Figure 5. ER Ca 2 overload in... | Rockefeller University Press

Figure 5.

ER Ca2+overload in VMP1-deficient T cells results in ER stress and secondary Ca2+overload in mitochondria. (A) Immunoblot analysis of peIF2α-S51, eIF2α, and VMP1 in total T cells freshly isolated from mice with indicated genotypes. Each lane represents an individual mouse. (B) Mitochondrial Ca2+ in T cells from mice with indicated genotypes was examined by flow cytometry with Rhod2. (C) CD4 T cells from mice with indicated genotypes were treated with DMSO or thapsigargin (TG; 1 nM) for 1 h. Mitochondrial Ca2+ was measured by flow cytometry with Rhod2. (D) CD4 T cells from mice with indicated genotypes were treated with DMSO or indicated concentrations of TG for 6 h; apoptosis was measured by flow cytometry. (E) CD4 T cells from mice with indicated genotypes were treated with DMSO or BZN (1 μM) for 1 h and mitochondrial Ca2+ was measured by flow cytometry with Rhod2. (F) CD4 T cells from mice with indicated genotypes were treated with DMSO or BZN (1 μM) for 6 h; apoptosis was measured by flow cytometry. (G) A model of VMP1 prevention of Ca2+ overload in ER and mitochondria. The balanced activities of VMP1 and SERCA determine the basal level of ER Ca2+ in T cells under steady state. In VMP1-deficent T cells, Ca2+ overload causes ER stress and secondary Ca2+ overload in mitochondria via MCU, which induces cell death. (A) Representative blots and statistics from one of two independent experiments. (B, C, and E) Representative plots and statistics of Rhod2 mean fluorescence intensity from one of two independent experiments. (D and F) Representative plots and statistics from one of three independent experiments. n = 3 mice per genotype in A, B, D, and E, n = 4 mice per genotype in C and F; each symbol represents an individual mouse; data represent mean ± SEM, one-way ANOVA in A–C and E, two-way ANOVA in F; ** <0.01, **** <0.001. Source data are available for this figure: SourceData F5.

ER Ca ²⁺ overload in VMP1-deficient T cells results in ER stress and secondary Ca ²⁺ overload in mitochondria. (A) Immunoblot analysis of peIF2α-S51, eIF2α, and VMP1 in total T cells freshly isolated from mice with indicated genotypes. Each lane represents an individual mouse. (B) Mitochondrial Ca²⁺ in T cells from mice with indicated genotypes was examined by flow cytometry with Rhod2. (C) CD4 T cells from mice with indicated genotypes were treated with DMSO or thapsigargin (TG; 1 nM) for 1 h. Mitochondrial Ca²⁺ was measured by flow cytometry with Rhod2. (D) CD4 T cells from mice with indicated genotypes were treated with DMSO or indicated concentrations of TG for 6 h; apoptosis was measured by flow cytometry. (E) CD4 T cells from mice with indicated genotypes were treated with DMSO or BZN (1 μM) for 1 h and mitochondrial Ca²⁺ was measured by flow cytometry with Rhod2. (F) CD4 T cells from mice with indicated genotypes were treated with DMSO or BZN (1 μM) for 6 h; apoptosis was measured by flow cytometry. (G) A model of VMP1 prevention of Ca²⁺ overload in ER and mitochondria. The balanced activities of VMP1 and SERCA determine the basal level of ER Ca²⁺ in T cells under steady state. In VMP1-deficent T cells, Ca²⁺ overload causes ER stress and secondary Ca²⁺ overload in mitochondria via MCU, which induces cell death. (A) Representative blots and statistics from one of two independent experiments. (B, C, and E) Representative plots and statistics of Rhod2 mean fluorescence intensity from one of two independent experiments. (D and F) Representative plots and statistics from one of three independent experiments. n = 3 mice per genotype in A, B, D, and E, n = 4 mice per genotype in C and F; each symbol represents an individual mouse; data represent mean ± SEM, one-way ANOVA in A–C and E, two-way ANOVA in F; ** <0.01, **** <0.001. Source data are available for this figure: SourceData F5.

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