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. 2021 Jul 17;11(11):3567–3584. doi: 10.1016/j.apsb.2021.07.012

Figure 4.

Figure 4

HA-1141 triggers non-canonical ER stress, ISR and ROS production. (A–F) HA-1141 triggers ER stress in a dose- and time-dependent manner. H358 cells were exposed to different concentration of HA-1141, tunicamycin (12 μmol/L) or MLN4924 (0.3 μmol/L) for 24 h or treated with HA-1141 (20 μmol/L) for the indicated periods of time. The relative mRNA level was analyzed by RT-PCR analysis (n = 3). Shown are the mean ± SEM. ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05, NS, no significance vs. control. Tun, tunicamycin, MLN, MLN4924. (G, H) HA-1141 induces non-canonical ER stress and ISR. H358 cells were treated with HA-1141 or tunicamycin at the indicated concentrations for 24 h, or with HA-1141 (20 μmol/L) or tunicamycin (3 μmol/L) for the indicated periods of time, followed by Western blotting analysis. (I) PKR knockdown rescues P-eIF2α induced by HA-1141. H358 cells were transfected with the indicated siRNAs for 72 h and then treated with DMSO or HA-1141 (20 μmol/L) for 24 h, followed by Western blotting analysis. (J) HA-1141 induces ROS production. H358 cells were treated with HA-1141 for 0.5 h and then labeled with DCFH-DA (0.1 μmol/L) for 15 min at 37 °C, followed by FACS analysis (n = 3). Shown are the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01 vs. control. (K) NAC rescues upregulation of ATF4 by HA-1141. H358 cells were pre-treated with NAC (10 mmol/L) for 2 h and then co-incubated with HA-1141 (20 μmol/L) or DMSO for 12 h, followed by Western blotting with the indicated antibodies. N8, NEDD8.