Journal: eLife

Article Title: Ribozyme activity modulates the physical properties of RNA–peptide coacervates

doi: 10.7554/eLife.83543

Figure Lengend Snippet: ( a ) The secondary structure of the ladder ribozyme and a schematic showing its function. The ribozyme is shown in red, whilst the substrate strands are shown in black. ( b ) A representative 8% urea PAGE stained with SYBR Gold showing the products of the R3C ladder system in solution and varying ratios of (Lys) 19-72 to R3C RNA (total monomer concentration = 1 mM, 10.5 µM substrate, 10.5 µM ribozyme) after a 2 hr reaction at 30°C in 50 mM Tris-HCl pH 8.6 and 10 mM MgCl 2 . The integrated lane profiles of the solution and 0.75:1 (Lys) 19-72 :RNA conditions are shown in blue and red, respectively ( c ) Variation in absorbance at 500 nm as a measure of coacervate formation upon addition of varying ratios of (Lys) 19-72 to the E L RNA after ligation for 3 hr at 30°C. Data points are an average of n = 3 independent replicates assembled from the same stock solutions. Error bars are standard deviations. ( d ) Example fluorescence microscopy image of (Lys) 19-72: RNA condensates at a ratio of 0.75:1 (Lys) 19-72 :RNA, imaged using 10% Cy5-tagged substrate strand. Scale bar = 20 μm. ( e ) Kinetics of chain elongation in solution (blue, first-order model), and with 0.75:1 (Lys) 19-72 :RNA (red, second-order model) at 30°C, pH 8.6, and 10 mM MgCl 2 . A total RNA monomer concentration of 1 mM was achieved by combining 9.5 µM substrate, 1 µM Cy5-tagged substrate and 10.5 µM ribozyme. Data points are an average of n = 3 independent replicates assembled from the same stock solutions. Error bars are standard deviations. ( f ) Chain extension rate constants for the R3C ladder ribozyme in solution (blue, first-order model) and in the presence of 0.75:1 (Lys) 19-72 :RNA (red, second-order model). Error bars are the standard errors for each parameter computed during non-linear regression. Equivalent data for condensates formed from the shorter (Lys) 5-24 peptide is shown in . Figure 1—source data 1. Unedited and uncropped gel image for , and labelled image showing key bands and conditions. Figure 1—source data 2. Numerical turbidity data for . Figure 1—source data 3. Unprocessed and uncropped fluorescence microscope image for 0.75:1 (Lys) 19-72 :RNA condensates imaged using 10% Cy5-tagged substrate strand. Figure 1—source data 4. Unedited and uncropped gel images for ribozyme kinetics . Lane identities are listed in the accompanying spreadsheet.

Article Snippet: Trizma base (Tris; Thermo Fisher Scientific, Waltham, MA), sodium hexametaphosphate ((NaPO 3 ) 6 , 611.77 g/mol; Sigma-Aldrich, St. Louis, MI), formamide (CH 3 NO, 45.04 g/mol; Sigma-Aldrich), ethylenediaminetetracetic acid disodium salt dihydrate (EDTA, C 10 H 14 N 2 Na 2 O 8 ·2H 2 O, 372.24 g/mol; Sigma-Aldrich), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O, 203.30 g/mol; (Merck, Darmstadt, Germany)), sodium hydroxide (NaOH, 39.997 g/mol; VWR, Radnor, PA), sodium chloride (NaCl, 58.44 g/mol; Sigma-Aldrich), urea (CH 4 N 2 O, 60.06 g/mol; Carl Roth, Karlsruhe, Germany), hydrochloric acid (HCl, 37%, 36.46 g/mol) (VWR), boric acid (H 3 BO 3 , 61.83 g/mol; Merck), ammonium persulfate (APS, (NH 4 ) 2 S 2 O 8 , 228.20 g/mol) (VWR), acrylamide (19:1 bisacrylamide; Thermo Fisher Scientific), tetramethylethylendiamine (TEMED, C 6 H 16 N 2 , 116.21 g/mol; Carl Roth), SYBR gold stain (Thermo Fisher Scientific), RNA oligomer length standard (low-range ssRNA ladder) (NEBm Ipswich, MA).

Techniques: Staining, Concentration Assay, Ligation, Fluorescence, Microscopy

Journal: eLife

Article Title: Ribozyme activity modulates the physical properties of RNA–peptide coacervates

doi: 10.7554/eLife.83543

Figure Lengend Snippet: The concatenation activity of the ribozyme was determined by reaction at 30, 37, or 45°C and with either equimolar (10.5 µM), twofold (8 µM ribozyme and 16 µM substrate) or fourfold (5 µM ribozyme and 20 µM substrate) substrate concentration relative to the ribozyme. The reaction buffer contained 10 mM MgCl 2 and 50 mM Tris-HCl pH 8.6, and the reaction was stopped after 2 hr. Excess substrate was found to inhibit the formation of long substrate concatenates at lower temperatures. Figure 1—figure supplement 1—source data 1. Unedited and uncropped gel image for , and labelled image showing key bands and conditions.

Article Snippet: Trizma base (Tris; Thermo Fisher Scientific, Waltham, MA), sodium hexametaphosphate ((NaPO 3 ) 6 , 611.77 g/mol; Sigma-Aldrich, St. Louis, MI), formamide (CH 3 NO, 45.04 g/mol; Sigma-Aldrich), ethylenediaminetetracetic acid disodium salt dihydrate (EDTA, C 10 H 14 N 2 Na 2 O 8 ·2H 2 O, 372.24 g/mol; Sigma-Aldrich), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O, 203.30 g/mol; (Merck, Darmstadt, Germany)), sodium hydroxide (NaOH, 39.997 g/mol; VWR, Radnor, PA), sodium chloride (NaCl, 58.44 g/mol; Sigma-Aldrich), urea (CH 4 N 2 O, 60.06 g/mol; Carl Roth, Karlsruhe, Germany), hydrochloric acid (HCl, 37%, 36.46 g/mol) (VWR), boric acid (H 3 BO 3 , 61.83 g/mol; Merck), ammonium persulfate (APS, (NH 4 ) 2 S 2 O 8 , 228.20 g/mol) (VWR), acrylamide (19:1 bisacrylamide; Thermo Fisher Scientific), tetramethylethylendiamine (TEMED, C 6 H 16 N 2 , 116.21 g/mol; Carl Roth), SYBR gold stain (Thermo Fisher Scientific), RNA oligomer length standard (low-range ssRNA ladder) (NEBm Ipswich, MA).

