Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein binds to and enters the cell through STEAP2 (A) Comparison of the cell-binding capacity of SARS-CoV-2 wild type (WT) N protein and Omicron N protein expressed in either E. coli or mammalian cells. 1 × 10 5 A549 cells were used to mixed with 1 μg WT N or Omicron N proteins. One hour after protein addition, allophycocyanin (APC) conjugated anti-His antibody was used to detect the cell binding capacity of WT N protein or Omicron N protein. The samples were analyzed by flow cytometry and data are shown as mean fluorescence intensity (MFI). (B) Antibody blocking assay. Aliquots of 10 μg of SARS-CoV-2 N protein were pre-mixed with 0, 1, 3, 10, 30, and 100 μg of normal mouse IgG or anti-N monoclonal antibody (NP-mAb-40) and incubated at 4°C overnight. The antibody/N protein complex was used for the A549 cell surface binding assay. The blocking capacity of anti-N antibody was normalized to N protein only control. (C) Membrane fractions of A549 and HPAEpiC cells were extracted and incubated with N protein conjugated beads for 3 h binding at 4°C, and pull-downed for LC-MS-MS analysis (upper panels). A549 and HPAEpiC cells were suspended and treated with N protein for 1 h on ice. After incubation, cells were crosslinked with 3 mM DTSSP for 1.5 h. Then, cells were lysed in RIPA lysis buffer, and N protein complex in the lysate was immunoprecipitated for LC-MS-MS analysis (lower panels). Y axis denotes −logP values while the X axis shows log2 fold change values. Orange dots highlight the statistically significant proteins, with p value < 0.05 (-Log p > 1.3) and fold change>2, and the enriched plasma membrane protein was labeled on the plot. Identified proteins were further sorted by HuMemProtDB. (D) To knock-down (KD) STEAP2 expression, HPAEpiC cells were infected with lentivirus carrying STEAP2 shRNA followed by puromycin selection for 14 days. The STEAP2 mRNA expression levels were assessed by qRT-PCR, and the relative KD efficiency of shSTEAP2 was compared to shLacZ control (left-hand side panel). N protein binding capabilities to HPAEpiC STEAP2 KD cells and shLacZ control KD cells were assessed by flow cytometry analysis, and data were shown as mean fluorescence intensity (MFI). (right-hand side panel). (E) Western blot analysis of STEAP2 in wild type (WT) and knock-out (KO) A549 cells were shown. N protein binding to A549 STEAP2 KO cells was assessed by flow cytometry analysis and shown as mean fluorescence intensity (MFI). Ccr (crotonyl-CoAcarboxylase/reductase, a bacterial protein) binding was used as a control. (F) SARS-CoV-2 N protein enters alveolar cells. HPAEpiC cells were treated with 10 μg SARS-CoV-2 N protein overnight and then stained with anti-N antibody. The localization of N protein (Red) was checked by fluorescence microscope and cell morphology was observed by dimensional interference contrast (DIC). Nuclei of cells were stained by DAPI (blue). (G) N protein entering cells by endocytosis and N protein co-localization with STEAP2. HPAEpiC alveolar cells were seeded on 8 well slides. Cells were pretreated with endocytosis inhibitors HCQ, or Dynasore. Then the cells were treated with N protein overnight. After treatment, the cells were stained by specific antibodies to detected N protein (red), endosome marker (EEA1) (green), and STEAP2 (yellow). Cells were observed under fluorescent microscopy (Invitrogen tech.). Scale bar: 50 μm. All data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; t test. See also Figure S5 .

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Comparison, Binding Assay, Flow Cytometry, Fluorescence, Antibody Blocking Assay, Incubation, Blocking Assay, Control, Membrane, Liquid Chromatography with Mass Spectroscopy, Lysis, Immunoprecipitation, Clinical Proteomics, Labeling, Knockdown, Expressing, Infection, shRNA, Selection, Quantitative RT-PCR, Protein Binding, Western Blot, Knock-Out, Staining, Microscopy, Marker

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein delivers nucleic acids into cells (A) N protein-RNA complex binding to the cell surface. Aliquots of 10 μg SARS-CoV-2 N protein were incubated with 1 μg of indicated RNAs for 1 h at 4°C, and added to A549 or HPAEpiC cultures. SARS-CoV-2 N protein only without RNA was used as a control. The samples were analyzed by flow cytometry and data are shown as mean fluorescence intensity (MFI). Data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; t test. (B) The observation of N protein-RNA enters into cells. HPAEpiC were seeded onto 8-well glass slides (40,000 cells/well). SARS-CoV-2 N protein 10 μg and 40 μg RNA-FAM (green) were mixed for 1 h at 4°C. cells were treated with SARS-CoV-2 N-RNA-FAM mixture for 1 h. The groups of non-treated cells and RNA-FAM only were as controls. After treatment, N protein was detected by anti-N antibody (Red). The localization of RNA-FAM was green. DAPI (blue) indicates cell nuclei. Scale bar: 15 μm. (C) Lattice light sheet microscopy time lapse imaging of N protein-RNA complex entering into HPAEpiC cells. SARS-CoV-2 N protein 10 μg was mixed with 40 μg RNA-FAM (fluorescein) for 1 h at 4°C and then treated with ice-cooled alveolar cells. The signals of RNA-FAM and Hochest 33,342 were monitored by lattice light sheet microscopy at different time points.

