Figure 2.

Neem leaf glycoprotein does not alter primary tumor cell properties. (A1) Schematic presentation of experiment design. Tumor cells were injected through t.v. followed by NLGP administration on the next day for once a week for 4 weeks, and mice were sacrificed following treatment completion (n = 3, for three times). (A2,A3) Representative photograph shows PBS- and NLGP-treated mice with its corresponding metastatic lungs (B16F10 and LLC), respectively. Dot plots indicate (B1,B3) mean lung weight ± SEM; (B2,B4) mean liver weight ± SEM of B16F10 and LLC bearing tumor host. (C1,C2) Representative hematoxylin-eosin–stained lung and liver sections of B16F10 and LLC mice. (D1,D2) B16F10 and LLC cells were treated with NLGP (1.5 μg/mL). Representative flow-cytometric dot plot overlaid, and histogram data represent the annexin V⁺ PI⁺, Ki67⁺, and PI⁺ cells from PBS- and NLGP-treated B16F10 and LLC cells (n = 1, repeat for three times). Neem leaf glycoprotein–treated and PBS tumor cell supernatant were analyzed for different cytokines by ELISA. Dot plots indicate mean amount of VEGF, TGF-β, and IL-2 for B16F10 (D3) and LLC (D4) cells, respectively (n = 1, repeat for three times; see Figures S2B.1,B.2; for statistical analysis). (D5,D6) Total RNA was isolated from NLGP- and PBS-treated tumor cells, and gene expression of VEGF, TGFβ, and IL-10 was analyzed against beta-actin as housekeeping gene (n = 1, repeat for three times).