Techniques: Activity Assay, Concentration Assay

Journal: eLife

Article Title: Ribozyme activity modulates the physical properties of RNA–peptide coacervates

doi: 10.7554/eLife.83543

Figure Lengend Snippet: ( a ) A representative 8% urea PAGE gel stained with SYBR gold showing the products of the R3C ladder system in solution and varying ratios of (Lys) 5-24 to R3C RNA (total monomer concentration = 1 mM) after a 2 hr reaction at 30°C in 50 mM Tris-HCl pH 8.6 and 10 mM MgCl 2 . The integrated lane profiles of the solution and 1.5:1 (Lys) 5-24 :RNA conditions are shown in blue and red, respectively. ( b ) Variation in absorbance at 500 nm as a proxy for coacervate formation upon addition of varying ratios of (Lys) 5-24 to the E L RNA after ligation for 3 hr at 30°C. Data points are an average of n = 3 independent replicates assembled from the same stock solutions with error bars reporting standard deviations. ( c ) Example fluorescence microscopy image of (Lys) 5-24 :RNA condensates at a ratio of 3:1 (Lys) 5-24 :RNA, imaged using 10% Cy5-tagged substrate strand. Scale bar = 20 μm. ( d ) Kinetics of chain elongation in solution (blue, first-order model), and with 3:1 (Lys) 5-24 :RNA (red, second-order model) at 30°C, pH 8.6, and 10 mM MgCl 2 . Data points are an average of n = 3 independent replicates assembled from the same stock solutions. Error bars represent standard deviations ( e ) Chain extension rate constants for the R3C ladder ribozyme in solution (blue, first-order model) and in the presence of 3:1 (Lys) 5-24 :RNA (red, second-order model). Error bars are the standard errors for each parameter computed during non-linear regression. Poor sample recovery led to an artificially reduced average substrate length at the t = 30 and t = 60 min time points for the reaction in the presence of (Lys) 5-24 . These points were therefore excluded when fitting data. Figure 1—figure supplement 2—source data 1. Unedited and uncropped gel image for , and labelled image showing key bands and conditions. Content identical to . Figure 1—figure supplement 2—source data 2. Numerical turbidity data. Content identical to . Figure 1—figure supplement 2—source data 3. Unprocessed and uncropped fluorescence microscope image for 3:1 (Lys) 5-24 :RNA condensates imaged using 10% Cy5-tagged substrate strand. Figure 1—figure supplement 2—source data 4. Unedited and uncropped gel images for ribozyme kinetics . Lane identities are listed in the included spreadsheet. Content identical to .

Article Snippet: Trizma base (Tris; Thermo Fisher Scientific, Waltham, MA), sodium hexametaphosphate ((NaPO 3 ) 6 , 611.77 g/mol; Sigma-Aldrich, St. Louis, MI), formamide (CH 3 NO, 45.04 g/mol; Sigma-Aldrich), ethylenediaminetetracetic acid disodium salt dihydrate (EDTA, C 10 H 14 N 2 Na 2 O 8 ·2H 2 O, 372.24 g/mol; Sigma-Aldrich), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O, 203.30 g/mol; (Merck, Darmstadt, Germany)), sodium hydroxide (NaOH, 39.997 g/mol; VWR, Radnor, PA), sodium chloride (NaCl, 58.44 g/mol; Sigma-Aldrich), urea (CH 4 N 2 O, 60.06 g/mol; Carl Roth, Karlsruhe, Germany), hydrochloric acid (HCl, 37%, 36.46 g/mol) (VWR), boric acid (H 3 BO 3 , 61.83 g/mol; Merck), ammonium persulfate (APS, (NH 4 ) 2 S 2 O 8 , 228.20 g/mol) (VWR), acrylamide (19:1 bisacrylamide; Thermo Fisher Scientific), tetramethylethylendiamine (TEMED, C 6 H 16 N 2 , 116.21 g/mol; Carl Roth), SYBR gold stain (Thermo Fisher Scientific), RNA oligomer length standard (low-range ssRNA ladder) (NEBm Ipswich, MA).

Techniques: Staining, Concentration Assay, Ligation, Fluorescence, Microscopy

Journal: eLife

Article Title: Ribozyme activity modulates the physical properties of RNA–peptide coacervates

doi: 10.7554/eLife.83543

Figure Lengend Snippet: Experiments were performed in solution at 30°C in 50 mM Tris-HCl pH 8.6, 10 mM MgCl 2 , and with the addition of either 1.5:1 (Lys) 5-24 :RNA or 0.75:1 (Lys) 19-72 :RNA. The reaction run using 10% Cy5-tagged substrate strand, and the reaction product were visualised on an 8% urea PAGE. The displayed gels correspond to the kinetic plots shown in and . Figure 1—figure supplement 3—source data 1. Source data contains unedited and uncropped gel images used to estimate yields, as well as labelled images showing key bands and conditions and is identical to .

Article Snippet: Trizma base (Tris; Thermo Fisher Scientific, Waltham, MA), sodium hexametaphosphate ((NaPO 3 ) 6 , 611.77 g/mol; Sigma-Aldrich, St. Louis, MI), formamide (CH 3 NO, 45.04 g/mol; Sigma-Aldrich), ethylenediaminetetracetic acid disodium salt dihydrate (EDTA, C 10 H 14 N 2 Na 2 O 8 ·2H 2 O, 372.24 g/mol; Sigma-Aldrich), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O, 203.30 g/mol; (Merck, Darmstadt, Germany)), sodium hydroxide (NaOH, 39.997 g/mol; VWR, Radnor, PA), sodium chloride (NaCl, 58.44 g/mol; Sigma-Aldrich), urea (CH 4 N 2 O, 60.06 g/mol; Carl Roth, Karlsruhe, Germany), hydrochloric acid (HCl, 37%, 36.46 g/mol) (VWR), boric acid (H 3 BO 3 , 61.83 g/mol; Merck), ammonium persulfate (APS, (NH 4 ) 2 S 2 O 8 , 228.20 g/mol) (VWR), acrylamide (19:1 bisacrylamide; Thermo Fisher Scientific), tetramethylethylendiamine (TEMED, C 6 H 16 N 2 , 116.21 g/mol; Carl Roth), SYBR gold stain (Thermo Fisher Scientific), RNA oligomer length standard (low-range ssRNA ladder) (NEBm Ipswich, MA).