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Binding Assay, Incubation, Control, Flow Cytometry, Fluorescence, Microscopy, Imaging

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein-assisted nucleic acid dispersion and expression in the co-culture environment (A)The co-culture system consisted of A549 as recipient cells, and 293T pre-transfected with two plasmids, one expressing GFP and the other expressing SARS-CoV-2 N protein or the pcDNA3.1 empty vector. (B–E) After 24 h co-culture of the donor cells and recipient cells, cell pool was stained with cytokeratin 18 (an A549 marker) and SV40 large T antigen (a 293T marker). A549 cells in the cell pool were gated from cytokeratin 18 positive and large T antigen negative. A549 GFP positive percentage was further assessed by flow cytometry analysis. Effects of SARS-CoV-2 N variants (B), treatment with RANTES (C), the p38 inhibitor SB203580 (D), or anti-N neutralizing antibody (E) were accessed by adding these effectors to the medium. Experiments are performed in three to five biological replicates. ∗, p value <0.05 (paired two-tailed student’s t -test). (F) SARS-CoV-2 N protein promotes gene delivery by cell-free diffusion to neighboring cells. A549 cells were plated in the lower chamber, while 293T donor cells co-transfected with plasmids expressing EGFP and indicated N proteins in the upper chamber. After 3 days of co-culture, GFP positive A549 cells were observed and counted. See also Figures S10 and .

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Dispersion, Expressing, Co-Culture Assay, Transfection, Plasmid Preparation, Staining, Marker, Flow Cytometry, Two Tailed Test, Diffusion-based Assay

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet:

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Bioprocessing, Recombinant, Magnetic Beads, Protease Inhibitor, Sequencing, Modification, SYBR Green Assay, shRNA

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: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a-d, Validation of mitotic exit in spindle-less cells by comparing H3-S10 dephosphorylation kinetics during unperturbed anaphase and spindle-less mitotic exit. a , Example images of wild-type HeLa cells during unperturbed anaphase fixed after time-lapse imaging, in metaphase, 6 min (maximally clustered), and 10 min after anaphase onset. Upper panel: chromatin labelled with H2B-mCherry, lower panel: H3-pS10 immunofluorescence. Single Z-slice shown. b , Quantification of H3-pS10 mean fluorescence and chromatin area in unperturbed mitosis, as shown in ( a ). Cell numbers: n = 9 (metaphase), n = 14 (3–6 min after anaphase onset), n = 8 (≥8 min after anaphase onset). Normalization to average value of metaphase time point. c , Example images of wild-type HeLa cells during spindle-less mitosis fixed after time-lapse imaging, in prometaphase (no flavopiridol), 10 min (maximally clustered), and 20 min after flavopiridol addition. Imaging as in ( a ). d , Quantification of H3-pS10 mean fluorescence and chromatin area in spindle-less mitosis, as shown in ( c ), demonstrates that histone 3-serine 10 was efficiently dephosphorylated in flavopiridol-induced mitotic exit and chromosomes cluster to a degree comparable to that of normal late anaphase. Cell numbers: n = 11 (nocodazole arrested pro-metaphase), n = 12 (10 min after flavopiridol addition), n = 13 (20 min after flavopiridol addition). Normalization to average value of prometaphase time point. e , Time-lapse microscopy of HeLa cell expressing IBB-EGFP and H2B-mCherry incubated in nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. f, Quantification of IBB-EGFP mean fluorescence within the chromosomal region, normalized to pre-flavopiridol time points, during spindle-less mitotic exit as in ( e ). n = 20 cells. g , Time-lapse microscopy of a HeLa cell expressing H2B-mCherry, progressing through reversine-induced mitotic exit in the absence of spindle. Yellow line indicates convex hull around chromosomes, single Z-slice shown. Time is relative to onset of clustering. h , Quantification of chromosome convex hull area of 11 cells as in ( g ). Individual cell curves were aligned based on half-maximum value of convex hull area. Normalization to average of first 4 time points. i , Live HeLa cell undergoing mitosis upon RNAi-mediated depletion of the spindle checkpoint protein Mad2 in the absence of a spindle. The cell line stably expresses H2B-mCherry and membrane marker AcGFP-Lap2β. Time relative to nuclear envelope breakdown (NEBD), single Z-slice shown. Representative example of 14 cells shown. Bars indicate mean in ( b, d ), lines and shaded areas indicate mean ± SD in ( f, h ). Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Biomarker Discovery, De-Phosphorylation Assay, Imaging, Immunofluorescence, Fluorescence, Time-lapse Microscopy, Expressing, Incubation, Stable Transfection, Membrane, Marker

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a , Chromosome organization during spindle-less mitotic exit. 3D-video of HeLa cell expressing H2B-mCherry imaged in the presence of nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. Yellow line indicates convex hull around chromosomes. b , Quantification of convex hull area of 16 cells as in ( a ), and inter-chromosomal area. c, HeLa cell expressing GEMs and H2B-mCherry, during spindle-less mitotic exit as in ( a ). Dashed lines indicate chromosomal area; single Z-slices shown. d, GEM particle count within chromosomal area as in ( c ), normalized to pre-flavopiridol time points (green). Chromosome convex hull area, normalized to pre-flavopiridol time points (magenta). n = 35 cells. Lines and shaded areas represent mean ± SD, dashed vertical lines indicate flavopiridol addition. Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Expressing

