Data Availability StatementAll data analyzed and generated through the current study are available from your corresponding author upon reasonable request. PCR.ER stress inhibitor 4-phenyl butyric acid (4-PBA) was used to explore the part of ER stress in GSK-J4 induced cell-cycle arrest and cell apoptosis. The combination effects of Decitabine and GSK-J4 on KG-1a cells proliferation and apoptosis were also evaluated by CCK8, circulation cytometry and immunoblot analysis. Results GSK-J4 reduced cell viability and caught cell cycle progression in the S phase by reducing the manifestation of CyclinD1 and CyclinA2 and increasing that of P21. Moreover, GSK-J4 enhanced the manifestation of apoptosis-related proteins (cle-caspase-9 and bax) and inhibited PKC-a/p-Bcl2 pathway to promote cell apoptosis. alpha-Cyperone In addition, ER stress-related proteins (caspase-12, GRP78 and ATF4) were improved markedly after exposure to GSK-J4. The effects of GSK-J4 on cell cycle, apoptosis and PKC-a/p-Bcl2 pathway were attenuated after treatment with ER stress inhibitor. Furthermore, decitabine could significantly inhibit the proliferation and induce the apoptosis of KG-1a cells after combined treatment with GSK-J4. Conclusion Taken together, this study provided evidence that ER stress could regulate the process of GSK-J4-induced cell cycle arrest, cell apoptosis and PKC-/p-bcl2 pathway inhibition and demonstrated a potential combinatory effect of decitabine and GSK-J4 on leukemic cell proliferation and apoptosis. test. The data were presented as mean??standard deviation (SD). em p /em -value? ?0.05 was considered statistically significant. Results GSK-J4 induced cell growth inhibition and cell cycle arrest Cell proliferation was monitored by using the CCK-8 assay. The CCK-8 data (Fig.?1a) showed that the viability of KG-1a cells was decreased in FLJ39827 a dose-dependent manner after treatment with 2, 4, 6, 8 and 10?M of GSK-J4 for 0, 24, 48, 72 and 96?h compared with the control group (p? ?0.05). To examine the effect of GSK-J4 on cell growth inhibition, the distribution of KG-1a cell phase was evaluated by flow cytometric. As shown in Fig.?1a, b, GSK-J4 led to a notable accumulation of S phase cells in a dose-dependent manner (p? ?0.05). After treatment with different concentrations of GSK-J4 for 48?h, the expression level of P21 was increased, while the expression levels of CyclinD1 and CyclinA2 were decreased significantly in a dose-dependent manner (p? ?0.05) (Fig.?1d, e). Open in a separate window Fig.?1 The effects of GSK-J4 on KG-1a cell proliferation and cell cycle distribution. a Cell viability was analyzed by the CCK-8 assay kit. b Cell cycle distribution was detected with flow cytometry. c The quantitative cell cycle distribution data. Values represent the mean??SD of three independent experiments. *p? ?0.05. d Western blotting was used to quantitatively analyze the expression levels of P21, CyclinD1 and CyclinA2. e Statistical analysis of the expression levels of P21, CyclinD1 and CyclinA2. -Actin was used as an internal control. Values stand for the suggest??SD of 3 independent tests.*p? ?0.05, **p? ?0.01 GSK-J4 induces KG-1a cell apoptosis To determine whether GSK-J4 make a difference KG-1a cell apoptosis, several apoptotic guidelines had been assessed by stream cytometry and European blotting. The movement cytometric data exposed how the apoptotic price of KG-1a cells in GSK-J4 treatment group was considerably increased set alongside the control group (p? ?0.05)(Fig.?2a, b). Furthermore, the outcomes of Traditional western blotting showed how the manifestation degrees of apoptosis-related protein (bax and cle-caspase9) had been significantly improved in GSK-J4 treatment organizations (p? ?0.05) (Fig.?2c, d). Open up in another windowpane Fig.?2 GSK-J4 induces KG-1a cell apoptosis. a The pace of cell apoptosis was detected by PI and annexin-V double-staining. b Statistical evaluation from the apoptotic price. Values stand for the suggest??SD of 3 independent tests.* em p /em ? ?0.05, ** em p /em ? ?0.01. c alpha-Cyperone Traditional western blotting was utilized to investigate the manifestation degrees of bax and cle-caspase9 in KG-1a cells after treatment with GSK-J4 for 48?h. d Statistical alpha-Cyperone evaluation from the manifestation degrees of Bax and cle-caspase9. -Actin was utilized as an interior control. Values stand for the suggest??SD of 3 independent tests.* em p /em ? ?0.05, ** em p /em ? ?0.01, *** em p? /em ?0.001 GSK-J4 triggered ER tension To examine whether GSK-J4 can result in ER tension, the protein manifestation degrees of ER stress-related molecules, such as caspase-12, GRP78 and ATF4, were detected by Western blotting. As is shown in Fig.?3a, b. The protein levels of caspase-12, GRP78 and ATF4 were increased significantly in KG-1a cells treated with GSK-J4 compared to the control group (p? ?0.05). To further confirm that GSK-J4 can stimulate ER stress, we detected the molecular indicators of ER stress in KG-1a cells after co-treatment with 4-phenyl butyric acid (4-PBA, the inhibitor of ER stress). The results of Western blotting indicated that the protein levels of caspase-12, GRP78 and ATF4 were remarkably lower in GSK-J4.