Techniques:

Journal: eLife

Article Title: Ribozyme activity modulates the physical properties of RNA–peptide coacervates

doi: 10.7554/eLife.83543

Figure Lengend Snippet: Ribozyme assays were performed at 45 °C in solution and in the presence of poly(L-lysine) (0.75:1 Lys 19-72 :RNA or 3:1 Lys 5-24 :RNA). The ribozyme reaction buffer contained 10 mM MgCl 2 and 50 mM Tris-HCl pH 8.6. The reaction was stopped after 3 hr and the extracted RNA was digested with RNase R, leaving only circular products. Circular bands are marked with an asterisk. The formation of circular products is observed in solution but is suppressed in the presence of poly(L-lysine). Figure 1—figure supplement 5—source data 1. Unedited and uncropped gel image, and labelled image showing key bands and conditions.

Article Snippet: Trizma base (Tris; Thermo Fisher Scientific, Waltham, MA), sodium hexametaphosphate ((NaPO 3 ) 6 , 611.77 g/mol; Sigma-Aldrich, St. Louis, MI), formamide (CH 3 NO, 45.04 g/mol; Sigma-Aldrich), ethylenediaminetetracetic acid disodium salt dihydrate (EDTA, C 10 H 14 N 2 Na 2 O 8 ·2H 2 O, 372.24 g/mol; Sigma-Aldrich), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O, 203.30 g/mol; (Merck, Darmstadt, Germany)), sodium hydroxide (NaOH, 39.997 g/mol; VWR, Radnor, PA), sodium chloride (NaCl, 58.44 g/mol; Sigma-Aldrich), urea (CH 4 N 2 O, 60.06 g/mol; Carl Roth, Karlsruhe, Germany), hydrochloric acid (HCl, 37%, 36.46 g/mol) (VWR), boric acid (H 3 BO 3 , 61.83 g/mol; Merck), ammonium persulfate (APS, (NH 4 ) 2 S 2 O 8 , 228.20 g/mol) (VWR), acrylamide (19:1 bisacrylamide; Thermo Fisher Scientific), tetramethylethylendiamine (TEMED, C 6 H 16 N 2 , 116.21 g/mol; Carl Roth), SYBR gold stain (Thermo Fisher Scientific), RNA oligomer length standard (low-range ssRNA ladder) (NEBm Ipswich, MA).

Techniques:

Journal: eLife

Article Title: Ribozyme activity modulates the physical properties of RNA–peptide coacervates

doi: 10.7554/eLife.83543

Figure Lengend Snippet: The activity of the active and inactive ligase ribozyme variants was tested in solution and in the presence of poly(L-lysine) (0.75:1 Lys 19-72 :RNA or 3:1 Lys 5-24 :RNA). The ribozyme reaction buffer contained 10 mM MgCl 2 and 50 mM Tris-HCl pH 8.6. The reaction was stopped after 2 hr. No ligation activity was detected in the presence of the inactive ribozyme in all conditions tested. Figure 2—figure supplement 1—source data 1. Unedited and uncropped gel image, and labelled image showing key bands and conditions.

Article Snippet: Trizma base (Tris; Thermo Fisher Scientific, Waltham, MA), sodium hexametaphosphate ((NaPO 3 ) 6 , 611.77 g/mol; Sigma-Aldrich, St. Louis, MI), formamide (CH 3 NO, 45.04 g/mol; Sigma-Aldrich), ethylenediaminetetracetic acid disodium salt dihydrate (EDTA, C 10 H 14 N 2 Na 2 O 8 ·2H 2 O, 372.24 g/mol; Sigma-Aldrich), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O, 203.30 g/mol; (Merck, Darmstadt, Germany)), sodium hydroxide (NaOH, 39.997 g/mol; VWR, Radnor, PA), sodium chloride (NaCl, 58.44 g/mol; Sigma-Aldrich), urea (CH 4 N 2 O, 60.06 g/mol; Carl Roth, Karlsruhe, Germany), hydrochloric acid (HCl, 37%, 36.46 g/mol) (VWR), boric acid (H 3 BO 3 , 61.83 g/mol; Merck), ammonium persulfate (APS, (NH 4 ) 2 S 2 O 8 , 228.20 g/mol) (VWR), acrylamide (19:1 bisacrylamide; Thermo Fisher Scientific), tetramethylethylendiamine (TEMED, C 6 H 16 N 2 , 116.21 g/mol; Carl Roth), SYBR gold stain (Thermo Fisher Scientific), RNA oligomer length standard (low-range ssRNA ladder) (NEBm Ipswich, MA).

Techniques: Activity Assay, Ligation

Journal: Redox Biology

Article Title: Specific delivery of delta-5-desaturase siRNA via RNA nanoparticles supplemented with dihomo-γ-linolenic acid for colon cancer suppression

doi: 10.1016/j.redox.2018.101085

Figure Lengend Snippet: Construction of tumor-targeting 3WJ nanoparticle. (A) Illustration for the design of a functional 3WJ RNA nanoparticle. (B) Dynamic light scattering characterization showing the hydrodynamic size of RNA nanoparticles. (C) Atomic force microscopy image of extended 3WJ-nanoparticles. (D) Cell-binding test via flow cytometry in HCA-7/C29 cells after being incubated with 100 nM 3WJ-Alexa 647 or 3WJ-EpCAM-Alexa 647 (in 100 µL PBS). Cells incubated with PBS only (without nanoparticles) served as controls. (E) Confocal microscopy analysis of 3WJ-EpCAM-Alexa 647 particles entering HCA-7/C29 cells. Cell skeletons were stained with Alexa 488 and cell nuclei with DAPI. (F) qRT-PCR analysis for D5D gene knockdown efficiency in HCA-7/C29 cells after treatment with 10 nM of 3WJ nanoparticles with different compositions. Data represent mean ± SD with three separate experiments. *: significant difference with p < 0.05.

Article Snippet: The apparent hydrodynamic sizes of the assembled RNA nanoparticles (40 μM in 1 ×Tris buffer) were measured by a Zetasizer nano-ZS (Malvern Instrument, LTD) at 25 °C.

