Journal: Nature Communications

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.1038/s41467-024-53904-z

Figure Lengend Snippet: A NPM1 structural features, including the secondary structure calculated from the oligomerization domain (OD) PDB 4N8M and the nucleic acid binding domain (NBD) PDB 2LLH , using DSSP (2 o Struc.; β-strands are indicated with arrows and α-helicies are indicated with cylinders). The CIDER linear net charge per residue (LNCPR) and linear hydropathy (Hydro.) are shown for the IDR. B p14 ARF structural features, including PSI-PRED secondary structure prediction (2 o Struc.), CIDER linear net charge per residue (LNCPR), and linear hydropathy (Hydro.). The amino acid sequence conservation (Cons.) is based on a multiple sequence alignment using MUSCLE. The bottom panel shows the Rosetta steric zipper propensity energy (R. Energy) calculated using ZipperDB. C CV-SANS curves for p14 ARF -NPM1 condensates, in 100% D 2 O buffer for full scattering intensity (NPM1 and p14 ARF detected; gray trace), in 45% D 2 O buffer where p14 ARF is contrast matched ([ 2 H]-NPM1 detected; green trace), and in 85% D 2 O buffer where [ 2 H]-NPM1 is contrast matched (p14 ARF detected; blue trace). Correlation peaks at ~200 Å and ~400 Å correspond to meso-scale organization of p14 ARF . All curves are offset for clarity. Scatter points represent the average, the error bars represent the uncertainty derived from the counting statistics of the SANS instrument, as described and cited in the Methods. D Schematic describing NPM1 with extended IDR conformations. E Schematic describing the spatial organization of p14 ARF within p14 ARF -NPM1 condensates. F 2D CC-DARR spectrum of [ 13 C, 15 N]-p14 ARF within the condensed phase. Select resonance assignments are labeled. G Secondary 13 C chemical shifts for [ 13 C, 15 N]-p14 ARF within the condensed phase. Assigned residues are highlighted in gray. The secondary structure prediction from panel B is shown for reference (2 o Struc.; top).

Article Snippet: The primary antibodies were used as follows: rabbit monoclonal anti-Cyclophilin B (Cell Signaling, 43603) at 1:1500–1:2000 dilution; rabbit monoclonal anti-GAPDH (Cell Signaling, 5174) at 1:2500 dilution; mouse monoclonal anti-NPM1 (ThermoFisher Scientific, 32-5200) at 1:1000 dillution; mouse monoclonal anti-p14 Arf (Cell Signaling, 2407) at 1:1000–1:15000 dilution; mouse monoclonal anti-GAPDH (Santa Cruz, sc-47724) at 1:2500 dilution; and rabbit polyclonal anti-p14 Arf (Novus, NB200-111) at 1:2000 dilution.

Techniques: Binding Assay, Cider Assay, Residue, Sequencing, Derivative Assay, Labeling

Journal: Nature Communications

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.1038/s41467-024-53904-z

Figure Lengend Snippet: A A CC-DARR spectrum acquired for [ 13 C, 15 N]-p14 ARF with 20 ms DARR mixing time shows resonances for T8 in two states, including in an expanded p14 ARF conformation (top), and in a collapsed p14 ARF conformation (bottom). B A CC-DARR spectrum acquired with 400 ms DARR mixing time shows additional cross-peaks indicating intramolecular contacts between T8 and H26. C The 2D-NHHC spectrum (gray) of p14 ARF (equal mixture of [ 13 C]-p14 ARF and [ 15 N]-p14 ARF ) shows that sidechains within the p14 ARF N-terminus make intermolecular contacts within the condensed phase with NPM1. HNCA (magenta) and 2D-HNCACX (blue) spectra for [ 13 C, 15 N]-p14 ARF are shown for reference. D Schematic describing possible modes of p14 ARF intra- and intermolecular interaction.

Article Snippet: The primary antibodies were used as follows: rabbit monoclonal anti-Cyclophilin B (Cell Signaling, 43603) at 1:1500–1:2000 dilution; rabbit monoclonal anti-GAPDH (Cell Signaling, 5174) at 1:2500 dilution; mouse monoclonal anti-NPM1 (ThermoFisher Scientific, 32-5200) at 1:1000 dillution; mouse monoclonal anti-p14 Arf (Cell Signaling, 2407) at 1:1000–1:15000 dilution; mouse monoclonal anti-GAPDH (Santa Cruz, sc-47724) at 1:2500 dilution; and rabbit polyclonal anti-p14 Arf (Novus, NB200-111) at 1:2000 dilution.

