Fig. 2

ATF2 promotes RCC cells proliferation in vitro. a CCK8 assay of ATF2 knockdown and control RCC cells at indicated times. b Plate colony formation assay of ATF2 knockdown and control ACHN cells in 10 cm dish for 3 weeks (n = 3). Average number of colonies (upper) and representative images (lower) were shown. c Flow cytometry analysis of cell cycle in ATF2 knockdown and control RCC cells. Representative cell cycle distributions were shown (left) and the histogram columns represent the average percentages of G0/G1, S and G2/M phases (right). d Flow cytometry analysis of apoptotic cells in ATF2 knockdown and control RCC cells (left) and the percentage of cells at the different apoptosis phases (right). The bar charts showed the increases in the early and late apoptotic indexes of RCC cells transfected with shATF2. e CCK8 assay of ATF2 overexpression and control RCC cells at indicated times. f Plate colony formation assay of ATF2 overexpression and control ACHN cells in 10 cm dish for 3 weeks (n = 3). Average number of colonies (upper) and representative images (lower) were shown. g Flow cytometry analysis of cell cycle in ATF2 overexpression and control RCC cells. Representative cell cycle distributions were shown (left) and the histogram columns represent the average percentages of G0/G1, S and G2/M phases (right). h & i Western blotting analysis of Cyclin B1 and Cyclin D1 in ATF2 knockdown (h), ATF2 overexpression (i) and control ACHN cells. β-Actin was used as an internal standard. j ChIP assay analysis of the enrichment of ATF2 at the proximal promoter region of Cyclin B1 and Cyclin D1 in the context of ATF2 overexpression. The enrichment of ATF2 (upper) on Cyclin B1 or Cyclin D1 promoter relative to input in 786-O cells and gel electrophoresis of PCR products from ChIP assay (lower). Results are presented as mean ± SEM from three independent experiments. *p < 0.05, **p <0.01 and *** p < 0.001