Techniques: Functional Assay, Microscopy, Binding Assay, Flow Cytometry, Incubation, Confocal Microscopy, Staining, Quantitative RT-PCR, Knockdown

Journal: Redox Biology

Article Title: Specific delivery of delta-5-desaturase siRNA via RNA nanoparticles supplemented with dihomo-γ-linolenic acid for colon cancer suppression

doi: 10.1016/j.redox.2018.101085

Figure Lengend Snippet: 3WJ nanoparticles and DGLA supplementation suppress xenograft tumor growth. (A) In vivo biodistribution analysis of 3WJ-EpCAM-Alexa 647 particles in organs and tumor tissues from mice bearing HCA-7 tumors at 8-h post-injection. (B) Ultrasound images of tumor growth in mice before and after 4-week treatments. Note, week 0 indicates the starting point of treatment, which is two weeks after cancer cell implantation. (C) Relative tumor volume measured twice a week using a digital caliper during the treatment. Data represent mean±SD with six tumor samples. *: significant difference with p < 0.05. Insert: photos of tumor tissues at the end of treatment.

Article Snippet: The apparent hydrodynamic sizes of the assembled RNA nanoparticles (40 μM in 1 ×Tris buffer) were measured by a Zetasizer nano-ZS (Malvern Instrument, LTD) at 25 °C.

Techniques: In Vivo, Injection

Journal: Redox Biology

Article Title: Specific delivery of delta-5-desaturase siRNA via RNA nanoparticles supplemented with dihomo-γ-linolenic acid for colon cancer suppression

doi: 10.1016/j.redox.2018.101085

Figure Lengend Snippet: 3WJ nanoparticles and DGLA supplementation promote 8-HOA formation in xenograft tumors. (A) Immunofluorescence analysis of D5D expression levels in tumor tissues. (B) LC/MS quantification of DGLA levels from tumor tissues. (C) LC/MS quantification of AA levels from tumor tissues. (D) DGLA/AA ratio from tumor tissues. (E) GC/MS quantification of 8-HOA from tumor tissues. All the quantification data represent mean±SD with six tumor samples. *: significant difference with p < 0.05.

Article Snippet: The apparent hydrodynamic sizes of the assembled RNA nanoparticles (40 μM in 1 ×Tris buffer) were measured by a Zetasizer nano-ZS (Malvern Instrument, LTD) at 25 °C.

Techniques: Immunofluorescence, Expressing, Liquid Chromatography with Mass Spectroscopy, Gas Chromatography-Mass Spectrometry

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 1. Nucleolar SIRT7 participates in multiple pathways in ribosomal biogenesis. A, B, HeLa cells treated with RNase A or buffer alone (Ctrl). Localizations of endogenous MYBBP1A, SIRT7, and RPA194 were detected via immunofluorescence microscopy. C, SIRT7 interacts with proteins involved in ribosome biogenesis. Rela- tive abundances of SIRT7-interacting proteins specific to ribosome biogenesis are indicated by the logarithm of the ratio of NSAF/PAX values. Node color and size correlate with NSAF/PAX ratios. Protein networks were assembled by STRING and visualized by Cytoscape. D, E, representative collision-induced dissociation MS/MS spectra of doubly charged tryptic peptides from mTOR (D) and GTF3C1 (E). Assigned b and y ion sequence fragments and prominent neutral losses from the precursor are labeled. BPI, base peak intensity.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Immunofluorescence, Microscopy, Tandem Mass Spectroscopy, Sequencing, Labeling

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 2. SIRT7 knockdown negatively regulates synthesis rate of rRNA. A, transcription inhibition in mitosis phase. Fluorescence mi- croscopy shows EU incorporation (EU-Alexan 488) into RNA 1 h after pulse-labeling. Cells undergoing mitosis exhibit chromosome con- densation (arrows). B, protein levels monitored via Western blotting following siRNA treatment (2 days). C, reduced synthesis rates of nascent RNA upon SIRT7 knockdown. Top: selected fluorescent images from EU-labeled HeLa cells treated with siRNA for 2 days. Relative EU levels, shown in parentheses, were calculated by normal- ization to the mean fluorescence value of control siRNA-treated cells. “Cell #” indicates the number of cells measured for each experiment. Bottom: normalized total cell fluorescence output from cells treated with siRNA targeting SIRT7. Mean fluorescence value (m) and stand- ard deviations (S.D.) for each condition are as follows: ctrl, m 69k, S.D. 44k; SIRT7, m 34k, S.D. 21k. p values were calculated using Student’s t test. Images were acquired using a 60 oil immer- sion lens; bar, 10 m. D, EU fluorescence distribution following pulse- labeling in cells treated with siRNA (si), with or without RNaseA, as analyzed via flow cytometry. E, pulse-and-chase experiment for measuring RNA stability. HeLa cells were treated with control or SIRT7 siRNA for 2 days and then EU labeled for 1.5 h. Then the cells were chased with different time points.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Knockdown, Inhibition, Fluorescence, Labeling, Western Blot, Control, Flow Cytometry

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 3. SIRT7 associates with ribosome units and regulates protein synthesis rate. A, co-localization of SIRT7-EGFP and RPL11 within nucleoli. A cell line stably expressing SIRT7-EGFP fusion pro- tein was transfected with RPL11 cDNA and monitored via immuno- fluorescence microscopy with a 60 oil immersion lens; bar, 10 m. B, co-fractionation of RPL5, RPL11, and SIRT7. Cells were treated with control and SIRT7 siRNA for 2 days. The distributions of ribo- some proteins and SIRT7 were analyzed by sucrose gradient. Pro- teins from individual fractions were precipitated and analyzed via Western blotting. C, SIRT7 knockdown inhibits protein synthesis. Protein synthesis rates after siRNA treatment for 3 days were moni- tored via pulse L-homopropargylglycine labeling followed by Click-iT reaction with a fluorescent dye.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Stable Transfection, Expressing, Transfection, Fluorescence, Microscopy, Fractionation, Control, Western Blot, Knockdown, Labeling

