Fig. 2

HOXD9 induces proliferation, cell cycle progress and apoptotic inhibition of NSCLC cells. NCI-H661 cells were transfected with HOXD9 overexpressing plasmid (exHOXD9) or empty vector, while NCI-H292 cells were transfected with two small interfering RNAs targeting HOXD9 (siHOXD9^#1, siHOXD9^#2) or none-specific sequence (siNC). A After 48 h of transfection, HOXD9 mRNA expression in two NSCLC cells (NCI-H661 and NCI-H292) was evaluated by real time-PCR assay. B After 48 h of transfection, HOXD9 protein expression in two NSCLC cells was evaluated by western blot assay. C After 48 h of transfection, NSCLC cells were seeded into 96-well microplates and analyzed with CCK-8 reagents at indicated time. D After 48 h of transfection, NSCLC cells were subjected into the EdU incorporation assay. The new generation cells were stained via EdU (red). DAPI stained nuclei in blue. Scale bar = 50 μm. Quantification of EdU-positive cells was performed to assess cell proliferation. E Cell cycle progression in two NSCLC cells was evaluated by flow cytometry analysis. F Apoptosis in NCI-H292 cells was evaluated by flow cytometry analysis. The apoptotic rate was quantified by adding the percentages of early apoptotic cells (Annexin V⁺/PI⁻ in Q4 quadrant) and late apoptotic cells (Annexin V⁺/PI⁺ in Q2 quadrant). G Expression of protein markers involved in apoptosis and cell cycle regulation in NCI-H292 cells was evaluated by western blot assay. * p < 0.05; ** p < 0.01; *** p < 0.001. Data in (A), (C), (D), (E) and (F) are presented as mean values ± SD. The blots were cropped and the original uncropped images of blots were shown in supplementary materials