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a, b, Effect of actin depolymerization on chromosome clustering. a , Live HeLa cells stably expressing actin-EGFP and H2B-mCherry were imaged in absence (control) or presence of latrunculin B. Nocodazole was present in both conditions. b , Quantification of chromosome convex hull area during a flavopiridol induced mitotic exit in the presence of nocodazole, in the absence (control) and presence of latrunculin B as shown in ( a ), normalized to average area pre-flavopiridol addition. Cell numbers: n = 13 (control), n = 22 (latrunculin B). c , Timing of chromosome clustering relative to nuclear envelopment. Live mitotic HeLa cell expressing H2B-mCherry and the inner nuclear envelope protein AcGFP-LAP2β were imaged in presence of nocodazole; flavopiridol was added at t = 0 min to induce mitotic exit. d, Quantification of chromosome area by convex hull (yellow line in H2B channel in ( c )) and quantification of AcGFP-LAP2β accumulation at the surface of the chromatin region, within a rim of 1.6 μm width (yellow ROI in Lap2β channel in ( c )). n = 21 cells. e-h , Kinetochore tracking in cells progressing through spindle-less mitotic exit. e , Live mitotic HeLa cell stably expressing H2B-mCherry and CENP-A-EGFP imaged in the presence of nocodazole before and after flavopiridol addition, time-lapse = 10 s; Z-projection. f , 240 s long kinetochore trajectories starting 240 s before and at 400 after flavopiridol addition. g , Representative mean squared displacement (MSD) analyses of kinetochore tracks (CENP-A–EGFP) of nocodazole-treated mitotic HeLa cells before and after flavopiridol addition. h , Diffusion coefficients derived from mean square displacement (MSD) analyses of trajectories as in ( f ). Significance was tested by a two-tailed ratio paired t-test (****P = 2.9 × 10 −11 ). n = 13 cells. Bars and lines indicate mean, shaded areas indicate ± SD, dashed vertical lines refer to flavopiridol addition. Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Stable Transfection, Expressing, Control, Diffusion-based Assay, Derivative Assay, Two Tailed Test

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: Live cell microscopy of wild-type HeLa cells stably expressing H2B-mCherry synchronized to metaphase by MG132 treatment. a , Upper panel: negative control treated with dimethyl sulfoxide (DMSO) solvent, lower panel: mitotic exit induced through acute flavopiridol addition at t = 0 min. Chromosome arms extend out of the metaphase plate before flavopiridol addition, but densely cluster on the metaphase plate 8 min after flavopiridol addition. b , Quantification of chromosome convex hull area of 24 control and 25 flavopiridol treated cells, as in ( a ). c , Upper panel: acute mitotic spindle depolymerization by addition of nocodazole at t = 2 min leads to chromosome movement out of the metaphase plate; lower panel: mitotic exit was induced by flavopiridol (0 min), briefly before nocodazole-mediated spindle disassembly (2 min), preventing movement of chromosomes out of the metaphase plate. d , Chromosome convex hull area measurements of 27 nocodazole and 23 flavopiridol and nocodazole treated cells, as shown in ( c ). Values normalized to average of all frames prior to first drug treatment. Showing single Z-slices, lines and shaded areas indicate mean ± SD, dashed vertical lines indicate drug addition, scale bars 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Microscopy, Stable Transfection, Expressing, Negative Control, Solvent, Control

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a-c, Effect of BAF depletion on chromosome clustering. a , Live mitotic HeLa cells stably expressing H2B-mCherry and GEMs were imaged 72 h after siRNA transfection, in the presence of nocodazole; flavopiridol was added at t = 0 min to induce mitotic exit. White dashed lines indicate chromosome areas, single Z-slice shown. b , Quantification of chromosome convex hull area, normalized to pre-flavopiridol addition. c , GEM particle count within chromosomal area normalized to pre-flavopiridol addition. n = 23 cells (siBAF), n = 23 cells (siControl). d , Immunoblot analysis of BAF and actin 72 h after siRNA transfection, showing one of two biological replicates. For gel source data, see . e , Localization of BAF-EGFP in live mitotic HeLa cell imaged in the presence of nocodazole; flavopiridol was added at t = 0 s to induce mitotic exit. f , Quantification of chromosome convex hull area and BAF-EGFP accumulation at the surface of the chromatin region as in ( e ), normalized to pre-flavopiridol. n = 21 cells. g , Localization of Ki-67, EGFP tagged on endogenous loci in live HeLa cell progressing from metaphase to anaphase (anaphase onset = 0 min), DNA was stained with SiR-Hoechst, Z-projection. h, Quantification of chromosome convex hull area and EGFP-Ki-67 mean fluorescence on chromosomes, normalized to pre-anaphase, for 41 cells as in ( g ). i , Localization of Ki-67 during spindle-less mitotic exit. Time-lapse microscopy of mitotic HeLa cell as in ( g ), in the presence of nocodazole; flavopiridol was added (t=0 min) to induce mitotic exit. Z-projection. j , Quantification of chromosome convex hull area and EGFP-Ki-67 mean fluorescence on chromosomes of 27 cells as in ( i ). Shaded areas indicate ± SD, scale bars 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Staining, Fluorescence, Time-lapse Microscopy

