Table 1.
Primers used in quantitative real-time PCR.
Figure 1.
A. SDS-PAGE analysis of rPoCoro 1A (Lane 1). Lane M, protein molecular weight marker. B. Anti-rPoCoro 1A polyclonal antibodies determination by western blotting. Lane 1, rPoCoro 1A. Lane M, protein molecular weight marker.
Figure 2.
Basal expression of porcine Coro1A in different tissues.
The expression of Coro1A was firstly normalized to the expression of GAPDH and then compared relative to the expression of Coro1A in heart, which was set as 1.
Figure 3.
Expression analysis of porcine Coro1A in PK-15 cells stimulated with LPS, poly (I:C) and H.parasuis.
A: LPS-induced expression of porcine Coro1A in PK-15 cells. PK-15 cells were cultured with 1 µg/ml LPS for 48 h. B: Poly (I:C)-induced expression of porcine Coro1A in PK-15 cells. PK-15 cells were cultured with 10 µg/ml poly (I:C) for 48 h. C: H.parasuis-induced expression of porcine Coro1A in PK-15 cells. PK-15 cells were cultured with 107 of CFU H.parasuis for 48 h. Relative expression of porcine Coro1A was detected by Q-PCR and normalized to the expression of GAPDH. The fold increase is expressed as the mean of three replicates with SEM by comparison with the control (0 h). Q-PCR was performed using primers described in table 1. Results are from the calculated average ± SD of three different cell samples in the same treatment. *P<0.05.
Figure 4.
Reduction of NF-κB activation when porcine Coro1A was overexpressed.
(A) PK-15 cells were co-transfected with the pNF-κB-luc reporter plasmid (0.2 µg), pRL-TK plasmid (0.05 µg), along with 0.6 µg of the expression plasmid encoding porcine Coro1A protein. Selected cells were stimulated by 20 ng/ml TNF-α at 18 h post-transfection, and cell extracts were prepared for the dual-luciferase activity at 6 h after this treatment. ***P<0.001 as compared with vector control. (B) An increasing quantities of porcine Coro1A expression plasmid (0, 0.2, 0.4, 0.8 µg) was co-transfected with pNF-κB-luc and pRL-TK into PK-15 cells. Cells were harvested at 24 h after transfection and analysed for luciferase activity. (a) P<0.05 compared with the vector group, (b) P<0.05 compared with 0.2 µg porcine Coro1A protein transfection group, (c) P<0.05 compared with 0.4 µg porcine Coro1A protein transfection group. Values for the samples were normalized using Renilla luciferase values and expressed as relative fold change in NF-κB-regulated gene expression compared with vector group, and each untreated empty vector control value was set as a basis level of 1. Data represent means of three replicates and results are representative of at least three independent experiments. (C) Overexpression of porcine Coro1A inhibits the degradation of IκBα and nuclear translocation of p65. PK-15 cells were transfected with the indicated amount (0, 2, 4 µg) of porcine Coro1A expression plasmid. Cell total protein extracts, cytoplasmic extracts and nuclear extracts were prepared at 24 h post-transfection and subjected to western blot analysis with antibodies specific for endogenous IκBα, p65 or p-p65. A polyclonal anti-Coronin 1A antibody was used to confirm the expression of Coro1A. The β-actin (for porcine Coro 1A, IκBα, total p65, p-p65 samples) and Histone H3 (for nuclear-p65 samples) were respectively used as a control for sample loading. Similarly, cells were treated with TNF-α (20 ng/ml) at 18 h post-transfection, and cell total protein extracts, cytoplasmic extracts and nuclear extracts were prepared for the western blot analysis at 6 h after this treatment. Western blot analyses were repeated in three independent experiments with similar results and a representative blot was selected. (D) Band densitometry was performed on the western blot shown in figure 4C left. The β-actin (for porcine Coro 1A, IκBα, total p65, p-p65 samples) and Histone H3 (for nuclear-p65 samples) were respectively used for normalization (*P<0.05, **P<0.01, Student's T-test). (E) Band densitometry was performed on the western blot shown in figure 4C right. The β-actin (for porcine Coro 1A, IκBα, total p65, p-p65 samples) and Histone H3 (for nuclear-p65 samples) were respectively used for normalization (*P<0.05, **P<0.01, Student's T-test). (F) PK-15 cells were transfected with 4 µg of plasmid encoding porcine Coro1A as well as empty vector pcDNA-3.1. At 24 h post-transfection, total RNA was extracted and the relative expression of IL-6, IL-8, and COX-2 genes were evaluated by Q-PCR. Results are expressed as decreasing mRNA levels relative to those in cells transfected with the empty vector and were normalized to the expression of housekeeping gene GAPDH. All data represent the means and standard deviation of three independent experiments. **P<0.01 as compared with empty vector control.
