Figure 4.. Terminal differentiation of T EFF cells is dependent upon Pofut1 and associated with

(A) Differentially expressed genes in sgPofut1- compared to sgNTC-transduced P14 cells at day 7.5 post-infection (p.i.). Upregulated (orange) or downregulated (blue) transcripts [false discovery rate (FDR) < 0.05] are highlighted. Selective MP- and TE-associated genes are labelled. (B) Enrichment plots of cell cycle-related signatures. NES, normalized enrichment score. (C) Flow cytometry (left) and quantification (right) of BrdU incorporation. (D) UMAP plot of published scRNA-seq dataset of P14 cells at day 8 p.i. (Chen et al., 2019). Each dot corresponds to an individual cell. The number and frequency of cells in each of the color-coded clusters (clusters 13) are indi cated. (E) Violin plots of Klrg1, Cxcr3 or Il7r expression in clusters 13 from (D). (F) Gating str ategy (left) and quantification (right) of the proportions of TE (KLRG1hiCXCR3loCD127lo), MP (KLRG1loCXCR3hiCD127hi) and TINT (CXCR3hiCD127lo) cells among WT P14 cells. (G) PCA plot of TE, MP and TINT cells [gating strategy in (F)] at day 7.5 p.i., with the percentage of variance shown. (H) Quantification of the relative frequency of BrdU+ cells in MP and TINT cells compared to TE cells. (I) Diagram of the in vivo differentiation assay (left), flow cytometry of KLRG1 versus CXCR3 expression (middle), and quantification of TINT, TE and CXCR3hiCD127hi cells (right). Only representative plots of KLRG1 versus CXCR3 are shown (TE population is largely defined by KLRG1hiCXCR3lo cells, which constitute ~ 95% of TE cells). (J) Quantification of TE, MP and TINT cells in the indicated P14 cells. (K) UMAP plot of Pofut1-dependent signature [downregulated genes as identified in (A)] in published scRNA-seq dataset from (D) (Chen et al., 2019). (L) UMAP plot of scRNA-seq data from sgNTC- (in black, left) and sgPofut1- (in red, right) transduced P14 cells (from dual-color transfer system) at day 7 p.i. Gray shadow indicates location of all cells; the number of analyzed cells in each group is indicated. (M) UMAP plot of the expression of Klrg1 (left), Cxcr3 (middle) and Il7r (right) in scRNA-seq data described in (L). (N) Flow cytometry of KLRG1 versus CXCR3 expression (left) and quantification (right) of TE cells in the in vivo differentiation assay similar as (I), except for the use of both wild-type and Pofut1-null TINT groups as the pretransfer cells. Data are from one (A, B, D, E, G, and KM), representative of two (C, H, and N), or compiled from at least two (I, J, and N) independent experiments, with 4 (A, C, G, H, and I), 17 (F), 11 (J), or 3 (L and N) biological replicates per group. *P < 0.05, **P < 0.01, and ***P < 0.001; NS, not significant; two-tailed paired Students t-test (C), two-tailed unpaired Students t-test (I, J, and N), or one-way analysis of variance (ANOVA) (F and H). Data are presented as mean s.e.m. See also Figures S4S6 and Tables S3S6.

Go here to see the original:
In vivo CRISPR screening reveals nutrient signaling processes ... - PubMed

Comments are closed.