mRNA expression analysis

Species specific analysis of HGF and MET gene expression in wild-type C57BL/6 mice and homozygous humanized B-hHGF/hMET mice by RT-PCR. The RNAs of the liver, lung and kidney were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hHGF/hMET mice(H/H; H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human HGF and MET primers. Human HGF and MET mRNA were detectable only in homozygous B-hHGF/hMET mice but not in wild-type mice. Mouse Met mRNA was detectable in wild-type C57BL/6 mice.

Protein expression analysis in serum

Strain specific HGF expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hHGF/hMET mice by ELISA. The serum was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hHGF/hMET mice(H/H; H/H) (female, n=3, 9-week-old). Expression level of mouse and human HGF was analyzed by ELISA(anti-mouse HGF ELISA kit: R&D, MHG00; anti-human HGF ELISA kit: Abcam, ab275901). Mouse HGF was only detectable in wild-type C57BL/6 mice. Human HGF was exclusively detectable in homozygous B-hHGF/hMET mice. Values are expressed as mean ± SEM. ND: not detectable.

Protein expression analysis

Western blot analysis of MET protein expression in homozygous B-hHGF/hMET mice. Liver, lung and kidney lysates were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hHGF/hMET mice (H/H, H/H), and then analyzed by western blot with anti-m/hMET antibody (CST, 3127S). 60 μg total proteins were loaded for western blotting analysis. MET was detected in the liver, lung, and kidney of wild-type mice and homozygous B-hHGF/hMET mice, as the antibody cross-reacted with both mouse and human MET.

Frequency of leukocyte subpopulations in spleen

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hHGF/hMET mice (female, 9-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hHGF/hMET mice were similar to those in C57BL/6 mice, demonstrating that humanization of HGF and MET do not change the frequency or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in blood

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6 mice and homozygous B-hHGF/hMET mice (female, 6-week-old, n=3). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hHGF/hMET mice were similar to those in C57BL/6 mice, demonstrating that humanization of HGF and MET do not change the frequency or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in lymph node

Frequency of leukocyte subpopulations in lymph node by flow cytometry. Leukocytes were isolated from wild-type C57BL/6 mice and homozygous B-hHGF/hMET mice (female, 6-week-old, n=3). A. Flow cytometry analysis of the leukocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hHGF/hMET mice were similar to those in C57BL/6 mice, demonstrating that humanization of HGF and MET do not change the frequency or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.