Extended Data Fig. 4. ZIKV infection of osteoblastic-like cells increase osteogenic gene expression.

(a-b) Mock- and ZIKV-infected U2OS cells were harvested at 2 and 4 dpi respectively for osteogenic gene expression. (c) Band intensity of pSMAD1/5/9 from U2OS whole cell lysate. (d) At day 1–4, IgG or nAb-treated and mock or ZIKV-infected U2OS cells (n=6) were harvested for RNA extraction and viral load against ZIKV NS1 RNA was quantified using qRT-PCR (N.D.; Not detected) (e) Human primary fetal brain pericytes are infected with ZIKV PRVABC59 or PBS (mock control) that were treated with 2 μg/mL of mouse IgG isotype control or human BMP2/4 neutralizing antibody. At 4 dpi, cells were harvested for osteogenic gene expressions, normalized to GAPDH and presented as fold changes relative to mock controls (n=4/group). Data in a, b, d and e are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Data are analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparisons (a), Mann-Whitney U-test (b), or two-tailed unpaired Student t-test (e). *P< 0.05, **P< 0.01, ***P< 0.001. Exact P values in b compared between Mock and ZIKV H/PF/2013 (BMP2 P= 0.0002; RUNX2 P= 0.0011; OSX P= 0.0002; ALPL P= 0.0002; DMP1 P= 0.0002 and PDPN P= 0.0002). Exact P values in e compared between IgG-treated and Nab-treated group (BMP2 P= 0.0054; RUNX2 P= 0.0005; OSX P= 0.0085; DMP1 P= 0.0099 and PDPN P= 0.0005). Data are representative of two independent experiments.