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 4. SIRT7 knockdown preferentially suppresses protein syn- thesis rates and inhibits proliferation. A, preferential inhibition of protein synthesis by SIRT7. Top: comparison of RNA and protein synthesis rates following siRNA treatment for 2 days. Bottom: knock- down efficiencies of individual siRNA were monitored via Western blotting. B, HeLa cells were transfected with SIRT7 siRNA and differ- ent plasmids (GFP, wild type, 111) for 2 days. The protein synthesis rates were measured using an HPG labeling method (top panel). The levels of SIRT7 were confirmed by Western blotting (bottom panel). WT: SIRT7 wild type; S111A: SIRT7 deacetylation-impacted mutant. GFP served as a control for overexpression. C, after siRNA treatment for 2 days, HeLa cells were treated with nucleotide releasing buffer for the detection of ATP levels. ADP levels were sequentially measured by adding ADP converting enzyme. The ratio of ADP to ATP in each condition was calculated. Camptothecin (CPT) was used as a control. D, E, cell proliferation rates following siRNA treatments for 2 and 3 days, respectively. p values were calculated using Student’s t test.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Knockdown, Inhibition, Comparison, Western Blot, Transfection, Labeling, Mutagenesis, Control, Over Expression

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 5. SIRT7 regulates the functions of mTOR and Pol III com- plexes. A, validation of SIRT7 interaction by reciprocal immunopre- cipitation using antibodies against GTF3C1 and mTOR. B, C, after SIRT7 siRNA treatment for 2 days, GTF3C1 and mTOR were immu- nopurified and analyzed via Western blotting using anti-acetyl-Lys antibody. D, HeLa cells were treated with SIRT7 siRNA for 2 days. The nucleus and nucleolus were enriched and analyzed via Western blot- ting using antibody specific to acetyl-lysine (Acetyl-K). E, SIRT7 knockdown affects mTOR pathway. Phosphorylated S6 (top panel) and LC3B protein (bottom panel) abundances were monitored via Western blotting in cells treated with siRNA. Numbers represent S6-P/S6 total (top panel) and LC3B/tubulin (bottom panel) ratios. F, STRING network of protein–protein interactions between RNA Pol III subunits and TFIIIC2 complex members. Colored nodes, reflecting relative abundance (NSAF, %), represent proteins co-isolated with SIRT7-EGFP from purified nuclei. “White nodes” added by STRING were colored gray, depicting the connectivity between RNA Pol III and

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Biomarker Discovery, Western Blot, Knockdown, Protein-Protein interactions, Isolation, Purification

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 6. Model for potential roles of SIRT7 in ribosome biogen- esis. Role of SIRT7 in Pol I, Pol III, and mTOR. SIRT7 majorly localizes within nucleoli and forms multiple complexes with ribosomes, mTOR, and TFIIIC2. Both mTOR and SIRT7 can interact with TFIIIC2 to regulate Pol III transcription. The SIRT7–mTOR complex may regulate an autophagy pathway that affects cell growth and proliferation.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Calcium-sensing Receptor Modulates Cell Adhesion and Migration via Integrins

doi: 10.1074/jbc.m111.265454

Figure Lengend Snippet: FIGURE 2. Cellular localization and cell surface expression of the CaSR and 1 integrin in rMTC 44-2 cells. A, cells grown in subthreshold (1.8 mM) Ca2 were immunolabeled with extracellular epitope mouse ADD anti-CaSR antibody (1:500) and a rabbit anti-1 integrin antibody (1:400) and were viewed using a confocal microscope (100). CaSR expression was present in discrete puncta distributed throughout the cell (green). 1 integrin was also expressed throughout the cell (red) and showed extensive overlap with the CaSR expression (yellow). Photomicrographs are representative of similar results obtained in three experiments. B, cells were preincubated with 0.5 mM Ca2 for 5 h prior to addition of buffer (control) or 10 mM Ca2 for 10 min, and cell surface proteins were covalently biotinylated and subjected to streptavidin-agarose chromatography. Cytoplasmic proteins appeared in the Flow Through fraction, whereas cell surface proteins were eluted in the Eluate. The CaSR, 1 integrin, and 3 subunits of the Na/K-ATPase (plasma membrane marker) were found in the cell surface fraction, whereas IP3 receptor (intracellular membrane marker) was found in the intracellular fractions. The CaSR immunoblot analysis (using the ADD monoclonal antibody) showed that the 130-kDa mature form of the CaSR was recovered primarily in the cell surface fraction as expected, whereas the 120-kDa immature form was recovered in the “flow-through” (n 3). C, band intensities present in the eluted fractions of the CaSR, 1 integrin, and 3 subunit blots were analyzed and are presented as a percent of control (n 3).

Article Snippet: After 24 h in culture, cells were equilibrated with Ca2 assay buffer (20 mM HEPES, 146 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2, 1 mg/ml bovine serum albumin, and 1 mg/ml glucose (pH 7.4)), loaded with 6 M Ca2 -sensitive fluorescent dye Fluo-4 AM (Invitrogen), and incubated in the dark for 1 h at room temperature (24, 25).

Techniques: Expressing, Immunolabeling, Microscopy, Control, Chromatography, Clinical Proteomics, Membrane, Marker, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Calcium-sensing Receptor Modulates Cell Adhesion and Migration via Integrins

doi: 10.1074/jbc.m111.265454

Figure Lengend Snippet: FIGURE 7. Effects of intracellular signaling inhibitors on Ca2 release, ERK activation, and CaSR-mediated cell adhesion and migration. A, effects of the PLC inhibitor U73122 and the inactive isomer U73343 on CaSR-induced increase in [Ca2]i. B, effects of U73122 on NPS R-568 potentiation of cell adhesion. C, effects of U73122 on cell migration. D, BAPTA inhibition of [Ca2]i release. E, effects of BAPTA on NPS R-568 enhancement of cell adhesion. F, BAPTA inhibition of the NPS R-568-mediated potentiation of cell migration. G, effects of the ERK inhibitor PD 98059 on ERK activation. H, effects of PD 98059 on cell adhesion. I, PD 98059 enhancement of NPS R-568 potentiation of cell migration. J, ROCK inhibitor Y-27632 had no effect on cell adhesion. K, effects of Y-27632 on cell migration. Each bar represents the mean S.E. of 3–5 independent experiments. *, p 0.05.

Article Snippet: After 24 h in culture, cells were equilibrated with Ca2 assay buffer (20 mM HEPES, 146 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2, 1 mg/ml bovine serum albumin, and 1 mg/ml glucose (pH 7.4)), loaded with 6 M Ca2 -sensitive fluorescent dye Fluo-4 AM (Invitrogen), and incubated in the dark for 1 h at room temperature (24, 25).

