd). The structure of the reference hRAD51 structure used in the superposition is drawn as a spacefill model. Two filament structures of different pitch and rise are obtained by modelling a filament based exclusively on one or the other dimer type found in the crystal structure of the hRAD51‐ATP filament (see also and ). Seven hRAD51 protomers corresponding approximately to one helical turn are shown in both cases, drawn as molecular surfaces in light blue.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques:

Journal: The EMBO Journal

Article Title: Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

doi: 10.15252/embj.201798162

Figure Lengend Snippet: Based on the experiments involving stretching cycles such as shown in Figs A–I, , and G and H, we propose a cartoon model with the following conformational states and possible transitions. Because of the large hysteresis in the data shown in Fig A–C, we propose that there are two ATP‐bound states: an ATP‐compact and an ATP‐extended state with a free‐energy difference of (4 ± 1) k B T. Because there is no significant hysteresis in the experiments shown in Fig G–I, we propose that there is only one ADP‐bound state, from which the hRAD51 NPF can disassemble: the ADP state. For switching between ATP‐bound and ADP‐bound states, additional energy is required. This is generated by the hydrolysis of one ATP molecule (providing ~25 k B T). Black: ssDNA, blue: ATP‐compact hRAD51 monomer, green: ATP‐extended hRAD51 monomer, purple: hRAD51 monomer in ADP state, red: ATP, yellow: ADP.

Article Snippet: Human full‐length hRAD51 was expressed in Escherichia coli and purified using a new protocol that exploits the affinity of hRAD51 for BRCA2 BRC‐repeat 4 ( https://www.addgene.org/105045/ ).

Techniques: Generated

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: VEGF-induced vascular permeability is reduced upon CRISPR/Cas9-mediated knockout of Stat3 in zebrafish. (A) VEGF-inducible zebrafish were crossed to Stat3 +/− (heterozygous) zebrafish to generate VEGF-inducible; Stat3 +/− double transgenic fish, which were intercrossed to generate VEGF-inducible; Stat3 −/− (KO) zebrafish. (B) CRISPR/Cas9-generated Stat3 KO zebrafish (bottom) display no overt vascular defects relative to wild-type (WT) zebrafish (top). The vascular system of 3 days post-fertilization (dpf) zebrafish was visualized by microangiography with 2000 kDa FITC-dextran. Representative images of at least three zebrafish per group are shown. Scale bars: 100 μm. (C) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf Stat3 +/+ (negative controls without VEGF induction; left) , VEGF-induced, Stat3 +/+ (middle) and VEGF-induced, Stat3 −/− (right) zebrafish. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at room temperature (RT). Scale bars: 50 μm. (D) Quantitative analysis of vascular permeability upon VEGF stimulation in WT Stat3 +/+ ( n =30) and KO Stat3 −/− ( n =9) zebrafish. Mean±s.e.m., unpaired, two-tailed Student's t -test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, CRISPR, Knock-Out, Transgenic Assay, Generated, Marker, Microscopy, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Endothelial cell-specific STAT3 knockout mice exhibit decreased VEGF-induced permeability. (A) Images of footpads from WT and endothelial cell-specific STAT3 knockout (STAT3 ECKO ) mice following tail vein injection with 1% Evans Blue dye and human recombinant VEGF-165 protein (2.5 µg/ml; left footpads) or PBS vehicle (right footpads) being injected into the root of the footpad. (B,C) Quantitation of Evans Blue leakage in Tie2-Cre negative; STAT3 flox/flox (WT) and Tie2-Cre positive; STAT3 flox/flox (STAT3 ECKO ) mice. n =7 mice in WT group and n =6 mice in STAT3 ECKO group. Each mouse was injected with PBS on the right anterior and posterior footpads and VEGF on the left anterior and posterior footpads. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. A.U., arbitrary units.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Knock-Out, Permeability, Injection, Recombinant, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Pharmacological inhibition of STAT3 stabilizes endothelial barrier integrity following VEGF stimulation in human endothelial cells. (A) Serum-starved human umbilical vein endothelial cells (HUVECs) were pretreated with DMSO (vehicle control) for 1 h, 30 µM AQ for 4 h, or 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 2 or 5 min. Lysates were immunoblotted. Densitometry was performed, and the values below the rows of bands represent the ratio of phosphorylated protein to respective total protein. (B) Human VEGF-165 recombinant protein (VEGF; 25 ng/ml) stimulation of HUVECs promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows; left column; DMSO vehicle control pretreatment for 1 h prior to VEGF stimulation). ZO-1 organization is maintained upon pretreatment with 30 μM AQ for 4 h (magenta arrows; middle column) or 10 μM PYR for 1 h (magenta arrows; right column) prior to VEGF stimulation. Nuclei were stained with DAPI (blue). (C) Serum-starved human pulmonary artery endothelial cells (HPAECs) were pretreated with 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 5 or 30 min. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained when HPAECs were pretreated with PYR (magenta arrows). Nuclei were stained with DAPI (blue). (D) VEGF (25 ng/ml) stimulation of human lung microvascular endothelial cells (HMVEC-Ls) promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained upon pretreatment with 20 μM PYR for 6 h prior to VEGF stimulation (magenta arrows). Nuclei were stained with DAPI (blue). At least two biological replicates were performed for each experiment depicted in A-D. Scale bars: 20 µm.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Inhibition, Recombinant, Staining

