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. 2021 Feb 5;22(4):1626. doi: 10.3390/ijms22041626

Figure 5.

Figure 5

Hiltonol significantly activated tumor suppressors, PKR and/or OAS, in human lung cancer cells. The mRNA expression levels of: (A) MDA5 and (B) TLR3, (C) PKR (p68 protein kinase) and (D) OAS (2’5’ oligoadenylate synthetase) were measured. (A) LSD value in A549, H292, H1299, and H358 are 0.485, 0.227, 0.862, and 2.028, respectively. (B) LSD value in A549, H292, H1299, and H358 are 0.177, 0.319, 0.127, and 0.576, respectively. (C) LSD value in A549, H292, H1299, and H358 are 4.991, 0.679, 6.071, and 9.478, respectively. (D) LSD value in A549, H292, H1299, and H358 are 0.178, 0.528, 0.371, and 0.327, respectively. (E) shows the fold-change in the mRNA levels of PKR and OAS in response to Hiltonol (or polyI:C). The significant changes in the PKR and/or OAS mRNA overexpression in lung cancer cell lines are in red. To measure the effects of PKR and/or OAS in lung cancer upon Hiltonol treatment, 24 h after siRNA-knockdown of PKR or OAS or PKR+OAS double knockdown, NSCLC cells were treated with Hiltonol (or PBS) for 48 h, followed by viability and proliferation studies. (F) Cell survival was assayed using MTT and CTB. For each treatment, cell counts were normalized with PBS-treated controls. In MTT assays, LSD value in H292 and H358 are 2.87 and 2.631, respectively. In CTB assays, LSD value in H292 and H358 are 3.102 and 2.801, respectively. (G) Cell proliferation was measured using Alamar Blue or Trypan Blue dye exclusion tests. For each treatment, cell counts on 48 h post-treatment of Hiltonol (or PBS) were normalized with the corresponding 0 h treatment controls. PKR (or OAS) -specific siRNA significantly restored Hiltonol-induced cancer cell death and inhibited proliferation in H292 and H358 cells (blue boxes). PKR and OAS double knockdown further improved recovery of cancer cell viability and proliferation during Hiltonol treatment (red boxes). Data are representative of means ±SD (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. In Alamar Blue assays, LSD value in H292 and H358 are 5.934 and 4.628, respectively. In Trypan Blue exclusion studies, LSD value in H292 and H358 are 4.978 and 1.743, respectively.