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a-d , Molecular organization of Ki-67 on the surface of mitotic chromosomes before and after flavopiridol addition. a , Ki-67 was tagged on the N-terminus by mCherry, on the C-terminus by EGFP and expressed in HeLa cells. Sister chromatid pairs oriented perpendicular to the imaging plane were imaged in live mitotic cells, lines indicate measurement regions for ( b ). b , Relative positions of mCherry and EGFP along an axis perpendicular to the chromosome surface. Fluorescence densities (dots) were measured along line profiles as in ( a ) and a sum of two Gaussian functions (lines) was separately fitted to the EGFP and mCherry channels, respectively, to determine peak positions (dashed lines). c , The radial displacement of EGFP relative to mCherry peaks was determined based on line profile measurements as in ( a, b ) for mCherry-Ki-67-EGFP (R-Ki-67-G: n = 34 chromosomes pre-flavopiridol, n = 46 chromosomes post-flavopiridol) and a construct where the fluorophores were linked in reverse order, EGFP-Ki-67-mCherry (G-Ki-67-R: n = 40 chromosomes pre-flavopiridol, n = 39 chromosomes post-flavopiridol). Bars represent mean, significance was tested by a two-tailed unpaired t-test (****P = 2.1 ×10 −8 , ****P = 2.3 × 10 −6 ). d, Model of Ki-67 organization on chromosome surfaces during early mitosis and during mitotic exit. e, Ki-67 KO cell expressing low levels of H2B-mNeonGreen. Representative example of 14 cells. f , Spindle-less mitotic exit in wild-type cells and in Ki-67 knockout (KO) cells overexpressing H2B-mNeonGreen to high levels to suppress the Ki-67 knockout individualization failure phenotype. Live cells were imaged in the presence of nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. Z-projection. g , Normalized chromosome convex hull area quantification of 24 wild-type and 22 Ki-67 KO cells as in ( f ). Lines and shaded areas represent mean ± SD. Scale bars 1 μm ( a ), all others 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Imaging, Fluorescence, Construct, Two Tailed Test, Expressing, Knock-Out

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a , Time-lapse microscopy of clustering-deficient HeLa Ki-67 KO cell stably expressing H2B-mCherry to high levels during a flavopiridol-induced mitotic exit in the presence of nocodazole. Transient expression of EGFP-Ki-67 to levels at least matching Ki-67 endogenous levels (lower panel). Single Z-slice shown. b , Chromosome convex hull area quantification, normalized to pre-flavopiridol time points, of 29 Ki-67 KO and 28 Ki-67 KO + EGFP-Ki-67 cells as in ( a ). c , Immunoblot analysis of H2B from cell lysates, isolated from wildtype cells (1) or cells transiently expressing H2B-mNeonGreen (2,3). Sample 3 was lysed after 48 h while sample 2 was additionally FACS sorted for the 10% brightest cells which we estimated to represent the population of cells that suppress the Ki-67 knockout individualization failure phenotype. For gel source data, see . n = 2 biological repeats. d, e , Immunofluorescence of acetylated H3 of Ki-67 KO cells arrested in nocodazole with or without trichostatin A. d , Representative examples. Single Z-slices shown. e , Quantification of acetylated H3 mean fluorescence intensity in Ki-67 KO cells arrested in nocodazole with and without trichostatin A. Bars represent mean, significance was tested with a two-tailed Kolmogorov-Smirnov test. (****P = 2.22×10 −16 ). n = 115 cells (Ki-67 KO), n = 111 cells (Ki-67 KO + TSA). f , Time-lapse microscopy of HeLa wild-type or clustering-deficient Ki-67 KO cells with or without transient re-expression of Ki-67 during a flavopiridol-induced mitotic exit in the presence of nocodazole. Cells were treated with trichostatin A 2 h before imaging to rescue the Ki-67 knockout individualization failure phenotype in Ki-67 KO cells. Representative examples stained with SiR-Hoechst, single Z-slices shown. g , Chromosome convex hull area, normalized to pre flavopiridol time points, quantification of 21 Ki-67 KO, 31 wild-type and 32 Ki-67 KO + EGFP-Ki-67 cells as in ( f ). Lines and shaded areas indicate mean ± SD ( b, g ), scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Time-lapse Microscopy, Stable Transfection, Expressing, Western Blot, Isolation, Knock-Out, Immunofluorescence, Fluorescence, Two Tailed Test, Imaging, Staining