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Suppression of STAT3 activity by pyrimethamine (PYR) inhibits VEGF-induced vascular permeability in zebrafish and mice. (A) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf zebrafish without induced VEGF pretreated with DMSO ( n =6) or 25 μM PYR ( n =5) or 3 dpf zebrafish with induced VEGF pretreated with DMSO ( n =4) or 25 μM PYR ( n =9) for 3 days. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at RT. Scale bars: 50 μm. (B) The quantitative analysis of vascular permeability without VEGF stimulation or upon VEGF stimulation in zebrafish pretreated with DMSO or PYR. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. (C) Representative images of footpads from mice treated with vehicle or PYR following tail vein injection with 1% Evans Blue and footpad injection of VEGF (2.5 μg/ml) or PBS vehicle. (D) Quantitation of Evans Blue dye leakage in C57BL/6 WT mice treated with vehicle or PYR. n =9 mice in the vehicle group and n =7 mice in the PYR group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Activity Assay, Permeability, Marker, Microscopy, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: JAK2 phosphorylates STAT3 to transduce VEGF/VEGFR-2 signaling and promote vascular permeability. (A) To perform a STAT3 GST pull-down of VEGFR-2 and JAK2, lysates of HUVECs stimulated with serum for 30 min were used as prey. GST fusion protein STAT3 expressed in 293F cells was used as bait. GST alone served as a negative control. Binding experiments were analyzed by SDS-PAGE and visualized by immunoblotting. GST-STAT3 and GST were each detected using an anti-GST antibody. Three biological replicates were performed and depicted findings are representative. (B) JAK2 phosphorylates STAT3 in vitro . In vitro kinase assays were performed using purified human STAT3 protein and kinase active JAK2 protein. The results shown here are representative of two independent experiments. (C) Representative images of footpads from C57BL/6 WT mice treated with vehicle or JAK2 inhibitor AG490. Following tail vein injection with 1% Evans Blue dye, human VEGF-165 protein (2.5 μg/ml) or PBS vehicle was injected into the root of the footpad. After 30 min, the mice were euthanized and the footpads were excised. (D) Quantitation of Evans Blue dye leakage in C57BL/6 mice treated with vehicle or AG490. n =4 mice per group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Two biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Transduction, Permeability, Negative Control, Binding Assay, SDS Page, Western Blot, In Vitro, Purification, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: STAT3 transcriptionally activates ICAM-1, a cell adhesion molecule that promotes vascular permeability. (A) Top: the pGL3-ICAM1-WT plasmid containing the human ICAM-1 promoter with a STAT3 binding site located at −115 to −107 bp. Bottom: the pGL3-ICAM1-SDM plasmid with a site-directed mutation (SDM) in the STAT3 binding site as indicated. (B) Dual luciferase assays were performed in HUVECs that were transfected with pGL3-ICAM1-WT or pGL3-ICAM1-SDM and empty vector or constitutively active STAT3. Firefly and Renilla luminescence was measured and plotted as a ratio. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. n =9 technical replicates. Depicted findings are representative of three independent experiments. (C) HUVECs that had been stably transduced with lentivirus encoding STAT3-specific shRNA or control shRNA were stimulated with human VEGF-165 protein (25 ng/ml) and the lysates were immunoblotted for ICAM1, p-STAT3 (Y705) and total STAT3. Depicted data are representative of three biological replicates. (D) RNA was harvested from VEGF; Stat3 +/+ or VEGF; Stat3 −/− 3 dpf embryos for quantitative PCR. stat3 transcripts are reduced in VEGF; Stat3 −/− ( n =5) compared to VEGF; Stat3 +/+ zebrafish ( n =7). Mean±s.e.m., unpaired, two-tailed Student's t -test. (E) The expression of icam-1 was assessed by real-time quantitative PCR using RNA derived from each zebrafish embryo in the absence of VEGF induction (Stat3 +/+ , n =3; Stat3 −/− , n =2) or 8 h following VEGF induction (Stat3 +/+ , n =4; Stat3 −/− , n =3) in the heat-inducible VEGF; Stat3 mutant zebrafish. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, Plasmid Preparation, Binding Assay, Mutagenesis, Luciferase, Transfection, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, Two Tailed Test, Expressing, Derivative Assay

