Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Cell proliferation of PANC-1 cells stably expressing empty expression vector, codon-optimized CDKN2A, one of three synonymous variants (p.L32L, p.G101G, p.V126V), or one of three pathogenic variants (p.L32P, p.G101W, p.V126D) over 14 days in culture. Cell proliferation values are given as mean of three repeats ± standard deviation normalized to PANC-1 cells that stably express empty vector. Statistically significant inhibition of cell proliferation inhibition in PANC-1 cells that stably express synonymous variants (*; p-value<0.001; Students t-test). ( B ) PANC-1 cells stably expressing codon-optimized CDKN2A transduced with a CellTag lentiviral library of 20 nonfunctional barcodes were cultured and representation (percent of reads supporting each barcode) before (day 9) and after a period of in vitro cell proliferation (day 45) was determined using next-generation sequencing. Percent values are given as the mean of three repeats ± standard deviation. Figure 1—figure supplement 1—source data 1. Raw data in . Figure 1—figure supplement 1—source data 2. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Standard Deviation, Inhibition, Transduction, Cell Culture, In Vitro, Next-Generation Sequencing

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: PANC-1 cell stably expressing 1 of 20 CDKN2A variants, 19 missense variants, and 1 synonymous variant, at residue p.V126 or p.R144 were cultured. Variant representation, as the percent of reads supporting the variant sequence, before and after a period in vitro cell proliferation determined by next-generation sequencing for the two residues, p.V126 ( A ) or p.R144 ( B ). CDKN2A variant p.V126D (*) was previously reported as pathogenic and increased representation during in vitro proliferation. CDKN2A variant p.R144C (**) was previously reported as benign variant and maintained representation during in vitro proliferation. Figure 1—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Stable Transfection, Expressing, Variant Assay, Residue, Cell Culture, Sequencing, In Vitro, Next-Generation Sequencing

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Functional classifications for 3120 CDKN2A variants, including 2964 missense variants and 156 synonymous variants. Variants were classified as functionally deleterious, indeterminate function, or neutral based on p-value using gamma generalized linear model (GLM). 525 (17.7%) variants were classified as functionally deleterious. ( B ) Log 2 p-value (gamma GLM) for 32 benchmark pathogenic variants, 6 benign variants, 31 variants of uncertain significance (VUSs) previously reported to have functionally deleterious effects, and 18 VUSs previously reported to have functionally neutral effects. ( C ) Heatmap with p-values (gamma GLM) for all 3120 CDKN2A variants assayed. Figure 2—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Functional Assay

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Distribution of log 2 p-value (gamma GLM) for all possible CDKN2A missense variants. ( B ) Distribution of log 2 p-value (gamma GLM) for benchmark pathogenic variants (red box), benchmark benign variants (blue box), variants of uncertain significance (VUSs) previously reported to have functionally deleterious effects (orange box), and VUSs previously reported to have functionally neutral effects (green box). ( C ) Dot plot showing log 2 p-value (gamma GLM) of all possible CDKN2A missense variants per residue. Figure 2—figure supplement 1—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Residue

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Dot plot showing log 2 normalized fold change of all possible CDKN2A missense variants by residue. ( B ) Log 2 normalized fold change for 32 benchmark pathogenic variants, 6 benign variants, 31 variants of uncertain significance (VUSs) previously reported to have functionally deleterious effects, and 18 VUSs previously reported to have functionally neutral effects. ( C ) Functional classifications for 3120 CDKN2A variants, including 2964 missense variants and 156 synonymous variants. Variants were classified as functionally deleterious, indeterminate function, or neutral based on log 2 normalized fold change. ( D ) Comparison of functional classification of all possible CDKN2A missense variants by log 2 p-value (gamma GLM) and log normalized fold change. Figure 2—figure supplement 2—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Residue, Functional Assay, Comparison

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Dot plot showing log 2 p-value (gamma GLM) for 560 CDKN2A missense variants assayed in duplicate. ( B ) Comparison of functional classifications for 560 CDKN2A missense variants assayed in duplicate. ( C ) Dot plot showing log 2 normalized fold change for 560 CDKN2A missense variants assayed in duplicate. Figure 2—figure supplement 3—source data 1. Raw data in . Figure 2—figure supplement 3—source data 2. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Comparison, Functional Assay

