Fig. 3

Targeting of CuS-TRPV1 to VSMCs membrane and opening of TRPV1 channels. a Representative TEM images of VSMCs incubated with 0.4 mg mL⁻¹ of CuS-TRPV1 or CuS for 2 h. Red arrows indicated localization of NPs in (i) whole cell section under low magnification, (ii) partially magnified cell membrane and (iii) magnified endosome or lysosome. Images are representative of three independent wells per group. Scale bar = 1 μm. b Representative IFC images of VSMCs incubated with the fluorescein-conjugated CuS-TRPV1 or CuS (0.4 mg mL⁻¹) for 2 h. Green fluorescence in the blue area indicated NPs targeting to the cell membrane. Mean membrane intensity of CuS (blue area: 6,520.78) was 23% of CuS-TRPV1 (blue area: 28,408.02). Data are shown as mean ± S.D. of three independent experiments, and analyzed by Student’s t-test. ***P < 0.001. c Fluorescence imaging of Ca²⁺ influx in VSMCs induced by CuS-TRPV1 heating. VSMCs were incubated with 0.4 mg mL⁻¹ of CuS-TRPV1 and irradiated by the 980 nm laser (5 W/cm²) for 1 cycle. Capsaicin (1 μM) was used as a positive control. Images represent three independent wells per group. Scale bar = 50 μm. d Western blot analysis of AMPK phosphorylation in VSMCs induced by CuS-TRPV1 heating-evoked Ca²⁺ influx. VSMCs were incubated with 0.4 mg mL⁻¹ CuS-TRPV1 and irradiated by the 980 nm laser (5 W cm⁻¹) for 10 cycles. Capsaicin (Cap, 1 μM) was used as a positive control and CuS (0.4 mg mL⁻¹) was used as a negative control. The relative levels of AMPK phosphorylation were normalized to total AMPK, with β-actin serving as a loading control. Data are shown as mean ± S.D. of three independent experiments, and analyzed by Student’s t-test. **P < 0.01 for CuS-TRPV1 +Laser vs. untreated group. ^#P < 0.05 for Cap vs. untreated group