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a , Schematic of genotyping strategy to endogenously mutate Ki-67’s PP1 binding motif RVSF to RASA. A newly generated SacII restriction site generated by CRISPR/Cas9 nickase as depicted was used to detect correctly mutated alleles. b , SacII restriction fragments were detected by gel electrophoresis following the assay depicted in ( a ), showing successful recombination of all three Ki-67 alleles present in HeLa cells for clone 43 and 96. Showing one example of 2 biological replicates. c-d , Spindle-less mitotic exit in wild-type cells and in homozygous Ki-67 RASA mutant cells. Live cells were imaged in the presence of nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. Quantification of chromosome convex hull area, normalized to pre-flavopiridol time points ( c ) and representative examples stained with SiR-Hoechst ( d ). Z projection. Lines and shaded areas indicate mean ± SD. n = 22 cells (wild-type), n = 23 cells (RASA) . e , Molecular organization of the Ki-67 RASA mutant on the surface of mitotic chromosomes before and after flavopiridol addition to taxol treated cells. Ki-67(RASA) was tagged by mCherry and EGFP on either protein end, respectively, and expressed in HeLa cells bearing the endogenous RASA mutation in all three copies of Ki-67. Bars represent mean, significance was tested by a two-tailed unpaired t-test (****P = 8.3 ×10 −8 , ***P = 0.00049). Chromosome numbers: n = 32 (R-Ki-67-G, Pre), n = 28 (R-Ki-67-G, Post), n = 37 (G-Ki-67-R, Pre), n = 34 (G-Ki-67-R, Post). f, g , Immunofluorescence of H3-pS10 during spindle-less mitotic exit in wild-type and Ki-67 KO cells. f , Representative examples of wild-type and Ki-67 KO cells before, 10 and 20 min after flavopiridol addition. Single Z-slice is shown. g , Quantification of H3-pS10 mean fluorescence intensity before (Wild-type n = 38 cells, Ki-67 KO n = 23 cells), 5 (Wild-type n = 61 cells, Ki-67 KO n = 61 cells), 10 (Wild-type n = 72 cells, Ki-67 KO n = 65 cells) and 20 min (Wild-type n = 73 cells, Ki-67 KO n = 55 cells) after mitotic exit induction with flavopiridol in wild-type and Ki-67 KO cells. Values normalized to average of wild-type 5 min time point. Showing combined data of two independent biological replicates. Bars represent mean, significance tested with a two-tailed Mann Whitney test (P = 0.72 for pre-flavopiridol time point, P = 0.96 for 5 min time point, P = 0.71 for 10 min time point and P = 0.26 for 20 min time point). Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Binding Assay, Generated, CRISPR, Nucleic Acid Electrophoresis, Mutagenesis, Staining, Two Tailed Test, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a, Time-lapse microscopy of clustering-deficient HeLa Ki-67 KO cell transiently expressing H2B-mCherry to high levels, and stably expressing IBB-EGFP, during a flavopiridol-induced mitotic exit in the presence of nocodazole. b , Quantification of IBB-EGFP mean fluorescence in chromosomal area (green) and quantification of chromosome convex hull area (magenta) in clustering-deficient cells as in ( a ), normalized to pre-flavopiridol. n = 15 cells. c, d , H2B-mCherry expression levels and chromosome area measurements for cells shown in – . , Quantification of H2B-mCherry total fluorescence for cells analyzed in (all data points normalized to mean of wild-type dataset). d , Chromosome convex hull area quantification (normalized to pre-flavopiridol area) for cells quantified in . n = 46 cells (wild-type), n = 29 cells (Ki-67 KO). e , Clustering-proficient HeLa cell imaged as in , but treated with leptomycin B. f , Quantification of total L10 fluorescence in nuclear regions as shown in ( e ), normalized to average of pre-flavopiridol addition. n = 22. g, h , Quantification of H2B-mCherry total fluorescence and chromosome convex hull area for cells analyzed in and in ( f ). Normalizations as in ( c, d ), cell numbers: n = 23 wild-type, n = 8 Ki-67 KO, n = 22 leptomycin B. Individual Ki-67 KO curves shown in . Significance tested with two-sided unpaired t-test (P = 0.28 in GEM cell lines wild-type vs Ki-67 KO, P = 0.12 in L10 cell lines wild-type vs LMB treated and P = 0.13 in L10 cell lines wild-type vs Ki-67 KO). Lines and shaded areas indicate mean ± SD, dashed vertical lines indicate flavopiridol addition. Scale bars 10 μm, showing single Z-slices.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Time-lapse Microscopy, Expressing, Stable Transfection, Fluorescence

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a, b, Time-lapse microscopy of spindle-less mitotic exit in clustering-proficient wild-type HeLa cell ( a ) and clustering-deficient Ki-67 KO HeLa cell ( b ). Cells stably expressed GEMs and transiently expressed H2B-mCherry; images show time point 1 min before and 20 min after flavopiridol-induced mitotic exit in presence of nocodazole. c, Quantification of GEM localization in 46 clustering-proficient wild-type and 29 clustering-deficient Ki-67 KO cells as in ( a, b ). Particle numbers were quantified in chromosomal area as in , and normalized to average of particle number pre flavopiridol. t = 0 min refers to addition of flavopiridol. Analyzed cells expressed H2B-mCherry at similar levels in both genetic backgrounds . d, e, Time-lapse microscopy of clustering-proficient wild-type cells and clustering-deficient Ki-67 KO cells ( e ) stably expressing ribosomal protein L10-EGFP and transiently expressing H2B-mCherry, before and after mitotic exit ind-ction in presence of nocodazole. f, Quantification of L10-EGFP total fluorescence within chromosomal area for conditions shown in ( d, e ). t = 0 min refers to flavopiridol addition, data normalized to average values of pre-flavopiridol addition. Analyzed cells expressed H2B-mCherry at similar levels in both conditions . n = 23 cells (wild-type), n = 8 cells (Ki-67 KO). Individual Ki-67 KO curves shown in . g, Model of nuclear assembly during mitotic exit. Lines and shaded areas represent mean ± SD, dashed vertical lines indicate flavopiridol addition. Single Z-slices shown; scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Time-lapse Microscopy, Stable Transfection, Expressing, Fluorescence

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A A concentration range of Alexa Fluor 647-conjugated CD3xHER2 bsAbs varying in affinity and epitope recognition site on either arm, were added on top of collagen gels containing BT474 (HER2+) tumoroids. Localization of bsAbs to the tumoroids was analyzed after 24 h by confocal microscopy. Images show maximum projections. Bar = 100 μm. B Localization of three different bsAbs combining a CD3 high affinity arm with the indicated HER2 arms in the BT474 tumoroid. White arrows indicate penetration of bsAbs into the tumoroids. Blue, Hoechst; red, Alexa Fluor 647-conjugated bsAb. Images were obtained from a single z-section through the center of the tumoroid. Bar = 50 μm. C Cartoon showing the location on the HER2 receptor of the epitopes recognized by the different HER2 arms and whether the interactions are high or low affinity.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Concentration Assay, Confocal Microscopy