Journal: Oncotarget

Article Title: Inhibitory effects of transcription factor Ikaros on the expression of liver cancer stem cell marker CD133 in hepatocellular carcinoma.

doi: 10.18632/oncotarget.2524

Figure Lengend Snippet: Figure 2: Ikaros represses CD133 expression in HCC. (A) The promoter activity of P1 after the transfection of Ikaros into Hep3B, Huh7, and PLC/PRF/5 (PLC) cells. **, p<0.01. (B) Binding of Ikaros on the CD133 gene promoter was assayed by chromatin immunoprecipitation (ChIP). ChIP was performed using antibodies to Ikaros in HCC-LY5, SMMC-7721, and MHCC-LM3 cells. A negative control with irrelevant antibody (IgG) was included for comparison. qPCR was performed on the bound fractions. (C) DNA pull- down assay to detect Ikaros binding to the P1 promoter using either wild-type (WT) or Ikaros mutant oligonucleotide sequences from the CD133 promoter in HCC-LY5, SMMC-7721, and MHCC-LM3 cells. (D) Immunoblot analysis of CD133 expression when Ikaros was overexpressed or silenced. (E) IHC analysis of Ikaros and CD133 in HCC samples. Representative images are shown. Scale bar, 100 μm. (F) The analysis of the correlation between Ikaros and CD133 in HCC tissue. **, p<0.01; r=-0.608; n=102. Data represent mean ± SD of three independent experiments with technical triplicates for each, and statistical analysis was performed using Student’s t test.

Article Snippet: Plasmid constructs, lentivirus production, and cell transfection Full-length human Ikaros (Origene) was subcloned into pWPXL (Addgene) at the BamH I and EcoR II sites.

Techniques: Expressing, Activity Assay, Transfection, Binding Assay, Chromatin Immunoprecipitation, ChIP-chip, Negative Control, Comparison, Pull Down Assay, Mutagenesis, Western Blot

Journal: Oncotarget

Article Title: Inhibitory effects of transcription factor Ikaros on the expression of liver cancer stem cell marker CD133 in hepatocellular carcinoma.

doi: 10.18632/oncotarget.2524

Figure Lengend Snippet: Figure 3: CtBP interacts with Ikaros as a transcription repressor complex. (A) Co-immunoprecipitation assay to detect Ikaros interaction proteins in HCC-LY5, SMMC-7721, and MHCC-LM3 cells. (B) Immunoblot analysis of co-immunoprecipitated mixture. (C) Confocal imaging analysis of CtBP and Ikaros colocalization in the nucleus. Red indicates Ikaros; green indicates CtBP; and blue indicates DAPI. Scale bar, 10 μm. (D) Binding of CtBP on the CD133 gene promoter was assayed by ChIP, which was performed using antibodies to CtBP in HCC-LY5, SMMC-7721, and MHCC-LM3 cells. A negative control with irrelevant antibody (IgG) was included for comparison. qPCR was performed on the bound fractions. (E) The promoter activity of P1 after the transfection of CtBP into Hep3B, Huh7, and PLC/ PRF/5 cells. **, p<0.01; *, p<0.05. Data represent mean ± SD of three independent experiments with technical triplicates for each, and statistical analysis was performed using Student’s t test.

Article Snippet: Plasmid constructs, lentivirus production, and cell transfection Full-length human Ikaros (Origene) was subcloned into pWPXL (Addgene) at the BamH I and EcoR II sites.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation, Imaging, Binding Assay, Negative Control, Comparison, Activity Assay, Transfection

Journal: Oncotarget

Article Title: Inhibitory effects of transcription factor Ikaros on the expression of liver cancer stem cell marker CD133 in hepatocellular carcinoma.

doi: 10.18632/oncotarget.2524

Figure Lengend Snippet: Figure 4: MAPKs/ETS1 pathway regulates the expression of Ikaros and CD133. (A) The promoter activity of Ikaros responding to ETS1 overexpressing in HCC-Y5, SMMC-7721, and MHCC-LM3 cells. **, p<0.01; *, p<0.05. The Ikaros promoter was analyzed using TFSEARCH (http://mbs.cbrc.jp/research/db/TFSEARCH.html). (B) Binding of ETS1 on the Ikaros gene promoter was assayed by ChIP which was performed using antibodies to ETS1 in HCC-LY5, SMMC-7721, and MHCC-LM3 cells. A negative control with irrelevant antibody (IgG) was included for comparison. qPCR was performed on the input and bound fractions. (C) DNA pull-down assay to detect ETS1 binding to the Ikaros promoter in HCC-LY5, SMMC-7721, and MHCC-LM3 cells. (D and E) Immunoblot analysis of Ikaros and CD133 expression when ETS1 overexpression or knockdown. (F) Immunoblot analysis of the expression of Erk1/2, pErk1/2, ETS1, pETS1, Ikaros, and CD133 when specific MAPKs inhibitors (U0126, PD98059) were added to Huh7, PLC/PRF/5, HCC-LY5, and SMMC-7721 cells. Data represent mean ± SD of three independent experiments with technical triplicates for each, and statistical analysis was performed using Student’s t test.