Techniques: Activation Assay, Migration, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Calcium-sensing Receptor Modulates Cell Adhesion and Migration via Integrins

doi: 10.1074/jbc.m111.265454

Figure Lengend Snippet: FIGURE 8. Proposed mechanistic scheme for CaSR-integrin interactions. The figure depicts one possible scenario where the CaSR may be associated with integrin heterodimers in a signaling complex. However, the actual protein-protein interaction may be indirect and involve additional proteins. Left, complex prior to CaSR stimulation; right, elevated extracellular cation concentrations activate the CaSR causing its Venus flytrap domain to undergo a conformational change. This protein domain movement together with increased [Ca2]i from Gq activation of PLC and the IP3 receptor could induce the integrin to flip it into anactivatedstatepromotingbindingtotheECMresultinginenhancedcelladhesionandmigration.TheXintherightpaneldenotesahypotheticalproteinthat may participate in facilitating the CaSR-mediated activation of integrins subsequent to increased [Ca2]i.

Article Snippet: After 24 h in culture, cells were equilibrated with Ca2 assay buffer (20 mM HEPES, 146 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2, 1 mg/ml bovine serum albumin, and 1 mg/ml glucose (pH 7.4)), loaded with 6 M Ca2 -sensitive fluorescent dye Fluo-4 AM (Invitrogen), and incubated in the dark for 1 h at room temperature (24, 25).

Techniques: Activation Assay

Journal: Nature cell biology

Article Title: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

doi: 10.1038/ncb3241

Figure Lengend Snippet: (a) Scheme of the human TPX2 constructs used in this study: full-length TPX2, N-terminally truncated TPX2 containing amino acids 274 - 747 (TPX2 ΔN ), and a minimal TPX2 construct containing amino acids 274 - 659 (TPX2 mini ). Regions known to interact with Aurora A (AurA), importin α (Imp α) are indicated, together with predicted coil coils (CC) and nuclear localisation signal (NLS). (b) Coomassie Blue-stained SDS-PAGE gel showing 1 μg of purified recombinant TPX2 constructs. (c, d) Single channel and merged TIRF microscopy images showing how mGFP-TPX2 (green in merge) at 5 nM (c) and 0.35 nM (d) binds either all along (c) or preferentially to the growing ends and the GMPCPP segment (d) of a growing Atto647N-labelled microtubule (magenta in merge) (“−“ and “+” indicate end binding, the GMPCPP “seed” is marked by an arrowhead). (e) Kymographs depicting the time course of binding of 5 nM mGFP-TPX2 all along a growing microtubule. Att647N-labelled tubulin concentration was 7.5 μM. (f) Kymographs depicting the time course of 0.35 nM mGFP-TPX2 binding to a growing microtubule end and the GMPCPP “seed”. Att647N-labelled tubulin concentration was 12.5 μM. (g, h) Kymographs showing binding of (g) 10 nM mGFP-TPX2 ΔN and (h) 33 nM mGFP-TPX2 mini to dynamic microtubules (merged channels on the left, mGFP-TPX2 on the right). Atto647N-labelled tubulin concentration is 12.5 and 15 μM, respectively. (i, j) Kymographs showing that neither (i) 1 nM full-length mGFP-TPX2 nor (j) 10 nM TPX2 mini binds to shrinking microtubules. Atto647N-labelled tubulin concentrations were 5 μM and 7.5 μM, respectively. For all kymograph pairs: merged channel - left, mGFP-TPX2 channel - right. Scale bars as indicated.

Article Snippet: Peak fractions were pooled and subjected to size exclusion chromatography using Superose 6 10/300 GL column (GE Healthcare) in TPX2 storage buffer (50 mM HEPES, 300 mM KCl, 2 mM MgCl 2 , 250 mM sucrose, 50 mM arginine, 50 mM glutamate, 5 mM 2-ME, pH 7.5).

Techniques: Construct, Staining, SDS Page, Purification, Recombinant, Microscopy, Binding Assay, Concentration Assay

Journal: Nature cell biology

Article Title: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

doi: 10.1038/ncb3241

Figure Lengend Snippet: (a) Modified box-and-whiskers graph for the microtubule growth speeds in the absence (control) and presence of different concentrations of full-length mGFP-TPX2 and mGFP-TPX2 mini , as indicated. Number of observed microtubule growth episodes per condition: control – n=150; mGFP-TPX2: 0.2 nM – n= 160, 1 nM – n=150, 5 nM – n=114; mGFP-TPX2 mini : 10 nM – n=153, 50 nM – n=121, 250 nM – n=154. All events are from one dataset each. (b) TIRF microscopy kymographs showing dynamic Atto647N-labelled microtubules in the absence (control) or presence of 5 nM mGFP-TPX2 or 250 nM mGFP-TPX2 mini . Scale bars as indicated. Bar graphs showing microtubule (c) catastrophe and (d) rescue frequencies, and box-and-whiskers graphs showing microtubule (e) growth and (f) depolymerisation speeds, for control, 5 nM mGFP-TPX2, and 250 nM mGFP-TPX2 mini , as indicated. Box-and-whiskers graphs show (g) microtubule lifetimes, (h) depolymerisation times and (i) depolymerisation lengths for the same conditions. Data for (c-i) were pooled from three datasets each. Total number of analysed events per condition: (c) catastrophes (total growth time in brackets): control – n=757 (457,280 s), mGFP-TPX2 –n= 209 (267,644 s), mGFP-TPX2 mini – n=385 (409,675 s); (d) rescues (total depolymerisation time in brackets): control – n=612 (22,062 s), mGFP-TPX2 – n=209 (10,708 s), mGFP-TPX2 mini – n=376 (13,130 s); (e) growth episodes: control – n=863, 5 nM mGFP-TPX2 – n=271, 250 nM mGFP-TPX2 mini –n=477; (f) depolymerisation episodes: control – n=756, 5 nM, mGFP-TPX2 – n=211, 250 nM mGFP-TPX2 mini –n=383. The same data were used in (g)-(i) (see ). Atto647N-labelled tubulin concentration was always 7.5 μM. Errors in bar graphs are SEM. For the modified box-and-whiskers plots the boxes range from 25 th to 75 th percentile, the whiskers span from 10 th to 90 th percentile, the horizontal line marks the mean value. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 (only displayed for comparisons with control); determined for the comparison of mean values analysing raw data (Tukey’s test in conjunction with One Way ANOVA).

