Figure 1. Genome-wide CRISPR screening... | Rockefeller University Press

Figure 1.

Genome-wide CRISPR screening reveals VMP1 promotes ER Ca2+release. (A) A cartoon illustrating SOCE and thapsigargin (TG)–Ca2+–NFAT–PD-1 signaling axis. (B) CRISPR screening strategy. (C) Screening result from Jurkat cells. Selected top hits are labeled. (D) Jurkat cells expressing indicated sgRNAs and Cas9 were treated with DMSO or TG (10 nM) for 48 h, and the expression of PD-1 was examined by flow cytometry. Mean fluorescence intensities of PD-1 staining are shown. (E) TG-induced SOCE in Jurkat cells expressing indicated sgRNAs and Cas9 was examined by flow cytometry. (F) ER Ca2+ and SOCE in HEK293T cells expressing indicated sgRNAs and Cas9 were examined by flow cytometry. (G) ER Ca2+ before and after TG (10 nM) treatment was monitored by flow cytometry. (H) HEK293T cells were transfected with indicated plasmids together with a BFP reporter. ER Ca2+ of BFPhigh cells was examined by flow cytometry. (I) HEK293T-G-CEPIA1er cells were transfected with indicated plasmids together with a BFP reporter. Fluorescence intensity of G-CEPIA1er in BFPhigh cells before and after TG (1 μM) treatment was measured by flow cytometry. (D and E) Representative plots from one of three independent experiments. (F–I) Representative plots and statistics from one of three independent experiments; n = 6 in F, n = 4 in G, n = 3 in H and I. Each symbol represents one sample; data represent mean ± SEM, two-tailed unpaired t test in F and H, two-way ANOVA in G and I; ** <0.01, *** <0.001, **** <0.0001.

Genome-wide CRISPR screening reveals VMP1 promotes ER Ca ²⁺ release. (A) A cartoon illustrating SOCE and thapsigargin (TG)–Ca²⁺–NFAT–PD-1 signaling axis. (B) CRISPR screening strategy. (C) Screening result from Jurkat cells. Selected top hits are labeled. (D) Jurkat cells expressing indicated sgRNAs and Cas9 were treated with DMSO or TG (10 nM) for 48 h, and the expression of PD-1 was examined by flow cytometry. Mean fluorescence intensities of PD-1 staining are shown. (E) TG-induced SOCE in Jurkat cells expressing indicated sgRNAs and Cas9 was examined by flow cytometry. (F) ER Ca²⁺ and SOCE in HEK293T cells expressing indicated sgRNAs and Cas9 were examined by flow cytometry. (G) ER Ca²⁺ before and after TG (10 nM) treatment was monitored by flow cytometry. (H) HEK293T cells were transfected with indicated plasmids together with a BFP reporter. ER Ca²⁺ of BFP^high cells was examined by flow cytometry. (I) HEK293T-G-CEPIA1er cells were transfected with indicated plasmids together with a BFP reporter. Fluorescence intensity of G-CEPIA1er in BFP^high cells before and after TG (1 μM) treatment was measured by flow cytometry. (D and E) Representative plots from one of three independent experiments. (F–I) Representative plots and statistics from one of three independent experiments; n = 6 in F, n = 4 in G, n = 3 in H and I. Each symbol represents one sample; data represent mean ± SEM, two-tailed unpaired t test in F and H, two-way ANOVA in G and I; ** <0.01, *** <0.001, **** <0.0001.

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