Fig. 5: ISO stimulates ER stress via the inhibition of AKT phosphorylation.

A, B Human osteosarcoma U2OS cells stably expressing GFP under the CHOP promoter (CHOP::GFP) were treated with the indicated agents (tunicamycin, TM (3 μM), TG thapsigargin (3 μM), isobacachalcone ISO (25 μM)) for 24 h. GFP nuclear translocation is shown in (A), and the average nuclear intensity of GFP was quantified in (B). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^**P < 0.01, ^***P < 0.001 vs. untreated control; Student’s t test). C, D U2OS cells stably expressing XBP1ΔDBD-venus (for monitoring venus expression upon alternative splicing of XBP1 mRNA) were treated as indicated for 16 h. XBP1s expression is shown in (C), and the average nuclear intensity was measured in (D). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^**P < 0.01, ^***P < 0.001 vs. untreated control; Student’s t test). E–L U2OS wild-type (WT) or knock-in for AKT^T308D/S473D cells were treated with TM (3 μM), TG (3 μM), ISO (25 μM) 24 h for assessing CHOP, 6 h for measuring peIF2α, and 16 h for monitoring ATF6 and XBP1s. After fixation, the cells were stained with corresponding primary antibodies followed by an AlexaFluor-568 secondary antibody. Nuclei were counterstained with Hoechst 33342. CHOP nuclear expression is shown in (E), and the average nuclear intensity of CHOP was quantified in (F). ATF6 nuclear translocation is shown in (G), and the average nuclear intensity of ATF6 was quantified in (H). PeIF2α was assessed by means of immunofluorescence staining (I), and the average cytoplasmic intensity of cells was depicted in (J). XBP1s activation is shown in (K), and the average nuclear intensity was measured in (L). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. untreated control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. WT; Tukey’s multiple comparisons test).