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Proportion of all possible 2964 CDKN2A missense variants in the day 9 cell pool (replicate 1 if duplicated). ( B ) Percent of functionally deleterious variants (black box), variants of indeterminate function, and functionally neutral variants (white box) by variant proportion in the day 9 cell pool (replicate 1 if duplicated). Left graph variants grouped as <2% and ≥2% in day 9 cell pool. Right graph, variants grouped as <2%, 1% intervals from 2% to 8%, ≥8% in the day 9 cell pool. Figure 2—figure supplement 4—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Variant Assay

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: Variant effect predictions for CDKN2A missense variants using CADD, PolyPhen-2, SIFT, VEST, AlphaMissense, ESM1b, and PrimateAI-3D. Predicted deleterious, damaging, or pathogenic effects (black box) and predicted neutral, tolerated, benign, or ambiguous effects (white box) presented as percent of missense variants with an available prediction. Number of missense variants with an available prediction for each in silico model given in parentheses. Accuracy shown as a red line. CADD: Combined Annotation Dependent Depletion; PolyPhen-2: Polymorphism Phenotyping v2; SIFT: Sorting Intolerant From Tolerant; VEST: Variant Effect Scoring Tool score. Figure 3—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Variant Assay, In Silico

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Schematic representation of CDKN2A with ankyrin repeats 1–4 represented. ( B ) Percent of functionally deleterious (black box), indeterminate function (gray box), and functionally neutral variants (white box) within ankyrin repeats and non-ankyrin repeat regions of CDKN2A. Ank; ankyrin repeat. ( C ) Dot plot showing distribution of percent functionally deleterious missense variants per residue. Figure 2—figure supplement 5—source data 1. Raw data in . Figure 2—figure supplement 5—source data 2. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Residue

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Number of algorithms predicting deleterious effect for 904 CDKN2A missense variants with predictions from seven algorithms. ( B ) Percent of functionally deleterious (black box) and indeterminate function or functionally neutral (white box) variants grouped by the number of algorithms predicting deleterious effect. ( C ) Number of algorithms predicting deleterious effect for 904 CDKN2A missense variants grouped by ankyrin repeats and non-ankyrin repeat regions. ( D–H ) Percent of functionally deleterious (black box) and indeterminate function or functionally neutral (white box) variants grouped by the number of algorithms predicting deleterious effect in Ank1 ( D ), Ank2 ( E ), Ank3 ( F ), Ank4 ( G ), and non-ankyrins repeat regions ( H ) of CDKN2A. Figure 3—figure supplement 1—source data 1. Raw data in . Figure 3—figure supplement 1—source data 2. Raw data in . Figure 3—figure supplement 1—source data 3. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques:

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Number of algorithms predicting deleterious effect for 2060 CDKN2A missense variants with predictions from five algorithms. ( B ) Percent of functionally deleterious (black box) and indeterminate function or functionally neutral (white box) variants grouped by the number of algorithms predicting deleterious effect. ( C ) Number of algorithms predicting deleterious effect for 2060 CDKN2A missense variants grouped by ankyrin repeats and non-ankyrin repeat regions. ( D–H ) Percent of functionally deleterious (black box) and indeterminate function or functionally neutral (white box) variants grouped by the number of algorithms predicting deleterious effect in Ank1 ( D ), Ank2 ( E ), Ank3 ( F ), Ank4 ( G ), and non-ankyrins repeat regions ( H ) of CDKN2A. Figure 3—figure supplement 2—source data 1. Raw data in . Figure 3—figure supplement 2—source data 2. Raw data in . Figure 3—figure supplement 2—source data 3. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques:

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Somatic missense variants in CDKN2A reported in COSMIC, TCGA, JHU, or MSK-IMPACT, by functional classification (deleterious – black box; indeterminate – gray box; neutral – white box). ( B ) Distribution of functionally deleterious missense somatic mutations CDKN2A reported in COSMIC, TCGA, JHU, or MSK-IMPACT by ankyrin (ANK) repeat. ( C ) Percent of missense somatic mutations in CDKN2A that were classified as functionally deleterious (black box), indeterminate function (gray box), or functionally neutral (white box) group by tumor type. Missense somatic mutations reported in COSMIC, TCGA, JHU, and MSK-IMPACT were combined. The number of missense somatic mutations for each tumor type given in parentheses. COSMIC; the Catalogue Of Somatic Mutations In Cancer, TCGA; The Cancer Genome Atlas, JHU; The Johns Hopkins University School of Medicine, MSK-IMPACT; Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets. Figure 4—source data 1. Raw data in . Figure 4—source data 2. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Functional Assay, Mutagenesis