Article Snippet: Peak fractions were pooled and subjected to size exclusion chromatography using Superose 6 10/300 GL column (GE Healthcare) in TPX2 storage buffer (50 mM HEPES, 300 mM KCl, 2 mM MgCl 2 , 250 mM sucrose, 50 mM arginine, 50 mM glutamate, 5 mM 2-ME, pH 7.5).

Techniques: Modification, Microscopy, Concentration Assay

Journal: Nature cell biology

Article Title: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

doi: 10.1038/ncb3241

Figure Lengend Snippet: (a) Right: Images of 33 nM mGFP-TPX2 mini (green in merge) at growing Atto647N-labelled microtubule ends (magenta in merge) at the indicated tubulin concentrations. Left: Averaged mGFP-TPX2 mini intensity profiles at the end regions of microtubules growing in the presence of 33 nM mGFP-TPX2 mini and the indicated Atto647N-labelled tubulin concentrations. (b) Averaged mGFP-TPX2 mini intensity profiles at the end regions of microtubules growing in the presence of 33 nM mGFP-TPX2 mini and 20 μM Atto647N-labelled tubulin for three different time windows after start of growth. (c) Averaged mGFP-TPX2 mini intensity profiles for the indicated mGFP-TPX2 mini concentrations at 25 μM Atto647N-labelled tubulin. (d) Cartoons depicting the assay conditions and TIRF microscopy kymographs showing single molecule binding events of 5 nM mGFP-TPX2 mini (green in merge) to the ends of growing Atto565-labelled microtubules (blue in merge) in the absence or presence of 195 nM Alexa647-labelled SNAP-TPX2 mini (magenta in merge). (e) Dissociation rate constant k off and association rate r on , respectively, as determined for the conditions in (d) from the dwell and waiting time distributions of the single mGFP-TPX2 mini molecule binding and unbinding events ( , ) as a function of the distance from the growing microtubule plus end. Note that the increase in k off values and decrease in r on values is less steep in the presence of excess Alexa647-labelled SNAP-TPX2 mini , agreeing with (4c). Lower panel: The normalised total fluorescence intensity of all mGFP-TPX2 mini binding events shows their steady state distribution. (f) Kymographs and instantaneous intensity line scans at different times, as indicated, showing strong temporal fluctuations of the fluorescence intensity of mGFP-TPX2 mini (25 nM, green in merge) at growing Atto647N-labelled microtubule ends (magenta in merge, 12.5 μM tubulin). (g) TIRF microscopy images showing 33 nM mGFP-TPX2 mini (green in merge, lower panels) accumulating strongly at the curved microtubule ends of Atto647N-labelled growing microtubules (magenta in merge, 30 μM tubulin). (h) Kymographs of 5 nM mGFP-TPX2 (green in merge) localising to the ends of Atto647N-labelled microtubules (magenta in merge, 25 μM tubulin) growing in the presence of the slowly-hydrolysable GTP analogue GTPγS. Scale bars as indicated.

Article Snippet: Peak fractions were pooled and subjected to size exclusion chromatography using Superose 6 10/300 GL column (GE Healthcare) in TPX2 storage buffer (50 mM HEPES, 300 mM KCl, 2 mM MgCl 2 , 250 mM sucrose, 50 mM arginine, 50 mM glutamate, 5 mM 2-ME, pH 7.5).

Techniques: Microscopy, Binding Assay, Fluorescence

Journal: Nature cell biology

Article Title: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

doi: 10.1038/ncb3241

Figure Lengend Snippet: (a) Scheme of the experimental setup for the ‘surface’ nucleation assay. (b) TIRF microscopy images from a time series showing Atto647N-labelled microtubules and/or tubulin particles on a glass surface with immobilised biotinylated Kin1 rigor mutant, chTOG or TPX2 after pre-incubation at the indicated concentrations. Insets on the right depict magnified images of the same surface at 7.5 min. (c) Time courses of the mean Atto647N-tubulin fluorescence intensities measured at the surface for the entire field of view for different chTOG, TPX2 and Kin1 rigor densities. Concentrations of biotinylated proteins used for surface incubation as indicated. The same Kin1 rigor curve is shown as a control for both chTOG and TPX2 plots. Note that the fluorescence signal typically represents the sum of several different tubulin species: soluble tubulin in the TIRF field (creating part of the background), immobilised individual tubulins (e.g. bound to chTOG), tubulin ‘stubs’ (bound to TPX2) and microtubules (bound to chTOG or TPX2). Raw intensities including background are shown. (d) Time series of TIRF microscopy images showing nucleation and growth of Atto647N-labelled microtubules on surfaces with immobilised biotinylated TPX2 (pre-incubated at 125 nM, top three rows) or, for controls, with a biotinylated Kin1 rigor (pre-incubated at 125 nM, bottom two rows) in either the absence or presence of 100 nM untagged chTOG, as indicated. Because of the high microtubule density at the TPX2 surface in the presence of chTOG, the same time series is also shown with reduced contrast for better visualisation of individual microtubules. Atto647N-labelled tubulin concentration was always 12.5 μM. Scale bars as indicated. t = 0 when the sample is placed at 30°C.

Article Snippet: Peak fractions were pooled and subjected to size exclusion chromatography using Superose 6 10/300 GL column (GE Healthcare) in TPX2 storage buffer (50 mM HEPES, 300 mM KCl, 2 mM MgCl 2 , 250 mM sucrose, 50 mM arginine, 50 mM glutamate, 5 mM 2-ME, pH 7.5).

Techniques: Microscopy, Mutagenesis, Incubation, Fluorescence, Concentration Assay

Journal: Nature cell biology

Article Title: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

doi: 10.1038/ncb3241

Figure Lengend Snippet: (a) Scheme of the experimental setup for the ‘solution’ nucleation assay. (b) Time series of TIRF microscopy images showing Atto647N-labelled microtubules that nucleated in solution in the presence of nucleation factors as indicated, followed after 1 min by binding to a neutravidin-coated surface via the biotinylated protein present as indicated. (c) TIRF microscopy images showing examples the concentration dependent nucleation efficiencies of chTOG- and TPX2-mediated microtubule nucleation in the ‘solution’ nucleation assay. (d) Time courses of mean Atto647N-tubulin fluorescence intensities measured for the entire field of view in ‘solution’ at the biotinylated chTOG and TPX2 concentrations as indicated. Note that the fluorescence signal typically represents the sum of several different tubulin species (see note in legend of ). Raw intensities including background are shown. Atto647N-labelled tubulin concentration was always 12.5 μM. Other protein concentrations and scale bar as indicated. t = 0 when the sample is placed at 30°C.

