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. 2019 Oct 8;11(10):1502. doi: 10.3390/cancers11101502

Figure 4.

Figure 4

Figure 4

Activation of malfunctional UPR underpins HA15 efficacy in MPM cells. (A,B) qRT-PCR of MESO-1 cells after treated with HA15 (10 uM) for the indicated time points. Data are presented as mean ± s.d. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.0001 by two-way ANOVA with Dunnett’s multiple comparisons test. (C) Western blots of MESO-1 cells after treatment with HA15 (20 uM) for the indicated time points. Quantification of the protein levels is shown under each band, with signal intensity measured by ImageJ and normalized to the loading control (β-actin). The value of the proteins in the vehicle group was set as 1. (D,E) FACS-based apoptotic assay of MESO-1 cells after treatment with indicated doses of HA15 for 72 h. Data are presented as mean ± s.d. (n = 3). ** p < 0.01, *** p < 0.005, and **** p < 0.0001 by two-way ANOVA with Dunnett’s multiple comparisons test. A representative FACS plot is shown (E) with early and late apoptosis quantified by the Annexin V+/PI- and Annexin V+/PI+ population, respectively. (F) MESO-1 cells transfected with the control (siCtrl) or CHOP siRNAs (siCHOP) were treated with indicated doses of HA15. Cell viability was measured 48 h after treatment (F). Data are presented as mean ± s.d. (n = 3). ** p < 0.01, and **** p < 0.0001 by two-way ANOVA with Dunnett’s multiple comparisons test. (G) Cell lysates were prepared from MESO-1 cells transfected with the control (siCtrl) or CHOP siRNAs (siCHOP) and analyzed by immunoblots. Protein quantification is shown underneath, whereby signal intensity was assessed by ImageJ and normalized to the loading control (β-actin), with the value of the DMSO group set as 1.