mscv-egfp  (Addgene inc)

Journal: Cell Genomics

Article Title: Phenotypic pleiotropy of missense variants in human B cell confinement receptor P2RY8

doi: 10.1016/j.xgen.2025.100981

Figure Lengend Snippet: Structural features of GGG recognition (A and B) Close-up view of the GGG binding site in P2RY8. (A) Expression mean Z scores mapped onto P2RY8 residues (sticks/ribbon) that form the GGG binding site (orange sticks/spheres). (B) Migration mean Z scores mapped onto P2RY8 residues (sticks/ribbon) that form the GGG binding site (orange sticks/spheres). (C) Plot comparing expression and migration mean Z scores by position; GGG-interacting positions are colored red. (D) Distribution of expression-adjusted migration scores for positions of specified categories. Whiskers extend to maximum and minimum, and boxes shows 25%, median, and 75%. Kruskal-Wallis test with Dunn’s multiple comparisons test; ns, nonsignificant. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (E) Migration of P2RY8-transduced WEHI-231 cells in the presence of 50 ng mL −1 CXCL12 with or without GGG or candidate P2RY8 ligands of varying concentrations, normalized to migration to CXCL12 alone. Representative results are from one of two independent experiments. G-S, glutathione; LTC 4 , leukotriene C 4 . See also .

Article Snippet: The WT P2RY8 MSCV was digested with BglII and NotI and K180I or W181M P2RY8 PCR products (from the constructs detailed in Individual variant validation above) were inserted using HiFi DNA Assembly Kit (NEB).

Techniques: Binding Assay, Expressing, Migration

Journal: Cell Genomics

Article Title: Phenotypic pleiotropy of missense variants in human B cell confinement receptor P2RY8

doi: 10.1016/j.xgen.2025.100981

Figure Lengend Snippet: DMS of P2RY8 across three phenotypes (A) Lentiviral vector schematic and DMS approach. OX56 is a peptide tag, and PRL is a preprolactin signal peptide. (B) Heatmaps of surface expression, migration, and proliferation Z scores; expression-adjusted migration and expression-adjusted proliferation scores; and accompanying line plots of mean scores for each position. (C) Plot comparing missense variant expression and migration Z scores colored by domain, table of variant counts, and boundaries at Z scores of 2 and −2. (D) Plot comparing mean expression and mean migration Z scores (averaged across all missense variants at each position), colored by domain. (E) Mean expression Z scores, mean expression-adjusted migration scores, and mean expression-adjusted proliferation scores for each position, partitioned by domain; lines are medians. Negative scores are LoF for expression, GoF for migration or proliferation. Welch ANOVA test with Dunnett’s T3 multiple comparisons test; for each assay TM compared to the five other domains; adjusted ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗p < 0.0001. C, C terminus; CTD, C-terminal domain; EC, extracellular; GoF, gain of function; H8, helix 8; IC, intracellular; LoF, loss of function; N, N terminus; NTD, N-terminal domain; TM, transmembrane; ρ, Spearman correlation coefficient. See also and .

Article Snippet: The WT P2RY8 MSCV was digested with BglII and NotI and K180I or W181M P2RY8 PCR products (from the constructs detailed in Individual variant validation above) were inserted using HiFi DNA Assembly Kit (NEB).

Techniques: Plasmid Preparation, Expressing, Migration, Variant Assay

Journal: Cell Genomics

Article Title: Phenotypic pleiotropy of missense variants in human B cell confinement receptor P2RY8

doi: 10.1016/j.xgen.2025.100981

Figure Lengend Snippet: Improving computational VEP using limited experimental data (A–C) Plots of missense variants comparing ESM1b scores and (A) expression Z score, (B) migration Z score, and (C) proliferation Z score, colored by domain. (D–F) Plots of positions comparing mean ESM1b score and (D) mean expression Z score, (E) mean migration Z score, and (F) mean proliferation Z score, colored by domain. (G) Plot of Spearman correlation between ESM1b score and variant expression, migration, and proliferation Z scores, partitioned by domain. (H) Plot comparing the distribution of variant ESM1b scores, partitioned by DMS phenotype. Whiskers extend to maximum and minimum, and boxes show 25%, median, and 75%. (I) Plot of Spearman correlation between fine-tuned ESM1b scores and DMS Z scores as training set size varies. Shows mean and SD. (J) Plot comparing Spearman correlation when using ESM1b score or fine-tuned ESM1b scores with 2 variants/position or 11 variants/position training sets, partitioned by domain. Shown are means and SD. (K) Plot comparing distribution of fine-tuned ESM1b scores for representative 2 variant per position training and representative 11 variant per position training, partitioned by DMS phenotype. Whiskers extend to maximum and minimum, and boxes shows 25%, median, and 75%. (L) Plot comparing Spearman correlations between human GPR68 DMS results and base ESM1b or ESM1b fine-tuned on P2RY8 expression results for 50 or 300 fine-tuning steps. (M) Plots showing difference in Spearman correlation between base ESM1b and 50-step fine-tuned ESM1b on GPR68 surface expression (L) or pH 5.5 signaling (R) (a value >0 indicates greater correlation with fine-tuning). In (G), (I), and (J), sign is inverted for migration and proliferation, so all three correlations have the same sign. GoF, Z score >2 for expression, < −2 for migration, proliferation; LoF, Z score < −2 for expression, >2 for migration, proliferation. See also and .