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Percent of missense somatic mutations in CDKN2A reported in either COSMIC, TCGA, JHU, or MSK-IMPACT that were classified as pathogenic or likely pathogenic (black box), variant of uncertain significance (VUS) (gray box), or benign or likely benign (white box) using American College of Medical Genetics (ACMG) interpretation guidelines. ( B ) Percent of missense somatic mutations in CDKN2A that were classified as pathogenic or likely pathogenic (black box), VUS (gray box), or benign or likely benign (white box) using ACMG interpretation guidelines grouped by mutation database. ( C ) Number of patients with a pathogenic or likely pathogenic missense somatic mutation grouped by mutation database. Patients with p.His83Tyr mutation (black box), patients with p.Asp84Asn mutations (gray box), and patients with other mutations highlighted. COSMIC: the Catalogue Of Somatic Mutations In Cancer; TCGA: The Cancer Genome Atlas; JHU: The Johns Hopkins University School of Medicine; MSK-IMPACT: Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets. Figure 4—figure supplement 1—source data 1. Raw data in . Figure 4—figure supplement 1—source data 2. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Variant Assay, Mutagenesis

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: Percent of missense somatic mutations in CDKN2A reported in either COSMIC ( A ), TCGA ( B ), JHU ( C ), or MSK-IMPACT ( D ) that were classified as functionally deleterious (black box), indeterminate (gray box), or functionally neutral (white box) in our CDKN2A functional assay grouped by tumor type. The number of missense somatic mutations for each tumor type given in parentheses. COSMIC: the Catalogue Of Somatic Mutations In Cancer; TCGA: The Cancer Genome Atlas; JHU: The Johns Hopkins University School of Medicine; MSK-IMPACT: Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets. Figure 4—figure supplement 2—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Functional Assay, Mutagenesis

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Synonymous and missense variants in CDKN2A reported in gnomAD. ( B ) 287 CDKN2A missense variants reported in gnomAD, by American College of Medical Genetics (ACMG) guideline classification. ( C ) 264 missense variants in CDKN2A reported in gnomAD, by functional classification (deleterious – black box; indeterminate – gray box; neutral – white box). ( D ) 395 missense variants in CDKN2A reported in ClinVar, by functional classification (deleterious – black box; indeterminate – gray box; neutral – white box).

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Functional Assay

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Recombinant, Plasmid Preparation, Expressing, Sequencing, Mutagenesis, Modification, Transfection, Software, Control

Journal: PLOS Biology

Article Title: Adenosine diphosphate released from stressed cells triggers mitochondrial transfer to achieve tissue homeostasis

doi: 10.1371/journal.pbio.3002753

Figure Lengend Snippet: (A) Representative confocal live cell images of Cox8-labeled (Dendra2) mitochondria in MLO-Y4 cell dendrites treated with vehicle, 0.2 μm or 2 μm ADP with 0.5 h or 2 h incubation. (B) Number of mitochondria per dendrites in MLO-Y4 cells. (C) Number of dendrites per cell. (D) Diagram of the Transwell 3D co-culture model (inner side: healthy osteocytes; outer side: stressed cells). (E and F) Live cell imaging and semiquantitative analysis showed attenuated ROS accumulation in the 0.2 μm ADP treatment group ( n = 75). (G) Representative confocal images of co-culture systems of stressed cells (F-actin labeled: mCherry) and healthy cells (Cox8 labeled: Dendra2), which are stimulated by ADP. (H) Number of transferred mitochondria per unstressed cell with or without stimulation of ADP ( n = 15). (I) Number of transferred mitochondria per stressed cell, with or without stimulation of ADP ( n = 15). (J) OCR of the coculture model containing 75%, 50% healthy cells with Rhot1 being knocked down using shRNA ( n = 3). (K–M) Basal, maximal, and spare respiratory capacity of models containing different ratios of healthy cells with or without ADP stimulation. Oligo stands for oligomycin A. OCR stands for oxygen consumption rate. Rhot1 stands for Rhot1-knocked-down cells. FCCP stands for mitochondrial oxidative phosphorylation uncoupler; Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; h stands for hour. The data underlying the graphs shown in the figure can be found in . ADP, adenosine diphosphate; ROS, reactive oxygen species.