Techniques:

Journal: Nature Communications

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.1038/s41467-024-53904-z

Figure Lengend Snippet: A The 2D 1 H- 15 N TROSY-HSQC spectrum of [ 13 C, 15 N]-NPM1 within the p14 ARF -NPM1 condensed phase displays resonances from the NPM1 IDR. B Linear net charge per residue (LNCPR) for the NPM1 IDR. Nuclear spin relaxation for [ 2 H, 15 N]-NPM1 in solution (blue scatter points) and condensed phase [ 13 C, 15 N]-NPM1 (red scatter points), including C 1 H- 15 N heteronuclear NOEs, D R 1 , and E R 2 transverse relaxation, which shows a restriction of NPM1 IDR backbone motions on the ps-ns timescale. The error bars for R 1 and R 2 transverse relaxation plots represent the standard errors from curve fitting, as described in Methods. F The contributions from exchange broadening, R ex . G 15 N-CPMG relaxation dispersion profiles for condensed [ 13 C, 15 N]-NPM1 measured at 800 MHz, including A186, T199, and A201, fit to a two-state model. Scatter points represent the decay rates, and the error bars represent the estimated systematic error, as described in Methods. H Schematic describing NPM1 IDR conformational exchange within condensates with p14 ARF .

Article Snippet: The primary antibodies were used as follows: rabbit monoclonal anti-Cyclophilin B (Cell Signaling, 43603) at 1:1500–1:2000 dilution; rabbit monoclonal anti-GAPDH (Cell Signaling, 5174) at 1:2500 dilution; mouse monoclonal anti-NPM1 (ThermoFisher Scientific, 32-5200) at 1:1000 dillution; mouse monoclonal anti-p14 Arf (Cell Signaling, 2407) at 1:1000–1:15000 dilution; mouse monoclonal anti-GAPDH (Santa Cruz, sc-47724) at 1:2500 dilution; and rabbit polyclonal anti-p14 Arf (Novus, NB200-111) at 1:2000 dilution.

Techniques: Residue, Dispersion

Journal: Nature Communications

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.1038/s41467-024-53904-z

Figure Lengend Snippet: A p14 ARF structural features, including PSI-PRED4.0 secondary structure prediction (2 o Struc.), CIDER linear net charge per residue (LNCPR) and linear hydropathy (Hydro.). The CIDER analysis for p14 ARF ΔH1-3 is shown below. B Zoomed in regions from confocal fluorescence micrographs of NPM1-AF488 in condensates with p14 ARF (top) and p14 ARF ΔH1-3 (bottom). Scale bars = 10 µm. C Index of dispersion for NPM1 in condensates with p14 ARF (gray boxes, whiskers and trace; derived from n = 6, 6, 6, 7, 6, 6, 7, 6, 6, 6, 7 images) and p14 ARF ΔH1-3 (blue boxes and whiskers and trace, where n = 5, 4, 6, 6, 8, 6, 6, 6, 6, 6, 6 images). Whiskers extend from the box to the furthest point within 1.5x the inter-quartile range. The black arrow highlights the increased NPM1 saturation concentration, ΔC sat , upon substitution of p14 ARF hydrophobic residues to Gly/Ser spacer residues. The gray arrow highlights the reentrant phase transition, which occurs at elevated p14 ARF concentrations. D ΔG tr for NPM1 in condensates with p14 ARF (gray boxes, whiskers and trace, where n = 696, 42, 61, 159, 225, 285, 333, 276, 306, 227, 773 condensates) and p14 ARF ΔH1-3 (blue boxes, whiskers and trace, where n = 2561, 1787, 29, 31, 82, 92, 134, 153, 166, 145, 162 condensates). Whiskers extend from the box to the furthest point within 1.5x the inter-quartile range. The C sat for NPM1 increases when p14 ARF hydrophobic residues are substituted. The gray arrow highlights the destabilization of NPM1 during the reentrant phase transition. E CV-SANS curves for p14 ARF ΔH1-3-[ 2 H]-NPM1 condensates collected at 50% D 2 O, where p14 ARF ΔH1-3 is contrast matched ([ 2 H]-NPM1 detected; green trace), at 85% D 2 O where [ 2 H]-NPM1 is contrast matched (p14 ARF ΔH1-3 detected; blue trace), and p14 ARF ΔH1-3-NPM1 condensate at 100% D 2 O for full scattering intensity (NPM1 and p14 ARF ΔH1-3 detected; gray trace). All curves are offset for clarity. Scatter points represent the average, the error bars represent the uncertainty derived from the counting statistics of the SANS instrument, as described and cited in the Methods. F Schematic describing condensed NPM1 with extended IDR conformations. G Schematic describing condensed p14 ARF ΔH1-3 in an extended conformation. H FRAP of NPM1-AF488 within condensates shows that substitution of p14 ARF hydrophobic residues to Gly/Ser spacer residues restores NPM1 mobility. Scale bars = 1 µm. I FRAP recovery curves for p14 ARF -NPM1 and p14 ARF ΔH1-3-NPM1 condensates with fits, as described in Methods (statistical significance was assessed by two-sided Wilcoxon rank-sum test, n = 10 curves for each condition, the p -value is shown in the figure). J NPM1-AF488 D App values extracted from the FRAP recovery curves in panel I (statistical significance was assessed by two-sided Wilcoxon rank-sum test, n = 10 D App values for each condition, the p -value is shown in the figure).