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Maximum projection images showing tumoroid (blue), T-cells (green) and loss of viability detected by PI (red) 6 days after exposure of collagen embedded BT474 tumoroids to a mixture of T-cells and bsAbs with either non-targeting CD3 arm or non-targeting HER2 arm. Bottom panel shows tumoroids treated with 10 μg/mL Cisplatin triggering near complete tumoroid killing versus tumoroids grown in absence of T-cells and bsAbs (negative control; NC) or T-cells only. Bar = 100 μm. B Quantification of image data as shown in A . Data were normalized to cisplatin condition. Graphs show mean and SEM of 3 independent experiments, each performed in triplicate (3 individual wells each containing 1 collagen embedded BT474 tumoroid). C Western blot showing loss of HER2 upon CRISPR-Cas9 mediated knockout in BT474 cells. Tubulin serves as a loading control. D Representative maximum projection images at 6 days after exposure of collagen embedded WT, shCTR, or sgHER2 BT474 tumoroids to a mixture of T-cells and 1 μg/mL CD3 wt xHER2 Herceptin bsAbs. Blue, tumor nuclei; green, T-cells; red, PI. bar = 100 μm. E Quantification of image data as shown in D . Data were normalized to cisplatin condition. Graph shows mean and SEM of 3 independent experiments, each performed in triplicate. P -value calculated using two-way ANOVA followed by Bonferroni’s multiple comparisons test. ns non-significant; *** P < 0.001.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Negative Control, Western Blot, CRISPR, Knock-Out, Control

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Maximum projection images showing tumoroid (blue), T-cells (green) and loss of viability detected by PI (red) 6 days after exposure of collagen embedded BT474 tumoroids to a mixture of T-cells and bsAbs for one representative experiment of at least 4 biological replicates. Blue, tumor nuclei; green, T-cells; red, PI. Bar = 100 μm. B Quantification of the image data as shown in A . Tumoroids grown in absence of T-cells and bsAbs are shown as negative control (NC). Tumoroids treated with 10 μg/mL Cisplatin are shown as positive control triggering near complete tumoroid killing. Results are normalized to cisplatin condition. Graphs show mean and SEM of 5 (CD3 wt xHER2 variants) or 4 (CD3 Low xHER2 variants) independent experiments, each performed in triplicate (3 individual wells each containing 1 collagen embedded BT474 tumoroid). P -value calculated using two-way ANOVA followed by Bonferroni’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to NC.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Negative Control, Positive Control

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Maximum projection images showing T-cell recruitment to the tumoroid and tumor killing with 12 h intervals from day 2 to day 5 after exposure of collagen embedded BT474 tumoroids to a mixture of T-cells and 0.1 μg/mL of the indicated bsAbs. Blue, tumor nuclei; green, T-cells; red, PI. Bar = 100 μm. B Quantification of time-lapse image data with 1-h intervals as shown in A . Green line represents the number of T-cells localized in the tumoroid. Red line represents the percentage of PI positive tumor cells, indicating loss of viability. Mean and SEM of three tumoroids is shown.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques:

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Cartoon illustrating experimental setup: BT474 tumoroids were printed below the higher surface of collagen gels with a diagonal upper surface. A mixture of T-cells and bsAbs was added on top of the lower surface. Maximum projection images are shown for bsAbs with a CD3 high affinity arm in combination with a high affinity HER2 arm either recognizing epitope 169 or epitope 153. BsAbs were used at 1 μg/mL. Time lapse starts upon initial contact of T-cells with tumoroids (indicated by red circle; 36 h after adding T-cells and bsAbs). Confocal image stacks were captured every hour. A time span of 40 h is shown with an 8-h interval, followed by an image displaying the final time point (day 5) where T-cell mediated tumoroid killing is near complete. Blue, tumor nuclei; green, T-cells; Red, PI. B , C 3D T-cell tracking analysis of time-lapse image data as shown in A . B MSD analysis of two typical T-cells in the vicinity of the tumoroid. The MSD was determined for time-lag from 1 to 25 h. Red color indicates the presence of CD3 wt xHER2 169 and blue color indicates CD3 wt xHER2 153 . Insets depict the 3D track of the two T-cells. The MSD for the two cells were characterized by D B = 0.x ± 0.x um 2 /h, V B = 0.x ± 0.x um/h, and D R = 0.x ± 0.x um 2 /h, V R = 0.x ± 0.x um/h, respectively. C MSD analysis of the total population of T-cells in the vicinity of the tumoroid over increasing time-lag from 1 to 30 h. In total 25 and 35 trajectories were analyzed for CD3 wt xHER2 169 and CD3 wt xHER2 153 , respectively. Insets depict median and SD for the parameters diffusion (D), velocity (v), and diffusive fraction (f D ) for the population analysis for the indicated bsAbs. Note that f D is lower for CD3 wt xHER2 169 bsAb. P -value calculated using multi comparison Dunn’s test following non-parametric Kruskal–Wallis test. * P < 0.05. D Confocal images of a single z-section through the center of a tumoroid exposed to T-cells and either CD3 wt xHER2 169 or CD3 wt xHER2 153 bsAbs. Initial contact of T-cells with the tumoroid (36 h after adding T-cells and bsAbs) and the same area 8 h later is indicated by the white circle. Red arrow indicates T-cell infiltration occurring only with the CD3 wt xHER2 169 bsAb. Blue, tumor nucleus; red, PI; green, T-cell. Bar = 100 μm.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Cell Tracking Assay, Diffusion-based Assay, Comparison