Article Snippet: The WT P2RY8 MSCV was digested with BglII and NotI and K180I or W181M P2RY8 PCR products (from the constructs detailed in Individual variant validation above) were inserted using HiFi DNA Assembly Kit (NEB).

Techniques: Expressing, Migration, Variant Assay

Journal: Cell Genomics

Article Title: Phenotypic pleiotropy of missense variants in human B cell confinement receptor P2RY8

doi: 10.1016/j.xgen.2025.100981

Figure Lengend Snippet: Validation of variants with independent effects on expression and function (A) Left: plot of positions comparing expression and migration mean Z score; those with a high expression-adjusted migration scores are colored red. Right: expression-adjusted migration score for each position, partitioned by domain; lines are medians. (B) Plot of positions comparing migration and proliferation mean Z scores, those with a high migration-adjusted proliferation score are colored red. (C) Plots of missense variants comparing expression and migration Z scores (left) or migration and proliferation Z scores (right), with select variants labeled. (D) P2RY8 surface expression normalized to WT P2RY8; lines are means. Shown are pooled results from 9 experiments, total 5–26 biological replicates per condition. (E) Plot of DMS expression Z score and WT-normalized expression for select variants. Circles are those in D, with corresponding colors, and crosses are variants assayed previously. , (F) Migration of cells toward CXCL12 in the presence of GGG relative to DMSO (vehicle); each dot is a biological replicate, and lines are means. Shown are pooled results from 9 experiments, total 4–23 biological replicates per condition. (G) Plot of DMS migration Z score and migration indices for variants shown in (F), with corresponding colors. (H) Proliferation over 13 days relative to untransduced cells; each point is a biological replicate, lines are means. Shown are pooled results from 8 experiments, total 5–21 biological replicates per condition. (I) Plot of DMS proliferation Z score and proliferation results for variants as shown in (H), with corresponding colors. (D, F, and H) One-way ANOVA test with Dunnett’s multiple comparisons test, each variant compared to the WT; adjusted ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (E, G, and I) Two-tailed p values; r, Pearson correlation coefficient. See also .

Article Snippet: The WT P2RY8 MSCV was digested with BglII and NotI and K180I or W181M P2RY8 PCR products (from the constructs detailed in Individual variant validation above) were inserted using HiFi DNA Assembly Kit (NEB).

Techniques: Biomarker Discovery, Expressing, Migration, Labeling, Variant Assay, Two Tailed Test

Journal: Cell Genomics

Article Title: Phenotypic pleiotropy of missense variants in human B cell confinement receptor P2RY8

doi: 10.1016/j.xgen.2025.100981

Figure Lengend Snippet: Structure of activated ligand-bound human P2RY8 (A and B) Cryo-EM density map (A) and ribbon model (B) of active human P2RY8 bound to GGG (orange). P2RY8 is fused to miniGα 13 (green) and bound to G β 1 (gray). (C–E) Ribbon model of P2RY8, colored to indicate DMS mean scores for (C) expression, (D) expression-adjusted migration, and (E) expression-adjusted proliferation. Gray, no data available. (F–H) Expression-adjusted migration scores for individual residues with close-up views of (G) the D(E)-R-Y motif and (H) the G protein interface. (I) Plot comparing mean expression Z scores and mean migration Z scores by position; Gα 13 -interacting positions are colored red. See also .

Article Snippet: The WT P2RY8 MSCV was digested with BglII and NotI and K180I or W181M P2RY8 PCR products (from the constructs detailed in Individual variant validation above) were inserted using HiFi DNA Assembly Kit (NEB).

