Background
Methods
MHV-3 induced fulminant hepatitis Balb/cJ mouse model
Plasmids and hydrodynamic delivery
Gene | Primer |
---|---|
Mouse KCTD9 (for cloning) | anti-sense 5’ ATAAGAATGCGGCCGCGCACCTCTGGGAACAAGC 3’ |
sense 5’ CCCAAGCTTGTGCGTGCGTGAGGAGTGT 3’ | |
mKCTD9-shRNA1 | anti-sense 5’ AAGCTTAAAAATTGACCGAAGTCCTGAGTATCTCTTGAATACTCAGGACTTCGGTCAAAAACAAGGCTTTTC 3’ |
mKCTD9-shRNA2 | anti-sense 5‘AAGCTTAAAAAGCCAGATGACAGGGATTAATCTCTTGAATTAATCCCTGTCATCTGGCAAACAAGGCTTTTC 3’ |
shRNA-Negative Control | anti-sense 5’ AAGCTTAAAAAGACGCAACGGAAGTGTTAATCTCTTGAATTAACACTTCCGTTGCGTCAAACAAGGCTTTTC 3’ |
mKCTD9-shRNA | sense 5’ GTAGGATCCATCCGACGCCGCCATCTC 3’ |
mKCTD9 (for qPCR) | anti-sense 5’ ACGATACTTCACAACAACAAGG 3’ |
sense 5’ CCTGGCTTCTTCTAGCACAC 3’ | |
GAPDH (for qPCR) | anti-sense 5’ CGGATTTGGTCGTATTGGG 3’ |
sense 5’ CTCGCTCCTGGAAGATGG 3’ |
Cell lines and transfection
Preparation of mouse liver mononuclear cells
Quantitative PCR
Western blot analysis
Immunocytochemistry and immunohistochemistry
Flow cytometry
Cytotoxic assays
Statistical analysis
Results
KCTD9 expression significantly elevated in intrahepatic lymphocytes of MHV-3-FHF mice
Brain | Thymus | Heart | Lung | Kidney | Stomach | Small intestine | ||
---|---|---|---|---|---|---|---|---|
0 h | 2.455 ± 0.170 | 2.331 ± 0.558 | 2.615 ± 0.079 | 3.411 ± 0.142 | 2.131 ± 0.111 | 2.358 ± 0.140 | 2.409 ± 0.395 | |
48 h | 2.938 ± 0.306 | 2.890 ± 0.027 | 2.804 ± 0.030 | 3.123 ± 0.168 | 2.541 ± 0.091 | 2.713 ± 0.460 | 2.940 ± 0.012 | |
t value | −2.392 | −1.734 | −3.893 | 2.251 | −3.933 | −1.283 | −2.325 | |
p value | 0.075 | 0.225 | 0.018 | 0.088 | 0.017 | 0.269 | 0.081 | |
Colon | Testis | Ovary | Muscle | Bone Marrow | Liver | Spleen | PBMC | |
0 h | 2.480 ± 0.172 | 3.420 ± 0.195 | 2.596 ± 0.249 | 1.945 ± 0.142 | 3.575 ± 0.999 | 2.118 ± 0.054 | 2.193 ± 0.017 | 1.331 ± 0.575 |
48 h | 2.373 ± 0.175 | 3.774 ± 0.140 | 2.805 ± 0.144 | 2.461 ± 0.197 | 2.870 ± 0.209 | 2.786 ± 0.389 | 1.971 ± 0.030 | 3.112 ± 0.602 |
t value | 1.002 | −2.563 | −1.257 | −3.633 | 1.199 | −2.946 | 11.195 | −3.706 |
p value | 0.365 | 0.062 | 0.277 | 0.022 | 0.297 | 0.042 | < 0.001 | 0.021 |