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. 2019 Oct 23;53(1):e12706. doi: 10.1111/cpr.12706

Figure 5.

Figure 5

WA‐induced apoptosis and G2/M arrest of GBM cells by ATF4‐ATF3‐CHOP axis. Cells were treated with 3 μmol/L WA for the indicated time and harvested for qRT‐PCR and Western blotting. For siRNA silencing, U251 cells were separately transfected with siRNAs of HMOX1, DNAJB1, ATF3, ATF4, XBP1 and CHOP for 48 h, then treated with 3 μmol/L WA for an additional 24 h, and finally analysed by MTT, Western blotting and Flow cytometry. A, Genes associated with ER stress were determined by qRT‐PCR. *P < .05, **P < .01 and ***P < .001 represented significant differences between U251 cells and U87 cells. B, Proteins associated with ER stress were identified by Western blotting. C, Cell viability of U251 cells treated by 3 μmol/L WA was measured by MTT assay after transfection with siRNAs of HMOX1, DNAJB1, ATF3, ATF4, XBP1 and CHOP, respectively. **P < .01 represented significant differences between the siRNA‐treated group and the corresponding negative control group. D, Apoptosis of U251 treated with 3 μmol/L WA was measured by Fow cytometry after transfection with siRNAs of ATF3, ATF4 and CHOP, respectively. *P < .05 and **P < .01 represented significant differences between the siRNA‐treated group and the corresponding negative control group. E, Mitochondrial membrane potentials of U251 treated with 3 μmol/L WA were measured by Flow cytometry after transfection with siRNAs of ATF3, ATF4 and CHOP, respectively. *P < .05 and **P < .01 represented significant differences between the siRNA‐treated group and the corresponding negative control group. F, The cell cycle stage of U251 treated with 3 μmol/L WA was determined by Flow cytometry after transfection with siRNAs of ATF3, ATF4 and CHOP, respectively. **P < .01 and ***P < .01 represented significant differences between the siRNA‐treated group and the corresponding negative control group. G, The changes of p21, Bim, Bad and cleaved‐PARP1, caspase 3/7/9 and cleaved caspase 3/7/9 proteins in U251 cells treated with 3 μmol/L WA were determined by Western blotting after transfection with siRNAs of ATF3, ATF4 and CHOP, respectively. H, The overall mechanism of WA activity in GBM cells. WA, Withaferin A