Article Snippet: Plasmid constructs, lentivirus production, and cell transfection Full-length human Ikaros (Origene) was subcloned into pWPXL (Addgene) at the BamH I and EcoR II sites.

Techniques: Expressing, Activity Assay, Binding Assay, Negative Control, Comparison, Pull Down Assay, Western Blot, Over Expression, Knockdown

Journal: Oncotarget

Article Title: Inhibitory effects of transcription factor Ikaros on the expression of liver cancer stem cell marker CD133 in hepatocellular carcinoma.

doi: 10.18632/oncotarget.2524

Figure Lengend Snippet: Figure 5: Ikaros inhibits the tumor growth and self-renewal of CD133+ HCC cells. (A) A representative image of a CSC sphere in self-renewal assays (left panel). The mean values of Huh7, CD133+/- PLC/PRF/5 cells sphere from three independent experiments are shown in the right panel. (Scale bar, 100 μm) (B) The size of tumors from NOD/SCTD mice injected with Ikaros-overexpressing or control cells (left panel). The weight of tumors from NOD/SCTD mice injected with Ikaros-overexpressing or control cells in the right panels. **, p<0.01; *, p<0.05. (C) Flow cytometric analysis of the percentage of CD133+ cells when Ikaros was overexpressed in Huh7 and PLC/PRF/5 cells. (D) Flow cytometric analysis of the percentage of CD133+ cells when Ikaros was knockdown in HCC-LY5 and SMMC-7721 cells. (E) Immunoblot analysis of expression of CD133, CK18, and CK19 when Ikaros was enforced to express or silenced. Data represent mean ± SD of three independent experiments with technical triplicates for each, and statistical analysis was performed using Student’s t test.

Article Snippet: Plasmid constructs, lentivirus production, and cell transfection Full-length human Ikaros (Origene) was subcloned into pWPXL (Addgene) at the BamH I and EcoR II sites.

Techniques: Injection, Control, Knockdown, Western Blot, Expressing

Journal: Oncotarget

Article Title: Inhibitory effects of transcription factor Ikaros on the expression of liver cancer stem cell marker CD133 in hepatocellular carcinoma.

doi: 10.18632/oncotarget.2524

Figure Lengend Snippet: Figure 6: The clinical significance of Ikaros in HCC. (A) The IHC analysis of Ikaros in HCC samples. Scale bar, 100 μm. (B) Immunoblot analysis of Ikaros expression in HCC samples. (C) The overall survival analysis of HCC patients with Ikaros expression. *, p<0.05; n=102; Log-rank p=0.045. (D) Immunoblot analysis of the expression of Erk1/2, ETS1, Ikaros, and CD133 in HCC samples. (E) The function model of Ikaros in HCC. MAPK pathway regulates the activity of ETS1 via phosphorylation of ETS1. ETS1 is able to up- regulate Ikaros expression, and Ikaros interacting with CtBP inhibits CD133 expression via directly binding to CD133 P1 promoter.

Article Snippet: Plasmid constructs, lentivirus production, and cell transfection Full-length human Ikaros (Origene) was subcloned into pWPXL (Addgene) at the BamH I and EcoR II sites.

Techniques: Western Blot, Expressing, Activity Assay, Phospho-proteomics, Binding Assay

Journal: Oncogene

Article Title: GATA3 interacts with and stabilizes HIF-1α to enhance cancer cell invasiveness.

doi: 10.1038/onc.2017.8

Figure Lengend Snippet: Figure 1. GATA3 is overexpressed in HNSCC and high GATA3 expression is associated with poor survivals. (a) GATA3 mRNA is overexpressed in HNSCC. GATA3 mRNA levels are higher in HNSCC (n = 41) compared with normal oral mucosa (n = 13). The data are retrieved from Ginos Head- Neck in the Oncomine database (https://www.oncomine.org). (b) Western blot analysis of GATA3 expression in paired HNSCC tumours (T) and adjacent non-tumour mucosa tissues (N) from 12 patients. Left: Western blots showing GATA3 expression and GAPDH control. Right: GATA3 expression was quantified and analysed by paired Student’s t-test. **Po0.01. (c) Representative images of immunohistochemistry of GATA3 in paired adjacent non-tumour mucosa (N) and tumour tissues (T) from HNSCC patients (n = 15). Scale bars, 50 μm. (d) Scoring of immunohistochemistry of GATA3 in HNSCC tumour tissues. The intensity of GATA3 staining is scored from 0 to 3 and grouped into low (score = 0, 1) and high (score = 2, 3) expression. (e) Kaplan–Meier survival analysis. Patients with follow-up period over 18 months are included (n = 144). Left and right panels indicate disease-free and overall survivals of patients with low and high GATA3 expression, respectively.