Figure 5.
Porcine Coro1A inhibit H.parasuis induced NF-κB activation.
(A) PK-15 cells were transfected with pNF-κB-luc (0.2 µg) and pRL-TK (0.05 µg), then cells were control-inoculated, live or heat-killed H.parasuis (107 CFU) inoculated. (a) P<0.05 compared with the control group, (b) P<0.05 compared with the inactivated group. (B) PK-15 cells were transfected with 2 µg of plasmid encoding porcine Coro1A as well as empty vector pcDNA-3.1, along with pNF-κB-luc (0.2 µg) and pRL-TK (0.05 µg). At 24 h post-transfection, cells were treated with live or heat-killed H.parasuis (107 CFU), and cell extracts were prepared for the luciferase reporter assays at 12 h after this treatment. Values for the samples were normalized using Renilla luciferase values. Inactivated H.parasuis-inoculated cells transfected with empty vector value and live H.parasuis-inoculated cells transfected with empty vector value were respectively set as a basis level of 1. (a) P<0.01 as compared with inactivated H.parasuis-inoculated cells transfected with empty vector. (b) P<0.01 as compared with live H.parasuis-inoculated cells transfected with empty vector. (C) PK-15 cells were transfected with the varying amount (0, 2, 4 µg) of pcDNA-Coro1A. At 24 h post-transfection, cells were inoculated with live H.parasuis (107 CFU) for 12 h. Western blot analysis with antibodies specific for endogenous IκBα, p65 or p-p65 were performed. A polyclonal anti-Coronin 1A antibody was used to confirm the expression of Coro1A. The β-actin (for porcine Coro 1A, IκBα, total p65, p-p65 samples) and Histone H3 (for nuclear-p65 samples) were respectively used as a control for sample loading. Western blot analyses were repeated in three independent experiments with similar results and a representative blot was selected. (D) Band densitometry was performed on the western blot shown in figure 5C. The β-actin (for porcine Coro 1A, IκBα, total p65, p-p65 samples) and Histone H3 (for nuclear-p65 samples) were respectively used for normalization (*P<0.05, **P<0.01, Student's T-test). (E) PK-15 cells were transfected with 4 µg of pcDNA-Coro1A as well as empty vector pcDNA-3.1. At 24 h post-transfection, cells were inoculated with live H.parasuis at 107 CFU and then harvested at 12 h for RNA extraction. The levels of IL-6, IL-8 and COX-2 mRNAs were measured by Q-PCR analysis. Results are expressed as decreasing mRNA levels relative to those inoculated H.parasuis cells transfected with the empty vector *P<0.05 as compared with H.parasuis-inoculated cells transfected with empty vector.
Figure 6.
Quantitative expression of porcine Coro1A in five tissues from pigs with Glässer's disease.
Increased in vivo gene expression of porcine Coro1A in lungs, spleen, lymph nodes of pigs with Glässer's disease. Relative expression of porcine Coro1A was detected by qPCR and normalized to the expression of GAPDH. The fold increase is expressed as the mean of three replicates with SEM by comparison with the control. The significance of difference for the expression compared to the control was calculated using Student's T-test. ***p<0.001; **p<0.01; *p<0.05.