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. Author manuscript; available in PMC: 2020 Jul 15.
Published in final edited form as: Nature. 2020 Jan 15;577(7792):689–694. doi: 10.1038/s41586-019-1912-x

Extended Data Fig 3. VEGF-C-mRNA validation in vitro and in vivo uptake/expression tropism.

Extended Data Fig 3.

a cDNA sequence of VEGF-C used for mRNA production. b 5-methyl cytosine and pseudo uridine were used to substitute all of cytosine and uridine in the mRNA. c VEGF-C-mRNA was transfected into HEK293T cells and cell lysate and media were collected to detect full length, and secreted intermediate and processed peptides. d VEGF-C-mRNA (5 μg) was delivered in vivo into the cisterna magna of mice using a JETPEI polyplex system. Six hours after, mice CSF was collected using a capillary tube, filtered with an amicon filter and the wash-through was used to run a western blot (each lane is n = 10 animals pooled). e Raw western blot images of Extended Data Fig 4c (gel 1: column 1, control; 2, Cy5-GFP-mRNA; 3, VEGF-C-mRNA; 4, control; 5, Cy5-GFP-mRNA; 6, VEGF-C-mRNA) (gel 2: column 1, control; 2, Cy5-GFP-mRNA; 3, VEGF-C-mRNA) and Extended Data Fig 4d (gel 3: column 1–3 Cy5-GFP-mRNA; column 4–6 Cy5-GFP-mRNA; 7–9 recombinant human VEGF-C in increasing concentration). Experiments were repeated twice independently with similar results. f VEGF-C-mRNA and Cy5 labeled GFP-mRNA was mixed at a 1:1 ratio and delivered in vivo with JETPEI. 15 minutes later, mice were euthanized and whole skull cap was imaged to observe the distribution of mRNA particles. Experiments were repeated independently twice with similar results. g-hVEGF-C-mRNA and Cy5 labeled GFP-mRNA were mixed at a 1:1 ratio and delivered in vivo with JETPEI. 24 h later brains, meninges and lymph nodes of treated mice were collected for flow cytometry to measure % Cy5 positive cells in each compartment (control, n = 6; Cy5-mRNA, n = 9; data are pooled from two independent experiments). i CSF, Meninges, Brain and Serum were collected at 2 month-time point (AAV-CTRL, AAV-VEGF-C), 24 h-time point (GFP-mRNA, VEGF-C-mRNA), or days 7 and 28 after tumor inoculation and ELISA was performed to detect VEGF-C (CSF; AAV-VEGF-C, n = 6; other groups n = 3; 5 animals were pooled for each sample) (Meninges; AAV-VEGF-C, n = 6; d7 tumor, n =3; other groups n = 5). (Brain; AAV-CTRL, GFP-mRNA, n = 6; AAV-VEGF-C, VEGF-C-mRNA, n = 5; d7 tumor, n = 3; d28 tumor, n = 7) (Serum; n = 3). Data are mean ± S.D. *P < 0.05; **P < 0.01; ***P <0.001; ****P<0.0001 (two-tailed unpaired Student’s t-test)