Journal: Molecular medicine reports

Article Title: Overexpression of salusin‑β downregulates adipoR1 expression to prevent fatty acid oxidation in HepG2 cells.

doi: 10.3892/mmr.2023.13141

Figure Lengend Snippet: Figure 2. Packaging and identification of the recombinant lentiviruses. (A) Schematic representation of the process used for lentivirus synthesis. (B) Representative images of the cytopathic effect in 293T cells. On day 4 of lentivirus packaging, small lesions appeared in the corresponding plasmid‑transfected groups, whereby the transfected cells fused to form syncytia (indicated by black arrows). Scale bar, 100 µm. (C) Transmission electron microscopy images of 293T cells transfected with recombinant lentiviral vectors and control cells. Red arrows indicate lentiviral particles. Scale bar, 0.5 µm. (D) Gel electrophoresis of PCR target gene products from the lentiviral particles harvested from the 293T cell culture medium. (E) Gel electrophoresis and semi‑quantitative PCR of salusin‑β mRNA from 293T cells after lentivirus‑mediated transduction of the shRNA against salusin‑β or a sequence without a hairpin structure for 24 h. (F) Relative mRNA expression levels of salusin‑β in the different groups of transfected 293T cells. Data are presented as the mean ± standard error of the mean (n=3). ***P<0.001 vs. sh‑Mock group. Control, negative control group without lentiviral treatment; pHAGE, empty lentiviral treatment group used as a control for pHAGE‑Salusin‑β; sh‑Mock, lentiviral treatment group containing a non‑targeting shRNA sequence as a control for sh‑Salusin‑β; pHAGE‑Salusin‑β, lentiviral treatment group for overexpression of Salusin‑β; pLKO.1‑sh‑Salusin‑β1‑3 and sh‑Salusin‑β1‑3, lentiviral treatment group for interference of Salusin‑β. sh/shRNA, short hairpin RNA; puro, puromycin resistance; AMPr, ampicillin resistance; HIV‑1, human immunodeficiency virus type 1; pol, polymerase; VSV‑G, vesicular stomatitis virus G.

Article Snippet: Lentivirus vectors pHAGE (cat. no. 118692; Addgene, Inc.) and pLKO.1 (cat. no. 8453; Addgene, Inc.), envelope plasmids psPAX2 (cat. no. 12260; Addgene, Inc.) and pMD2G (cat. no. 12259; Addgene, Inc.), the pLKO.1‐sh‐Mock plasmid (containing a non‐mammalian targeting shRNA sequence 5'‐CCG GCA ACA AGA TGA AGA GCA CCA ACT CGA GTT GGT GCT CTT CAT CTT GTT GTT T TTG‐3'; cat. no. SHC002; Sigma‐Aldrich; Merck KGaA) and Top10 competent cells (cat. no. B528412; Sangon Biotech Co., Ltd.) were donated by the Basic Medicine Laboratory at Wuhan Huazhong University of Science and Technology (Wuhan, China).

Techniques: Recombinant, Transfection, Transmission Assay, Electron Microscopy, Control, Nucleic Acid Electrophoresis, Cell Culture, Transduction, shRNA, Sequencing, Expressing, Negative Control, Over Expression, Virus

Journal: Molecular medicine reports

Article Title: Overexpression of salusin‑β downregulates adipoR1 expression to prevent fatty acid oxidation in HepG2 cells.

doi: 10.3892/mmr.2023.13141

Figure Lengend Snippet: Figure 3. Salusin‑β downregulates adipoR1 expression in 293T cells. (A) Electrophoresis of the semi‑quantitative PCR products of salusin‑β and adipoR1 mRNA from 293T cells transfected with lentivirus for 24 h. The control group was not infected with lentivirus and the experimental groups were transfected with pHAGE, pHAGE‑Salusin‑β, pLKO.1‑sh‑Mock, or pLKO.1‑sh‑Salusin‑β lentivirus. (B) Relative mRNA expression levels of salusin‑β and adipoR1 in the different groups of transfected 293T cells. (C) Protein expression levels of salusin‑β and adipoR1 in 293T cells transfected with lentivirus for 24 h were analyzed by western blotting. (D) Semi‑quantification of the protein expression levels of salusin‑β and adipoR1. Data are presented as the mean ± standard error of the mean (n=3). ***P<0.001 vs. control group. AdipoR1, adiponectin receptor 1; sh, short hairpin RNA.

Article Snippet: Lentivirus vectors pHAGE (cat. no. 118692; Addgene, Inc.) and pLKO.1 (cat. no. 8453; Addgene, Inc.), envelope plasmids psPAX2 (cat. no. 12260; Addgene, Inc.) and pMD2G (cat. no. 12259; Addgene, Inc.), the pLKO.1‐sh‐Mock plasmid (containing a non‐mammalian targeting shRNA sequence 5'‐CCG GCA ACA AGA TGA AGA GCA CCA ACT CGA GTT GGT GCT CTT CAT CTT GTT GTT T TTG‐3'; cat. no. SHC002; Sigma‐Aldrich; Merck KGaA) and Top10 competent cells (cat. no. B528412; Sangon Biotech Co., Ltd.) were donated by the Basic Medicine Laboratory at Wuhan Huazhong University of Science and Technology (Wuhan, China).

Techniques: Expressing, Electrophoresis, Transfection, Control, Infection, Western Blot, shRNA

Journal: Molecular medicine reports

Article Title: Overexpression of salusin‑β downregulates adipoR1 expression to prevent fatty acid oxidation in HepG2 cells.

doi: 10.3892/mmr.2023.13141

Figure Lengend Snippet: Figure 4. Salusin‑β downregulates adipoR1 expression in HepG2 cells. (A) Protein expression levels of salusin‑β and adipoR1 in HepG2 cells transfected with lentivirus for 24 h were analyzed by western blotting. (B) Semi‑quantification of the protein expression levels of salusin‑β and adipoR1. Data are presented as the mean ± standard error of the mean (n=3). ***P<0.001 vs. control group. AdipoR1, adiponectin receptor 1; sh, short hairpin RNA.

Article Snippet: Lentivirus vectors pHAGE (cat. no. 118692; Addgene, Inc.) and pLKO.1 (cat. no. 8453; Addgene, Inc.), envelope plasmids psPAX2 (cat. no. 12260; Addgene, Inc.) and pMD2G (cat. no. 12259; Addgene, Inc.), the pLKO.1‐sh‐Mock plasmid (containing a non‐mammalian targeting shRNA sequence 5'‐CCG GCA ACA AGA TGA AGA GCA CCA ACT CGA GTT GGT GCT CTT CAT CTT GTT GTT T TTG‐3'; cat. no. SHC002; Sigma‐Aldrich; Merck KGaA) and Top10 competent cells (cat. no. B528412; Sangon Biotech Co., Ltd.) were donated by the Basic Medicine Laboratory at Wuhan Huazhong University of Science and Technology (Wuhan, China).

Techniques: Expressing, Transfection, Western Blot, Control, shRNA

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