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Schematic illustration of the transwell assay. BT474 cells were seeded in the lower chamber of a transwell plate, with or without the addition of unlabeled T-cells and with or without CD3 wt xHER2 169 bsAbs. Cell Tracker CMFDA-labeled T-cells were added to the upper chamber. The number of green-fluorescent T-cells migrating to the lower chamber was analyzed after 48 h using confocal microscopy and flow cytometry. A condition using 100 ng/mL CXCL12 was used as positive control. B Bright field images (showing a mixture of unlabeled T-cells, infiltrating CMFDA-labeled T-cells, as well as tumor cells) and green fluorescence channel images (showing labeled T-cells) taken in the lower chamber at 48 h for the indicated conditions. Red dotted line outlines islands of tumor cells. Red arrow indicates a T-cell cluster. Bar = 100 μm. C Flow cytometry analysis of cell populations from the lower chamber at 48 h. Horizontal axis shows CMFDA signal (negative for unlabeled T-cells co-cultured with tumor cells in bottom chamber; positive for CMFDA-labeled T-cells recruited from the upper chamber); vertical axis shows CD3 levels. Flow cytometry analysis ( D ) and bar graph showing mean and SEM from 3 to 4 biological replicates ( E ), depicting cell counts in Q2 (CMFDA:CD3 double positive T-cells migrated from the upper to the lower chamber) for the indicated conditions. P -value calculated using one-way ANOVA followed by Bonferroni’s multiple comparisons test. ns, non-significant; ** P < 0.01; *** P < 0.001. F T-cell recruitment triggered by conditioned media (CM) from the indicated conditions. Graph shows cell counts of CMFDA-labeled infiltrated T-cells collected from the lower chamber. Mean and SEM from 2 to 4 biological replicates. P -value calculated using one - way ANOVA followed by Bonferroni’s multiple comparisons test. ns non-significant; * P < 0.05; ** P < 0.01. G Bright field images (showing a mixture of unlabeled T-cells, infiltrating CMFDA-labeled T-cells, as well as tumor cells) taken in the lower chamber at 48 h for the indicated conditions. Red dotted line outlines islands of tumor cells. Red arrows indicate T-cell clusters. Bar = 100 μm. H Bar graph showing mean and SEM from 3 biological replicates, depicting cell counts in Q2 (CMFDA:CD3 double positive T-cells migrated from the upper to the lower chamber) for the indicated conditions. P -value calculated using one-way ANOVA followed by Bonferroni’s multiple comparisons test. ns non-significant; ** P < 0.01.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Transwell Assay, Labeling, Confocal Microscopy, Flow Cytometry, Positive Control, Fluorescence, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 1. Generation of human Tspan15-expressing MEFs as an immunogen and validation of resulting mouse anti-human Tspan15 mAbs. (A) ADAM10-knockout MEFs (–) and ADAM10- knockout MEFs stably overexpressing FLAG-tagged Tspan15 (+) were lysed in 1% Triton X-100 lysis buffer and subjected to anti-FLAG (top panel) and anti-α-tubulin (bottom panel) western blotting. (B) Wild-type (WT) and Tspan15-knockout (KO) Jurkat human T cells were analysed by flow cytometry with tissue culture supernatant for each of the four mouse anti-human Tspan15 hybridomas (1C12, 4A4, 5D4 or 5F4; solid line), or with mouse IgG1 as a negative control (dotted line). Histograms are representative of two independent experiments. (C) HEK-293T cells were transfected with FLAG-tagged human TspanC8 expression constructs (except for Tspan10, which was of mouse origin) or an empty vector control (–),

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Biomarker Discovery, Knock-Out, Stable Transfection, Lysis, Western Blot, Flow Cytometry, Negative Control, Transfection, Construct, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 3. Tspan15 mAbs 1C12 and 4A4 partially inhibit ADAM10/Tspan15 activity. (Ai) Wild-type (WT), ADAM10-knockout (A10 KO) and Tspan15-knockout (T15 KO) HEK-293T cells were transfected with a VE-cadherin expression construct. Cells were treated with 10 μM DAPT to prevent post-ADAM10 proteolysis by γ-secretase, followed by 2 mM NEM for 30 minutes to activate ADAM10. Cells were lysed in 1% Triton X-100 lysis buffer and subjected to western blotting with an antibody against the cytoplasmic tail of VE-cadherin. No C-terminal fragment was detected in the absence of NEM (data not shown). (Aii) VE-cadherin cleavage data were quantitated to calculate the percentage cleaved. Data were arcsine- transformed and statistically analysed by a one-way ANOVA with a Dunnett’s multiple comparisons test (***p<0.001 compared to WT). Error bars represent standard error of the mean from three independent experiments. (B) Wild-type HEK-293T cells were transfected with VE-cadherin, treated with Tspan15 mAbs or MOPC-21 negative control mAb for 30 minutes, and stimulated with NEM as described for panel

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Activity Assay, Knock-Out, Transfection, Expressing, Construct, Lysis, Western Blot, Transformation Assay, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 4. Tspan15 and ADAM10 co-localise on the cell surface. (Ai) A549 cells were fixed and stained with anti-ADAM10 mAb (red) and either anti-Tspan15 mAb 5D4 (green) or anti-CD9 mAb 1AA2 (green). ADAM10, Tspan15 and CD9 on the basal membrane were imaged using TIRF microscopy. Images shown are representative of 48 fields of view from four independent experiments (scale bar 10 µm). (Aii) The degree of co-localisation between ADAM10 and Tspan15 or CD9 was determined using Manders’ coefficients to measure the proportion of overlapping pixels contained within total ADAM10 signal in the red channel (M1) and total Tspan15 or CD9 signal in the green channel (M2). Data were arcsine- transformed and statistically analysed by a one-way ANOVA with a Tukey’s multiple comparisons test to compare M1 and M2, within and between Tspan15 and CD9 (***p<0.001 for all pairwise comparisons). Error bars represent standard error of the mean.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Staining, Membrane, Microscopy, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 5. ADAM10 is the principal Tspan15-interacting protein in HEK-293T cells. Wildtype (WT) and Tspan15-knockout (KO) HEK-293T cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with Tspan15 mAb 1C12 cross-linked to protein G sepharose beads. Proteins were identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Proteomic profiles of WT and Tspan15 KO HEK-293T immunoprecipitates are presented in a volcano plot to identify differentially expressed proteins. The minus log10 transformed p-value of each protein was plotted against the log2 transformed protein label free quantification ratio between the Tspan15 co-immunoprecipitation of WT samples and the control co-immunoprecipitation of Tspan15 KO samples. Proteins with significant fold change (p<0.05) are depicted in red; blue dots represent proteins with no significant changes in expression. A permutation-based false discovery rate estimation was applied and visualised as hyperbolic curves in grey.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Knock-Out, Lysis, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Quantitative Proteomics, Control, Expressing