Article Snippet: Plasmid construction The human full-length GATA3 (NM_002051) cDNA clone was purchased from OriGene (SC108486, Rockville, MD, USA) and subcloned into pcDNA3.1/ mycHis plasmid using the restriction sites of EcoRI and XbaI.

Techniques: Expressing, Western Blot, Control, Immunohistochemistry, Staining

Journal: Oncogene

Article Title: GATA3 interacts with and stabilizes HIF-1α to enhance cancer cell invasiveness.

doi: 10.1038/onc.2017.8

Figure Lengend Snippet: Figure 2. GATA3 promotes tumour invasiveness in vitro and in vivo. (a) Western blot analysis of GATA3 expression in various cell lines as indicated. GAPDH was an internal control. (b) Knockdown and overexpression of GATA3 were confirmed by western blot analysis. For knockdown, OEC-M1 cells were transfected with non-targeting siRNA (siCtr) or two independent siRNAs against GATA3 (siGATA3-1 and siGATA3-2). For overexpression, FaDu and A375 cells were transfected with empty pcDNA3.1 (Mock) or GATA3/pcDNA3.1 (GATA3). (c) Effects of GATA3 on cell migration and invasion analysed by transwell migration and Matrigel invasion assays, respectively. Cells were seeded after transfection for 48 h. Normoxia (N, O2 = 21%) or hypoxia (H, O2 = 1%) is as indicated. Results are represented as mean ± s.d. from three independent experiments. *Po0.05, **Po0.01. (d) Effects of GATA3 on experimental metastasis in vivo. Upper panel, representative images of lungs and metastatic tumour nodules indicated by arrowheads. Lower panel, statistical analysis of tumour nodules. Mock or GATA3 overexpressing OEC-M1 and A375 stable transfectants (1 × 106 cells / per injection) were injected through tail veins into NOD-SCID mice (n = 6 and 5, respectively). Results are represented as mean ± s.d. Data are analysed by Student’s t-test. *Po0.05. (e) Expression of GATA3 and HIF-1α in subcutaneous tumour xenografts. Left, hematoxylin and eosin staining. The hollow arrow indicates the intact capsule separating the tumour from surrounding tissues. The black arrow indicates the invasion of tumour into skeletal muscles. M, skeletal muscle. T, tumour. Middle and right, immunohistochemical staining of GATA3 and HIF-1α. Scale bars, 50 μm.

Article Snippet: Plasmid construction The human full-length GATA3 (NM_002051) cDNA clone was purchased from OriGene (SC108486, Rockville, MD, USA) and subcloned into pcDNA3.1/ mycHis plasmid using the restriction sites of EcoRI and XbaI.

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Control, Knockdown, Over Expression, Transfection, Migration, Injection, Staining, Muscles, Immunohistochemical staining

Journal: Oncogene

Article Title: GATA3 interacts with and stabilizes HIF-1α to enhance cancer cell invasiveness.

doi: 10.1038/onc.2017.8

Figure Lengend Snippet: Figure 3. GATA3 expression increases HIF-1α protein levels. (a) Western blot analysis of GATA3 and HIF-1α under normoxia (N) or hypoxia (H) for overnight with or without a proteasome inhibitor, MG132 (20 μM), as indicated. GAPDH was an internal control. OEC-M1 cells were transfected with non-targeting siRNA or GATA3 siRNA (siGATA3-1 or siGATA3-2) and A375 cells were transfected with Mock or GATA3 plasmid. (b) Real-time RT-PCR analysis of HIF-1α mRNA expression. The total RNA was extracted from cells grown under normoxia or hypoxia for overnight. The results are represented as mean ± s.d. from three independent experiments. (c) Western blot analysis of GATA3 and P402A/ P564A HIF-1α mutant. GAPDH was an internal control. pLKO or shGATA3/pLKO (shGATA3) expressing OEC-M1 stable transfectants as well as Mock or GATA3 overexpressing A375 stable transfectants were transfected with empty vector or HA-P402A/P564A HIF-1α/pcDNA3.1 (HA-mtHIF-1α). (d) Effects of GATA3 on HIF-1α ubiquitination. OEC-M1 cells transfected with non-targeting siRNA or GATA3 siRNA (siGATA3-2) and A375 cells transfected with Mock or GATA3 plasmid were incubated with MG132 (20 μM) under hypoxia for 6 h. Cell lysates were immunoprecipitated with anti-HIF-1α antibody and then immunoblotted with anti-ubiquitin antibody. GATA3, HIF-1α and GAPDH in whole cell lysates (input) were shown. (e) Representative images of immunohistochemical staining of GATA3 and HIF-1α in serial sections of HNSCC tissues. Scale bars, 50 μm.

