Fig. 2

Chronic stress-induced autophagy is through Tim-3. BALB/c male mice (N = 5 per group) were sacrificed after 6 h or 12 h of chronic stress. Cellular lysates were extracted from mouse spleens. a The expression of Tim-3 was determined by Western blot. *P < 0.05, **P < 0.01 compared with indicated groups. b Total RNA was isolated from mouse spleens, and Tim-3 mRNA levels were determined by quantitative RT-PCR. c After 12 h of stress, mouse spleens were harvested and fixed in 4% buffered formalin. IHC was performed on adjacent sections with anti-Tim-3 Ab and hematoxylin was used as a counterstain (100×). d The primary splenocytes we isolated from stressed or unstressed mice were transfected with small interfering RNA against Tim-3 (Tim-3-Si) or negative control siRNA (NC-Si) and cellular lysates were obtained, then the Tim-3 and autophagy level were examined by Western blot analysis. *P < 0.05 compared with indicated groups