Figure 2.

PM Ca²⁺ Influx Channels in M1 Macrophages In Vitro

(A) BM macrophages were pulsed with medium alone (M0) or IFNγ (M1) for the indicated times and subjected to whole-cell patch clamp recordings. The I-V curves display presence of signature current for TRPC1 channels in M1 macrophages and for ORAI1 channels in M0 macrophages. Averages of 8–10 recordings at −80 mV and corresponding statistics are shown in bar graph (A′).

(B) Comparison of IV curves of WT and TRPC1^−/− macrophages cultured with medium alone (M0) for 24 hr, or IFNγ (M1) for 24 hr. The signature current for TRPC1 channels increased in IFNγ-exposed WT macrophages, but not in TRPC1^−/− macrophages. Average current density recordings from 8 to 10 cells at −80 mV and corresponding statistics are shown in bar graph (B′).

(C) BM macrophages transfected with control siRNA (siC) or siRNA specific for ORAI1 (siO1) were cultured under M0 and M1 conditions for 24 hr. I-V curves were compared in control and ORAI1 knockdown cells by whole-cell patch-clamp recordings. Statistics from 8–10 recordings are shown in bar graph (C′).

(D) I-V curves were compared between BM macrophage transfected with control siRNA (siC) and cells transiently transfected with siRNA against TRPC1 and ORAI1 together (siT1+siO1) and cultured under M0 and M1 conditions for 24 hr. Averages (8–10 recordings at −80 mV) and statistics are shown in bar graph (D′).

(E) Representative time course of TRPC1 and ORAI1 protein expression in response to IFNγ by BM macrophages after immunoprecipitation with anti-STIM1 antibody (Cell Signaling, 4916S), followed by immunoblotting as seen upon subjecting 30 μg protein to SDS-PAGE and anti-TRPC1 (Abcam, ab192031), anti-ORAI1 (Alamone Lab, ACC-060), or anti-STIM1 (Cell Signaling, 4916S; used as loading control). The bar graphs (E′) represent average pixel intensity of the respective protein bands from three independent experiments.

(F) TRPC1 and ORAI1 protein expression in BM macrophage pulsed with IFNγ for 0 min, 2 hr, or 6 hr by western blot as seen subjecting 30 μg protein to SDS-PAGE and using anti-TRPC1 (Abcam, ab192031), anti-ORAI1 (Alamone Lab, ACC-060), or β-actin (Cell Signaling, 4970S). The bar graphs (F′) represent the average pixel intensity of the respective TRPC1 and ORAI1 protein bands from three independent experiments.

*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (Student's t test).

See also Figures S2 and S4.