Article Snippet: Plasmid construction The human full-length GATA3 (NM_002051) cDNA clone was purchased from OriGene (SC108486, Rockville, MD, USA) and subcloned into pcDNA3.1/ mycHis plasmid using the restriction sites of EcoRI and XbaI.

Techniques: Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR, Mutagenesis, Ubiquitin Proteomics, Incubation, Immunoprecipitation, Immunohistochemical staining, Staining

Journal: Oncogene

Article Title: GATA3 interacts with and stabilizes HIF-1α to enhance cancer cell invasiveness.

doi: 10.1038/onc.2017.8

Figure Lengend Snippet: Figure 4. GATA3 physically interacts with HIF-1α. (a, b) Co-immunoprecipitation assays of GATA3 and HIF-1α in OEC-M1 and A375 cells. OEC- M1 cells were transfected with non-targeting siRNA or GATA3 siRNA (siGATA3-2) and A375 cells were transfected with Mock or GATA3 plasmid. Cells were incubated with MG132 (20 μM) and CoCl2 (800 μM) for 6 h. Upper panel, cell lysates were immunoprecipitated with IgG control antibody or anti-HIF-1α antibody and then immunoblotted with anti-GATA3 or anti-HIF-1α antibody. Lower panel, cell lysates were immunoprecipitated with IgG control antibody or anti-GATA3 antibody and then immunoblotted with anti-HIF-1α or anti-GATA3 antibody. HIF-1α, GATA3 and GAPDH in whole cell lysates (input) were shown. (c) GST pull-down assays. 293FT cells were transfected with F-, N- or C-HIF-1α/pCMV-Tag 4A (Flag-tagged) and incubated with CoCl2 (800 μM) for 4 h. Left panel, GST-GATA3 fusion protein pulled down F-HIF-1α and N-HIF-1α, but not C-HIF-1α. On the same membrane, C-HIF-1α could not be detected by the rabbit anti-HIF-1α polyclonal antibody (Cell Signaling Technology), which is known to recognize this region (Ser653, data not shown). The black arrowhead indicates F-HIF-1α. The asterisk indicates N-HIF-1α. The expression of F-HIF-1α and N-HIF-1α was confirmed by western blotting and shown in the middle panel. For unknown reason, C-HIF-1α could not be detected by anti-Flag antibody. We used rabbit anti-HIF-1α polyclonal antibody (Cell Signaling Technology) to confirm C-HIF-1α expression as shown in the right panel (hollow arrowhead). (d) The design of HIF-1α constructs is based on known functional domains and the pull-down assay results were summarized schematically. PAS, Per-ARNT-Sim.

Article Snippet: Plasmid construction The human full-length GATA3 (NM_002051) cDNA clone was purchased from OriGene (SC108486, Rockville, MD, USA) and subcloned into pcDNA3.1/ mycHis plasmid using the restriction sites of EcoRI and XbaI.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Incubation, Control, Membrane, Expressing, Western Blot, Construct, Functional Assay, Pull Down Assay

Journal: Oncogene

Article Title: GATA3 interacts with and stabilizes HIF-1α to enhance cancer cell invasiveness.

doi: 10.1038/onc.2017.8

Figure Lengend Snippet: Figure 5. GATA3 regulates expression of HIF-1 target genes under hypoxic conditions. (a) Real-time RT-PCR analysis of the expression of HIF-1α target genes, including PGK1, CA9, SLC2A1, SLC2A3 and VEGFA in OEC-M1 and A375 cells. (b) Western blot analysis of GATA3, HIF-1α, PGK1, CA9 and SLC2A1. GAPDH was an internal control. Total RNAs and cell lysates were extracted from cells grown under normoxia (N) or hypoxia (H), as indicated. (c) ChIP assays with anti-GATA3 or anti-HIF-1α antibody and SLC2A1 and VEGFA genes in OEC-M1 cells. Control or GATA3 knockdown cells were cultured under hypoxia for overnight and then harvested for ChIP assays. Isotype matched IgG was used as controls. The results are represented as mean ± s.d. from three independent experiments. *Po0.05, **Po0.01.

Article Snippet: Plasmid construction The human full-length GATA3 (NM_002051) cDNA clone was purchased from OriGene (SC108486, Rockville, MD, USA) and subcloned into pcDNA3.1/ mycHis plasmid using the restriction sites of EcoRI and XbaI.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Knockdown, Cell Culture

Journal: Nature

Article Title: Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.

doi: 10.1038/nature08792

Figure Lengend Snippet: Figure 2 | Telomere elongation in DKC1 mutant iPS cells. a, Telomere Southern blot of del37L fibroblasts (Fib) and iPS clones 1 and 2 as a function of passage. MM, molecular mass marker. b, Quantitative real-time PCR (qPCR) analysis of telomere length in del37L fibroblasts and iPS clones at indicated passages (p). Error bars represent s.e.m. c, Quantitative fluorescence in situ hybridization (qFISH) for telomere length in del37L cells. a.f.u., arbitrary fluorescence units. Inset, mean relative length values shown 6s.d.

