Figure 1.
LPS induced more serious damage in Pin1−/− mice.
15–20-week-old Pin1 WT (n = 5) and Pin1−/− (n = 5) mice were intraperitoneally injected with LPS at 10 µg/g body weight or PBS and their survival rates was monitored.
Figure 2.
Histopathology of the lungs of WT and Pin1−/− mice after LPS injection.
Representative histological sections with hematoxylin and eosin staining of lungs from surviving mice that were sacrificed 100 h after LPS injection intrapenetoreally. The left panels show the control lungs of WT (A) and Pin1−/− mice (C), and the right panels show the lungs of WT (B) and Pin1−/− mice (D) that were injected with LPS for 100 h. Scale bars, 30 µm. control; n = 5 per genotype, LPS-injected; n = 5 per genotype.
Figure 3.
Comparison of TNFα, IL-6 and TLR4 mRNA and Pin1 protein in the macrophages of WT and Pin1−/− mice after LPS injection.
(A) Peritoneal macrophages from WT and Pin1−/− mice were stimulated with 100ng/ml LPS for the indicated periods. Total RNA was extracted and then subjected to quantitative real-time PCR analysis using primers specific for TNFα and IL-6. (B) Peritoneal macrophages from WT and Pin1−/− mice were stimulated with various concentrations of LPS for 4 h.Total RNA was extracted and then subjected to quantitative real-time PCR analysis using primers specific for TNFα and IL-6. (C) Peritoneal macrophages from WT and Pin1−/− mice were stimulated with various concentrations of LPS for 4 h.Total RNA was extracted and then subjected to PCR analysis using primers specific for TLR4. (D) Peritoneal macrophages from WT mice were stimulated with 100ng/ml LPS for the indicated periods. At indicated time points, cell lysates were prepared and subjected to Western blotting analysis using anti-Pin1 and anti-tubulin as a control. mRNA levels were normalized to that of β-actin and then normalized to the relative mRNA level of WT at time 0 h or mRNA level of WT stimulated with PBS. Results are shown as means±SEM for 3 independent sets of experiments. Asterisk* denotes significant difference (p<0.05).
Figure 4.
Function of Pin1 in RAW264.7 stimulated with LPS.
(A) RAW264.7 was stimulated with 100ng/ml LPS for the indicated periods. At indicated time points, cell lysates were prepared and subjected to Western blotting analysis using anti-pSer16 Pin1 and anti-tublin as a control. (B) RAW264.7 was pretreated with Pin1-specific inhibitor, PiB and stimulated with 100ng/ml LPS for 4 h.Total RNA was extracted and then subjected to quantitative real-time PCR analysis using primers specific for TNFα and IL-6. (C) Pin1 overexpressed RAW264.7 was stimulated with 100ng/ml LPS for 4 h.Total RNA was extracted and then subjected to quantitative real-time PCR analysis using primers specific for TNFα and IL-6. mRNA levels were standarized by β-actin. Results were shown as means±SEM for 3 independent sets of experiments. Asterisk* denotes significant difference (p<0.05).
Figure 5.
Pull down assay of PU.1 by Pin1.
(A) The human macrophage cell line THP-1 treated with or without 10 ng/ml PMA were lysed with Lysis buffer. GST-Pin1and GST beads were incubated with the cell lysates treated with phosphatase previously or not. (B) Wild-type Pin1, Pin1 mutants in which Trp34 at the WW domain was mutated to Ala34 (Pin1W34A), and Pin1 in which Arg68 and Arg69 at the PPIase domain mutated to Ala68 and Ala69 (Pin1R68/69A) produced as GST-fusion proteins were bound to the glutathione-beads. PU.1 in which Thr92 or Ser119 were mutated to Ala were prepared and used instead of wild type of Pin1.
Figure 6.
Immunofluorescent Cell Staining.
COS7 cells were transfected with (A) HA-Pin1 or Flag-PU.1 and (B) HA-Pin1 and Flag-PU.1 together. After 48 h, cells were fixed with 4% paraformaldehyde, treated with HA probe and FLAG M2 antibodies and Alexa Fluor 488- and 595- conjugated secondary antibodies, and DAPI staining for nucleus. These cells were observed under fluorescence microscope (Biozero 8000, KEYENCE, Japan).