Article Snippet: The primary antibodies were used as follows: rabbit monoclonal anti-Cyclophilin B (Cell Signaling, 43603) at 1:1500–1:2000 dilution; rabbit monoclonal anti-GAPDH (Cell Signaling, 5174) at 1:2500 dilution; mouse monoclonal anti-NPM1 (ThermoFisher Scientific, 32-5200) at 1:1000 dillution; mouse monoclonal anti-p14 Arf (Cell Signaling, 2407) at 1:1000–1:15000 dilution; mouse monoclonal anti-GAPDH (Santa Cruz, sc-47724) at 1:2500 dilution; and rabbit polyclonal anti-p14 Arf (Novus, NB200-111) at 1:2000 dilution.

Techniques: Cider Assay, Residue, Fluorescence, Dispersion, Derivative Assay, Concentration Assay, Sublimation

Journal: Nature Communications

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.1038/s41467-024-53904-z

Figure Lengend Snippet: A Zoomed in regions from fluorescence microscopy images of live DLD-1 NPM1-G (clone B11) cells, before and after 48 h of doxycycline induced p14 ARF -iRFP expression. Scale bars = 2 µm. B Z-score analysis of NPM1-GFP and p14 ARF -iRFP levels in DLD-1 NPM1-G cells, showing that p14 ARF and NPM1 levels are anti-correlated (statistical significance was assessed by two-sided Mann–Whitney U-test, n = 2272, 122, 54 cells, p -values are shown in the figure) C FRAP curves with fits, as described in Methods, for cells sorted from the DLD-1 NPM1-G population shown in B. The curves on the left are from a cell expressing a high level of nucleolar NPM1 (clone F6; green trace) and a low level of nucleolar p14 ARF (clone F6; blue trace). The curves on the right are from a cell expressing a low level of nucleolar NPM1 (clone G2; green trace) and a high level of nucleolar p14 ARF (clone G2; blue traces). In unsorted DLD-1 NPM1-G cells, D The D App and E mobility for nucleolar NPM1-GFP and p14 ARF -iRFP (small green and blue transparent markers, respectively, n = 45 cells) are reduced as nucleolar p14 ARF -iRFP levels increase. Reductions also occur as the duration of p14 ARF -iRFP expression is extended (large opaque markers; scatter points represent the mean and error bars represent the standard deviation, where n = 20 cells). F A schematic describing the correlated reductions in p14 ARF and NPM1 dynamics and their assembly into large molecular weight complexes within the granular component (GC) of the nucleolus.

Article Snippet: The primary antibodies were used as follows: rabbit monoclonal anti-Cyclophilin B (Cell Signaling, 43603) at 1:1500–1:2000 dilution; rabbit monoclonal anti-GAPDH (Cell Signaling, 5174) at 1:2500 dilution; mouse monoclonal anti-NPM1 (ThermoFisher Scientific, 32-5200) at 1:1000 dillution; mouse monoclonal anti-p14 Arf (Cell Signaling, 2407) at 1:1000–1:15000 dilution; mouse monoclonal anti-GAPDH (Santa Cruz, sc-47724) at 1:2500 dilution; and rabbit polyclonal anti-p14 Arf (Novus, NB200-111) at 1:2000 dilution.

Techniques: Fluorescence, Microscopy, Expressing, MANN-WHITNEY, Standard Deviation, Molecular Weight

Journal: bioRxiv

Article Title: Elevated type I interferon signaling defines the proliferative advantage of ARF and p53 mutant tumor cells

doi: 10.1101/2024.11.11.623046

Figure Lengend Snippet: p53 and CDKN2A status affects overall survival of human breast and lung cancer patients and cells from these cancers are sensitive to JAK1 inhibition. A TCGA data from breast cancer patients was analyzed for overall survival in p53 mutant (134 cases) and p53 mutant with CDKN2A loss (197 cases). P =0.009. B Proteins isolated from mouse 4T1 breast cancer cells were separated by SDS-PAGE and immunoblotted with the indicated antibodies and compared to Δp53-Ras-shScr or –shARF cells. C Five-day proliferation assay performed with cells described in (B). D Representative image of foci assay performed with cells described in (B). Quantification of three independent measurements is shown with ****= P <0.001. Error bars represent standard deviation (SD) of n=3. E TCGA data from non-small cell lung cancer patients was analyzed for overall survival in p53 mutant (1446 cases) and p53 mutant with CDKN2A loss (1013 cases). P =2.32e-11. F Lung adenocarcinoma tissues were immunostained with antibodies recognizing p14ARF and ISG15. High and low expression for each protein is depicted from 24 total samples. G Proteins isolated from human lung cancer cells were separated by SDS-PAGE and immunoblotted with the indicated antibodies. H Representative image of foci assay performed with H838 and HCC827 cells that were treated with DMSO or 1μM baricitinib. Quantification of three biological replicates performed in triplicate is shown with indicated P values. Error bars represent standard deviation of n=3.

Article Snippet: Rabbit anti-p14ARF (Bethyl) and mouse anti-ISG15 (Santa Cruz) were used at a 1:200 dilution.

Techniques: Inhibition, Mutagenesis, Isolation, SDS Page, Proliferation Assay, Standard Deviation, Expressing