Article Snippet: The DKC1 retroviral vector was created by cloning a full-length DKC1 cDNA (OriGene) into pMIG-W (Addgene) and retrovirus was produced as described31.

Techniques: Mutagenesis, Southern Blot, Clone Assay, Marker, Real-time Polymerase Chain Reaction, Fluorescence, In Situ Hybridization

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Co-immunoprecipitation (co-IP) of ChAT with heat shock proteins HSC70, HSP70, and HSP90 is altered by mutation of N-terminal proline-rich motif in ChAT. (A) Immunoblots showing co-IP of ChAT with endogenous HSC70, HSP70 and HSP90 from HEK293 cells expressing either wild-type or P17A/P19A-ChAT. Control cells were transfected with empty vector. Using HEK293 cells, co-IP of P17A/P19A-ChAT with HSP70 (B) , HSP90 (C) and HSC70 (D) , respectively, is greater than that of wild-type ChAT ( *** p ≤ 0.001, Student's t -test, mean ± SEM, n = 4). (E) Co-IP of ChAT with endogenous HSC70 and HSP90 from mouse cholinergic SN56 cells expressing either wild-type or P17A/P19A-ChAT or CMS-related mutant proteins V18M- or A513T-ChAT. Control cells were transfected with empty vector. (F) Using SN56 cells, Co-IP of P17A/P19A-ChAT ( *** p ≤ 0.001) and V18M-ChAT ( * p ≤ 0.05), but not A531T-ChAT, with HSC70 is greater than that of wild-type ChAT (mean ± SEM, n = 5). (G) While there was a trend toward increased HSP90 interaction with P17A/P19A-ChAT ( p = 0.09), no significant differences were observed for HSP90 interaction with mutant ChAT compared to wild-type ChAT in SN56 cells (mean ± SEM, n = 5). Statistical analysis for (F) and (G) was performed by one-way ANOVA with Dunnett's post-hoc test. (H) Detection of in situ interactions of wild-type ChAT with endogenous HSC70 and HSP90 by proximity ligation assay (PLA) in SN56 cells. Formalin-fixed cells were first co-labeled with goat anti-ChAT together with either mouse anti-HSC70 or mouse anti-HSP90 primary antibodies, then incubated with oligonucleotide-linked secondary antibodies. Following DNA ligation and DNA amplification using the Duolink in Situ Orange Kit (Sigma), in situ ChAT-HSP interactions were imaged by confocal microscopy. Positive in situ ChAT-HSP interactions where visualized as fluorescent red dots while nuclei were stained with DAPI (blue). Control cells were either transfected with empty vector or had primary antibodies omitted from the assay (No 1° antibodies). Images are representative of 3 independent experiments; scale bars are 10 μm.

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Expressing, Transfection, Plasmid Preparation, In Situ, Proximity Ligation Assay, Labeling, Incubation, DNA Ligation, Amplification, Confocal Microscopy, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: ChAT interacts with the HSP-associated E3 ubiquitin ligase C-terminus of HSC70-interaction protein (CHIP). (A) Immunoblots showing co-IP of ChAT with FLAG-CHIP from SN56 cells co-expressing either wild-type or mutant ChAT protein with FLAG-tagged CHIP. Control cells were transfected with either empty vector or to express either wild-type ChAT or FLAG-CHIP alone. (B) Co-IP of ChAT with FLAG-CHIP is enhanced for P17A/P19A- ( *** p ≤ 0.001), V18M- ( *** p ≤ 0.001), and A513T-ChAT ( * p ≤ 0.05) as compared to wild-type ChAT (one-way ANOVA with Dunnett's post-hoc test, mean ± SEM, n = 5). (C) Co-IP of wild-type and mutant ChAT with endogenous CHIP following anti-ChAT co-IP from ChAT-expressing SN56 cells ( n = 3). (D) Detection of in situ ChAT interactions with endogenous CHIP by proximity ligation assay (PLA) in SN56 cells expressing wild-type ChAT. Formalin-fixed cells were first co-labeled with goat anti-ChAT together with rabbit anti-CHIP primary antibodies, then incubated with oligonucleotide-linked secondary antibodies. Following DNA ligation and DNA amplification using the Duolink In Situ Orange Kit (Sigma), in situ ChAT-CHIP interactions were imaged by confocal microscopy. Positive in situ ChAT-CHIP interactions were visualized as fluorescent red dots while nuclei were stained with DAPI (blue). Control cells were either transfected with empty vector or primary antibodies omitted from the assay (No 1° antibodies). Images are representative of 4 independent experiments; scale bars are 10 μm. (E) siRNA-mediated knock-down of CHIP has no effect on the steady-state protein levels of either wild-type or mutant ChAT. ChAT-expressing SN56 cells were co-transfected with 25 nM of either anti-CHIP siRNA or scramble-control siRNA for 72 h. Control cells were mock-transfected ( n = 4).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Expressing, Mutagenesis, Transfection, Plasmid Preparation, In Situ, Proximity Ligation Assay, Labeling, Incubation, DNA Ligation, Amplification, Confocal Microscopy, Staining