Techniques: Cryo-EM Sample Prep, Expressing, Migration

Journal: Cell Genomics

Article Title: Phenotypic pleiotropy of missense variants in human B cell confinement receptor P2RY8

doi: 10.1016/j.xgen.2025.100981

Figure Lengend Snippet: In vitro and in vivo delineation of heterogeneous variants effects (A) Migration of transduced Ly8 cells toward CXCL12 with varying concentrations of GGG relative to DMSO (vehicle) and normalized to untransduced cells; shown are means and SEMs. Pooled results are from 10 experiments, total 2–22 (mean 9.5, SD 4.9) replicates per condition. (B) Effect of GGG on proliferation over 7 days relative to untransduced cells; shown are means and SDs. Pooled results are from 5 experiments, total 6–12 replicates per condition. (C and D) Transduced Ly8 cells exposed to GGG or vehicle were assayed using phospho-flow cytometry; graphs show the ratio of pAkt + (C) or pErk + (D) cells after GGG vs. vehicle, normalized to GFP vector. Shown are means and SDs. Pooled results are from 4 experiments, total 6 replicates per condition. (E) BRET trimeric G protein activation assay: ratio of luminescence and GFP fluorescence normalized to DMSO (vehicle) condition. Shown are means and SDs. Pooled results are from 4 experiments, total 9–12 replicates per condition. (F) NanoBiT β-arrestin recruitment assay: luminescence relative to DMSO (vehicle) is plotted. Shown are means and SDs. Pooled results are from 4 experiments, total 12 replicates per condition. (G) P2RY8 surface expression of transduced Ly8 cells after 45 min GGG treatment. Pooled from 3 experiments, total 6–8 replicates per condition. (H) Ratio of GFP+ cell frequency among GC and follicular B cells in Peyer’s patches of irradiated CD45.1 mice reconstituted with bone marrow transduced with EV-GFP, WT-P2RY8-GFP, K180I-GFP, or W181M-GFP. Each point is a mouse. Mean and SD are shown. Pooled from 3 experiments, total 4–8 mice per condition. (I) Representative immunofluorescence micrographs. Polyclonal B cells transduced with K180I-GFP or W181M-GFP were co-transferred with WT-TagBFP into SRBC-immunized recipient mice. Green, anti-GFP; magenta, anti-TagBFP; yellow, anti-CR1 (GC label); and blue, immunoglobulin D (IgD; follicular B cell label). Scale bar, 100 μm, both images are of the same scale. (J) Ratio of transduced cells within the GC to those within the follicle (including GC). A given mouse is represented by one WT dot and one K180I or W181M dot. Two independent experiments, one recipient mouse per variant per experiment, at least 45 GCs analyzed per mouse. For (A) and (B), two-sided p value from z test comparison with WT with Benjamini-Hochberg multiple comparisons adjustment; E max and IC 50 were determined by non-linear regression (R). For (E) and (F) Curves were fit using log(agonist) vs. response, 3-parameter mode (GraphPad Prism). For (C), (D), (H), and (J), one-way ANOVA test with Dunnett’s multiple comparisons test, each variant compared to WT; adjusted p value: ns, p > 0.05, ∗∗ p <0.01, ∗∗∗ p <0.001, ∗∗∗∗ p <0.0001. See also .

Article Snippet: The WT P2RY8 MSCV was digested with BglII and NotI and K180I or W181M P2RY8 PCR products (from the constructs detailed in Individual variant validation above) were inserted using HiFi DNA Assembly Kit (NEB).

Techniques: In Vitro, In Vivo, Migration, Flow Cytometry, Plasmid Preparation, Activation Assay, Fluorescence, Expressing, Irradiation, Transduction, Immunofluorescence, Variant Assay, Comparison

Journal: Cell Genomics

Article Title: Phenotypic pleiotropy of missense variants in human B cell confinement receptor P2RY8

doi: 10.1016/j.xgen.2025.100981

Figure Lengend Snippet: Phenotypes of germline and lymphoma-associated P2RY8 variants (A) Plots of missense variants, comparing expression and migration (left) and migration and proliferation (right) of variants, with gnomAD variants colored by domain. (B) Plot comparing allele frequency for missense variants in gnomAD by position and mean migration Z score for each position. p value was calculated using algorithm AS 89 with Edgeworth series approximation. (C and D) Plots of missense variants, comparing expression and migration (left) and migration and proliferation Z scores (right), with (C) 48 variants reported in DLBCL or Burkitt lymphoma colored red and (D) non-hematologic cancer variants colored blue. (E) Cumulative density plots of expression, migration, and proliferation Z scores and expression-adjusted migration scores for all missense variants (black), gnomAD (green), DLBCL/Burkitt lymphoma (red), and non-hematologic cancer (blue). See also .

Article Snippet: The WT P2RY8 MSCV was digested with BglII and NotI and K180I or W181M P2RY8 PCR products (from the constructs detailed in Individual variant validation above) were inserted using HiFi DNA Assembly Kit (NEB).

Techniques: Expressing, Migration