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 6. Tspan15 protein expression requires ADAM10. (A) Tspan15 surface expression in wildtype (WT), Tspan15-knockout (KO) and ADAM10 KO Jurkat, HEK-293T and A549 cell lines were analysed

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Knock-Out

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 7. The requirement of Tspan15 for ADAM10 surface expression is cell type dependent. (A) ADAM10 surface expression in WT, ADAM10 KO and Tspan15 KO Jurkat, HEK-293T and A549 cells was measured by flow cytometry and quantitated as described in Figure 4A. (B) HUVECs were transfected with two different Tspan15 siRNAs or negative control siRNA and surface expression of ADAM10 was measured by flow cytometry and analysed as described in Figure 6A.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Flow Cytometry, Transfection, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 8. ADAM10 and Tspan15 form dynamic bimolecular fluorescence complementation (BiFC) complexes. (A) Schematic representation of ADAM10 tagged with the C-terminal half of superfolder GFP (sfGFP-C), Tspan15 tagged with the N-terminal half of superfolder GFP (sfGFP-N) and the predicted ADAM10/Tspan15 BiFC dimer. Solid ovals represent N-glycosylation. (B) HEK-293T cells were transfected with the ADAM10 and Tspan15 BiFC expression constructs, fixed and stained with Alexa Fluor® 647-conjugated Tspan15 mAb 5D4, and analysed by confocal microscopy. The image shown is representative of middle plane sections taken from two independent experiments (scale bar 10 µm). (C-D) Fluorescence correlation spectroscopy (FCS) measurements from the upper membrane of HEK-293T expressing the ADAM10/Tspan15 BiFC complexes were used to determine the average particle concentration (C) and diffusion co-efficient (D) of the complexes. (E) Fluorescence fluctuations from the FCS reads were also subjected to photon counting histogram (PCH) analysis to obtain the average molecular brightness (ε) of particles within the confocal volume. The FCS data were separated into groups that preferentially fit to a one-component or a two-component PCH model with dimmer and brighter subcomponents. Data were obtained from 43 individual measurements from three independent experiments. Error bars represent standard errors of the mean, N is the number of particles, and cpm is the counts per molecule. Data were log-transformed and statistically analysed by a one-way ANOVA followed by Tukey’s multiple comparisons test (***p<0.001).

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Fluorescence, Glycoproteomics, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Spectroscopy, Membrane, Concentration Assay, Diffusion-based Assay, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 9. A synthetic ADAM10/Tspan15 fusion protein is a functional scissor. (A) Schematic representation of the synthetic ADAM10/Tspan15 fusion protein that has the C-terminus of ADAM10

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Functional Assay

Journal: Molecular plant

Article Title: The WAK-like protein RFO1 acts as a sensor of the pectin methylation status in Arabidopsis cell walls to modulate root growth and defense.

doi: 10.1016/j.molp.2023.03.015

Figure Lengend Snippet: Figure 6. RFO1-GFP particle dwell time at the PM is altered by the perturbation of cell wall pectin methylation. (A) Representative spinning-disc confocal images (top) of a 5-day-old root elongating epidermal cell and kymographs (bottom) along the length of the dashed yellow lines from the corresponding video (n = 50 frames, 1 frame/s) of RFO1-GFP (rfo1-1 pRFO1RFO1-GFP) and RFO1-GFP iPMEIox (rfo1-1 pRFO1RFO1-GFP iPMEIox) seedlings treated with 12 mM estradiol for 2 h. RFO1-GFP iPMEIox seedlings were co-treated with Mock () or 10 mg/ml of dmPectin (+). Unless specified, all data derived from 180-s videos taken at 1 frame/s. Scale bar, 5 mm. (B) Dwell time frequencies of RFO1-GFP particles at the PM as depicted in (A). Means (m) are shown by dashed lines. For each condition, data represent N > 12 000 particles from a minimum of three cells per root and three roots. (C) Average RFO1-GFP particle dwell times at the PM. (D) Quantification of the total number of RFO1-GFP particles at the PM (left), and particles with dwell times >50 s (middle) and >100 s (right). (E) Average instantaneous diffusion coefficients of RFO1-GFP particles at the PM. (C–E) Data represent the mean ± SE of N > 25 cells per condition from three independent replicates using three cells per root, three roots per condition, and tracking R1072 particles/cell, obtained from videos as described in (A). One-way ANOVA with Dunnett’s multiple-comparison test, **p < 0.01, ****p < 0.0001.

Article Snippet: RFO1-GFP particles were imaged with a CSU-W1 Yokogawa spinning-disk head fitted to a Nikon Eclipse Ti-E-inverted microscope with a CFI PlanApo 3 100 NA 1.40 oil immersion objective and two iXon Ultra EM-CCD cameras (Andor, GB); a 31.2 lens between the spinning disk and camera was used.

Techniques: Methylation, Derivative Assay, Diffusion-based Assay, Comparison