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 1. Individuals with biallelic CDK4 variants display microcephaly and short stature. (A) Family pedigrees with segregation of CDK4 variants. (Square) Male, (circle) female, (filled symbols) individuals with microcephaly, (strikethrough) deceased. WT Reference (+), variants v1 and v2, and zygosity are indicated for each studied individual. (B) Diagram of CDK4 transcript (top) and protein (bottom); coding exons are depicted as black rectangles. Red lines indicate variant location. (SS) Splice site disrupted. (C) Altered splicing predictions for the c.218G > A substitution generated using Alamut. (Blue rectangles) Strength of splice donor predictions for individual splice algo- rithms, (blue triangle) predicted donor splice site. (D) Growth parameters at birth and at last assessment (postnatal). (W) Weight, (OFC) orbito–frontal circumference. Z-scores show standard deviations from population mean for age and sex. Dashed lines indicate a 95% con- fidence interval for the general population. Individual subject data points from families A (circles) and B (squares) are graphed, and mean values are plotted. (E) MRI scan of age-matched control (4 years 8 months) and affected individuals with a CDK4 variant. Coronal FLAIR projection shows simplified parietal and temporal gyri, reduced white matter volume, and the absence of brain malformations. Scale bars, 10 cm. (See also Supplemental Figure S1C for additional MRI projections.) (F) Photographs of all affected individuals.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Variant Assay, Generated, Control

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 3. Full-length CDK4 protein is undetectable in patient fibroblasts. (A,B) Immunoblots of total cell extracts obtained from expo- nentially growing control (C1 and C2) and patient (P1 and P2) fibroblasts without (A) and with (B) CDK4 complementation. α-Tubulin was used as the loading control. A rabbit monoclonal antibody to C-terminal CDK4 was used; a different mouse CDK4 antibody raised against full-length CDK4 was used in Figure 5A. A smaller ∼12 kDa molecular weight band was variably detected in P1 with this antibody (Sup- plemental Fig. S2D) that might correspond to the 46 amino acid truncated nonfunctional protein predicted from RNA studies. (C) CDK6 and Cyclin D1 levels were unchanged in patient fibroblasts compared with wild-type controls.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Molecular Weight

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 4. CDK4 mutations do not alter mitosis. (A) Percentage of mitotic cells (p-Histone H3 ser10-positive) in control (C1 and C2) and patient (P1 and P2) fibroblasts as measured by flow cy- tometry. Data points are from three independent experiments (two for C1); one-way ANOVA with Tukey post test; mean ± SEM. (B) Quantification of metaphase cells with more than two centrosomes, expressed as percentage. Numbers of cells analyzed were as follows: C1, 79; C2, 94; P1, 150; and P2, 101. Two-tailed t- test; mean ± SEM; measurements were pooled from two indepen- dent experiments. (C) Representative confocal images of control (C1 and C2) and patient (P1 and P2) fibroblasts fixed and stained for DAPI (gray), α-tubulin (green), and pericentrin (magenta). Scale bars, 5 µm.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Control, Two Tailed Test, Staining

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 5. CDK4 mutations impair G1-to-S progression and lead to reduced cell proliferation. (A) Western blot of control and patient-de- rived fibroblasts with and without WT CDK4 complementation. (B, left) Growth curves of control and patient-derived fibroblasts with and without WT CDK4 complementation. (Right) Bar graph showing quantification of doubling times; one-way ANOVA with Tukey post test. P-values are indicated; mean ± SEM. (C) Cell cycle distribution (G0/G1, S, and G2/M) derived from BrdU and DNA (DAPI) flow cytometry scatter plots show fewer cells in S phase (BrdU+) in patient-derived fibroblasts compared with controls. n = 3 independent experiments; mean ± SEM. Gates are shown on representative plots at the right. (D) Cell cycle distribution after complementation of patient-derived fibroblasts with CDK4. Reduced G0/G1 and increased S-phase populations consistent with rescue of a G1/S progression defect. n = 3 in- dependent experiments; mean ± SEM. (See also Supplemental Fig. S4A.) (E) Quantification of DNA synthesis rate (BrdU mean fluorescence intensity [MFI] of gated population in the red rectangle) from experiments depicted in C.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Derivative Assay, Flow Cytometry, DNA Synthesis, Fluorescence