Figure 3.

Neem leaf glycoprotein does not attenuate tumor cell migration. (A1) In vitro migration assay was performed in 8-μm pore Transwell insert with overnight serum starve tumor cells (B16F10 and LLC) against 10% heat-inactivated FBS in presence and absence of NLGP (1.5 μg/mL). Representative photographs of one of three independent experiments show crystal violet (0.01%)–stained migrated cells (after 16 h). Serum-free gradient was used as negative control. Dot plot (A2) indicates the mean number of migrated cells ± SEM of five separate fields from the same experiment. One-way analysis of variance (ANOVA) was used for statistical analysis followed by Tukey multiple-comparisons test. (B) A scratch wound was made in a full confluent B16F10 cell layer with a cell scratcher. Wound healing was monitored in a time-dependent manner in presence or absence of NLGP (1.5 μg/mL). Representative photograph shows one of three independent experiments. Wound closure was measured by using ImageJ by calculating the distance between the edge manually. Two-way ANOVA was used for statistical analysis followed by Bonferroni test. ***p < 0.001. (C1) Schematic experimental design. Tumor cells (B16F10 and LLC) were stained with CFSE followed by t.v. injection (n = 3, repeats for one). Mice were sacrificed following 6 and 18 h of tumor inoculation, and lungs were harvested. (C2) Representative dot plot image of flow-cytometric analysis of CFSE⁺ tumor cells in lungs after 6 and 18 h of tumor inoculation. (C3) Bar diagram indicates mean CFSE⁺ tumor cells ± SEM (n = 3). (D1,D2) Representative immune-fluorescence image of harvested lung sections at 10× magnification (n = 3, repeat for one). CFSE⁺ tumor cells are indicated by green color. Scale bar 200 μm. **p < 0.005.