Article Snippet: By adopting genetical modification of cells, we had developed a cell line of MLO-Y4 cells with Cox8, a subunit located in mitochondrion, labeled by Dendra2 fluorescent protein (plasmid: mito-dendra2, Addgene plasmid #55796; Vector: pHAGE Lentivirus vector) [ ].

Techniques: Labeling, Incubation, Co-Culture Assay, Live Cell Imaging, shRNA, Phospho-proteomics

Journal: PLOS Biology

Article Title: Adenosine diphosphate released from stressed cells triggers mitochondrial transfer to achieve tissue homeostasis

doi: 10.1371/journal.pbio.3002753

Figure Lengend Snippet: (A and B) The level of extracellular ADP and ATP in the bone fragment supernatant of 1-month and 18-month mice ( n = 3). (C) Immunostaining of ATP5β and confocal imaging of cortical bone from 1-month-old and 18-month-old mice. The sectional area is divided into 2 parts along the longitudinal axis from the outer layer with 20 μm width for the upper and middle parts. (D) Fluorescent intensity of ATP5β in cortical bone from 1-month and 18-month mice. (E) Respective fluorescent intensity of ATP5β in upper and middle regions of cortical bone from 1-month and 18-month mice. (F) Diagram of supernatant transfer, cell culture, and RNA-Seq analysis. (G) Selected mitochondrial function-related GO analysis. Genes were significantly up-regulated in osteocytes treated with supernatant from 18-month-old mice compared with those in osteocytes treated with supernatant from 1-month-old mice. (H) Volcano map of RNA-seq expression data of genes related to mitochondrial motility in bones from 1-month-old mice treated with bone fragment supernatant from 1-month-old or 18-month-old mice. Mfn1, Mfn2, Miro2, TRAK1, and Kif5c were significantly down-regulated in samples treated with bone fragment supernatant from 1-month-old mice ( n = 3). (I) Diagram of supernatant transfer, cell culture, and live cell imaging. Cortical bone fragments from both young and aging mice were pretreated with ADP647 and medium containing residual ADP647 that was collected following different time courses stimulated MLO-Y4 cells. (J) Representative confocal images of MLO-Y4 cells treated with medium from 1-month or 18-month mouse bone particles. (K and L) Quantification of ADP647 fluorescence intensity from 30 to 70 min in MLO-Y4 cells treated with bone fragment supernatants from 1-month-old and 18-month-old mice. (M) Representative confocal images of co-culture systems of stressed cells (F-actin labeled: mCherry) and healthy cells (Cox8 labeled: Dendra2), which are stimulated young and old osteocytes supernatant, respectively. (N) Number of transferred mitochondria per unstressed cell with stimulation of young and old osteocytes supernatants ( n = 20). (O) Number of transferred mitochondria per stressed cell, with stimulation of young and old osteocytes supernatants. Oligo stands for oligomycin A. Young stands for osteocytes supernatant from 1-month mice. Old stands for osteocytes supernatant from 18-month mice; ( n = 20). (P) Diagram of the 3D Transwell co-culture model. (Inner side: healthy osteocytes; outer side: stressed cells.) (Q and R) Representative live cell imaging and quantitation analysis showed attenuated ROS accumulation in the group treated with the supernatant from 18-month-old mice ( n = 75). The data underlying the graphs shown in the figure can be found in . ADP, adenosine diphosphate; GO, gene ontology; ROS, reactive oxygen species.

Article Snippet: By adopting genetical modification of cells, we had developed a cell line of MLO-Y4 cells with Cox8, a subunit located in mitochondrion, labeled by Dendra2 fluorescent protein (plasmid: mito-dendra2, Addgene plasmid #55796; Vector: pHAGE Lentivirus vector) [ ].

Techniques: Immunostaining, Imaging, Cell Culture, RNA Sequencing, Expressing, Live Cell Imaging, Fluorescence, Co-Culture Assay, Labeling, Cell Stimulation, Quantitation Assay