Figure 3.

TRPC1 Mediates IFNγ-Induced Ca²⁺ Influx in Peritoneal Macrophages In Vivo

(A) Schematic showing calcium imaging, electrophysiological recordings, and biochemical analysis performed on peritoneal macrophages (peritoneal macrophages) from IFNγ i.p. injected WT and TRPC1^−/− mice or mice injected with TRPC1 siRNA or ORAI1 siRNA to transiently knock down these proteins in vivo before the animals received IFNγ.

(B) Ca²⁺ entry triggered by Tg in peritoneal macrophages from WT or TRPC1^−/− mice that received IFNγ i.p. (M1). Analog plots of the fluorescence ratio (340/380 nm) from an average of 40–50 cells are shown. The bar graph (B′) indicates means ± SEM of the Ca²⁺ release (left peak in B) and store-operated Ca²⁺ entry (SOCE) (right peak in B).

(C) Ca²⁺ entry triggered by Tg in IFNγ-exposed peritoneal macrophages transiently deficient in TRPC1 (M1-siT1) or ORAI1 (M1-siO1), or control cells (M1-siC) obtained from mice that received non-targeting siRNA. Analog plots of the fluorescence ratio (340/380 nm) from an average of 40–50 cells are shown. The bar graph (C′) indicates means ± SEM of the Ca²⁺ release.

(D) I-V curves in peritoneal macrophages from WT or TRPC1^−/− mice that received IFNγ (M1) or vehicle (M0) i.p. Average of 8–10 recordings for current intensity at −80 mV is presented in the bar graph (D′).

(E) IFNγ-exposed peritoneal macrophages transiently deficient in TRPC1 (M1-siT1) or ORAI1 (M1-siO1), or control cells (M1-siC) were subjected to whole-cell patch-clamp recordings. Average of 8–10 recordings used for I-V relationships are shown in bar graph (E′).

(F) TRPC1-STIM1 and ORAI1-STIM1 complex formation in peritoneal macrophages from C57BL/6 mice i.p. injected with PBS (M0-PBS), thioglycolate (M0-Thio) and PBS, or thioglycolate and IFNγ (M1). Immunoprecipitation was performed on 250 μg of protein extracts from approx. 5 × 10⁶ cells using anti-STIM1 antibodies (Cell Signaling, 4916S) as seen after subjecting the immunoprecipitates to SDS-PAGE followed by immunoblot detection with anti-TRPC1 (Abcam, ab192031) and anti-ORAI1(Alamone Lab, ACC-060). Anti-STIM1 (Cell Signaling, 4916S) was used for loading control. Bar graph (F′) represents the average pixel intensity of the respective protein bands from three independent experiments.

(G) TRPC1 and ORAI1 protein expression in peritoneal macrophages from C57BL/6 mice i.p. injected with PBS (M0-PBS), thioglycolate and PBS (M0-Thio), or thioglycolate and IFNγ (M1) as seen subjecting 30 μg protein to SDS-PAGE and using anti-TRPC1 (Abcam, ab192031), anti-ORAI1 (Alamone Lab, ACC-060), or anti-GAPDH for western blot. Bar graph (G′) represents the average pixel intensity of the respective protein bands from three independent experiments.

*p ≤ 0.05, ***p ≤ 0.001 (Student's t test).

See also Figure S3.