Article Snippet: Peak fractions were pooled and subjected to size exclusion chromatography using Superose 6 10/300 GL column (GE Healthcare) in TPX2 storage buffer (50 mM HEPES, 300 mM KCl, 2 mM MgCl 2 , 250 mM sucrose, 50 mM arginine, 50 mM glutamate, 5 mM 2-ME, pH 7.5).

Techniques: Microscopy, Binding Assay, Concentration Assay, Fluorescence

Journal: Nature cell biology

Article Title: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

doi: 10.1038/ncb3241

Figure Lengend Snippet: (a) Times series of TIRF microscopy images of the ‘solution’ nucleation assay where microtubules that nucleated in solution in the presence of different concentrations of biotinylated TPX2 mini and in the additional presence of untagged chTOG, followed after 1 min by binding to a neutravidin-coated surface. (b) Time series of TIRF microscopy images of the ‘surface’ nucleation experiments with immobilised biotinylated TPX2 mini (pre-incubated at 500 nM) in the absence (top row) or presence (bottom row) of untagged chTOG. Atto647N-labelled tubulin concentration was always 12.5 μM. Other protein concentrations and scale bars as indicated. t = 0 when the sample is placed at 30°C.

Article Snippet: Peak fractions were pooled and subjected to size exclusion chromatography using Superose 6 10/300 GL column (GE Healthcare) in TPX2 storage buffer (50 mM HEPES, 300 mM KCl, 2 mM MgCl 2 , 250 mM sucrose, 50 mM arginine, 50 mM glutamate, 5 mM 2-ME, pH 7.5).

Techniques: Microscopy, Binding Assay, Incubation, Concentration Assay

Journal: Nature cell biology

Article Title: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

doi: 10.1038/ncb3241

Figure Lengend Snippet: (a) Time series of TIRF microscopy images showing microtubule nucleation and growth on surfaces with immobilised biotinylated rigor kinesin (pre-incubated at 125 nM) always in the presence of 100 nM chTOG and Atto647N-labelled tubulin, without and with additional 500 nM importin α/β complex (first and second row, respectively), 100 nM mGFP-TPX2 (third row) or both 500 nM importin α/β and 100 nM mGFP-TPX2 (fourth row) as indicated. Plots showing the time course of the mean Atto647N-labelled microtubule (magenta) and mGFP-TPX2 (green) intensities for the same experiments are shown next to each individual times series of images. Note that the mGFP-TPX2 signal declines at later time points likely due to depletion of TPX2 from solution due to binding to the efficiently nucleated growing microtubules. (b) Time series of TIRF microscopy images showing that compared to a control (left), inclusion of importin α/β (500 nM) inhibits stabilisation of Atto647N-labelled nucleation intermediates by surface-immobilised biotinylated TPX2 (pre-incubated at 125 nM) (right image pair). Plot showing the time course of the mean Atto647N-labelled tubulin intensities on the surface (right). Atto647N-labelled tubulin concentration was always 12.5 μM. Scale bars as indicated. t = 0 when the sample is placed at 30°C. (c) Model of synergistic TPX2 and chTOG-stimulated microtubule nucleation and growth. Different reactions, all promoting nucleation and growth, are regulated by the distinct and complementary activities of TPX2 and chTOG.

Article Snippet: Peak fractions were pooled and subjected to size exclusion chromatography using Superose 6 10/300 GL column (GE Healthcare) in TPX2 storage buffer (50 mM HEPES, 300 mM KCl, 2 mM MgCl 2 , 250 mM sucrose, 50 mM arginine, 50 mM glutamate, 5 mM 2-ME, pH 7.5).

Techniques: Microscopy, Incubation, Binding Assay, Concentration Assay

Journal: PLOS Biology

Article Title: GABAergic regulation of striatal spiny projection neurons depends upon their activity state

doi: 10.1371/journal.pbio.3002483

Figure Lengend Snippet: (A) The RiboTag construct AAV5-DOI-hSyn-RpI22I1-3Xflag-2A-eGFP was injected into the striatum of Adora2a-cre mice at P18 or at 6 months of age. (B) The coronal slice images demonstrate both the coverage and restriction to the striatum of the stereotaxically injected AAV carrying the RiboTag and eGFP genes (stereotaxic injection coordinates: ML = −1.85, AP = +0.74, DV = −3.50). Scale bars = 1 mm and 20 μm. Ten days later, the infected tissue (green fluorescence) was dissected out with the aid of fluorescence microscopy and qPCR was performed. (C) mRNA abundance (ΔCT) levels for the chloride cotransporters NKCC1 ( SLC12A1 ) and KCC2 ( SLC12A5 ) were determined by qPCR in striata from Adora2a-cre mice 4 weeks and 6 months of age. The data underlying the graphs shown in the figure can be found in dx.doi.org/10.5281/zenodo.10386854 . AAV, adeno-associated virus; qPCR, quantitative polymerase chain reaction; SPN, spiny projection neuron.

Article Snippet: TaqMan probes were used for PCR amplification of Hprt , Mm03024075_m1, Slc12a2 (NKCC1), Mm01265955_m1, Slc12a5 (KCC2), Mm00803929_m1, Slc4a3 (AE3) Mm00436654_g1, and Slc4a10 (NCBE) Mm00473827_m1.

Techniques: Construct, Injection, Infection, Fluorescence, Microscopy, Virus, Real-time Polymerase Chain Reaction

Journal: STAR Protocols

Article Title: In vivo regeneration of neo-nephrons in rodents by renal progenitor cell transplantation

doi: 10.1016/j.xpro.2021.100314

Figure Lengend Snippet:

Article Snippet: Slc12a1 (Rn00692576_m1) TaqMan Assay , Applied Biosystems , N/A.

Techniques: Recombinant, Blocking Assay, Gene Expression, TaqMan Assay, Software, Fluorescence, Microscopy