Fig 2: Functional mechanism of ATO‐induced inhibition of APL cell growth. We investigated the functional mechanism of different concentrations of ATO on the expression level of E2F1, cyclin E, and pRb in p53‐knockdown NB4 cells by western blotting. ATO did not significantly reduce the expression of E2F1 and cyclin E and accumulation of pRb in p53‐knockdown NB4 cells (3A). It phosphorylated pRb at S608 and T373 residues both in KG1a and NB4 cells (3B, C) and also regulated the association of E2F1, p53 and pRb in KG1a cells (3D), as characterized by western blotting with phosphor active antibodies and IP method. ImageJ bundled with Java 1.8.0_172; URL—https://imagej.nih.gov/ij/download.html. APL, acute promyelocytic leukemia; ATO, arsenic trioxide; E2F1, E2F Transcriptional factor‐1

Fig 3: Effect of ATO on the interaction between pRb and E2F1 in APL cells. CHIP assay was performed with protein lysate of both untreated and ATO treated NB4 cells and protein‐DNA complex was precipitated with specific E2F1 and pRb antibodies. Proteins were cleaved from complex using proteinase K treatment and CHIP DNA eluted, and real time PCR was performed using Taqman probes of both E2F1 and pRb. Our findings indicate that ATO reduced interaction of E2F1 (2A) by stimulating the interaction of pRb (2B) significantly with E2F1 promoter in KG1a cells. APL, acute promyelocytic leukemia; ATO, arsenic trioxide; CHIP, chromatin‐immunoprecipitation; E2F1, E2F Transcriptional factor‐1

Fig 4: ATO reduces expression of E2F and cyclin E, and upregulates the expression and accumulation of pRb in APL cells. APL cell lines were treated with different concentrations (0, 2, 4, 6 and 8 μg/ml) of ATO for 24 h. After incubation, cell lysates were prepared in RIPA buffer and the expression profiles of E2F1, cyclin E, and pRb were analyzed by western blotting. ATO reduced the expression levels of E2F1 and cyclin E and activated pRb expression in NB4 (1A), KG1a (1B), & HL‐60 (1C) cells. We also checked both expression and localization of E2F1 and pRb in both ATO‐treated and untreated KG1a by confocal imaging. ATO reduced the expression of E2F1 and increased accumulation of pRb significantly in KG1a cells (Figure 1(D,E), i–v)). ImageJ bundled with Java 1.8.0_172; URL—https://imagej.nih.gov/ij/download.html. APL, acute promyelocytic leukemia; ATO, arsenic trioxide; E2F1, E2F Transcriptional factor‐1; RIPA, radioimmunoprecipitation assay buffer