Fig. 6. GCN2-mediated integrated stress response contributes to the production of proinflammatory SASP components.
a Volcano plot showing upregulated genes related to the ISR signaling (green) and mitogen-activated protein kinase cascade (MAPK) signaling pathway (blue) in WT typhoid toxin-treated macrophages compared to PltBS35A mutant toxin-treated macrophages using an arbitrary threshold of P < 0.05 and fold change >1.5. b Expression of genes involved in the ISR signaling and MAPK signaling pathway by RNA-seq. c Immunoblot analyses demonstrate the activation of MAPK signaling pathways in THP-1-derived macrophages treated with typhoid toxin. Phosphorylated JNK, p38, ERK1/2, p65, and β-actin (loading control) were monitored at various time points. d THP-1-derived macrophages exposed to typhoid toxin were treated with p38 MAPK (SB202190) (1 μM) and JNK MAPK (SP600125) (5 μM) inhibitors for 16 h. Total mRNA was analyzed using RT-qPCR. Data are presented as mean ± s.d (n = 3) (t-tests; *P < 0.05, ns, not significant). e A model (Created with BioRender.com) of the ISR signaling. f The ISR signaling pathway in THP-1-derived macrophages exposed to typhoid toxin was analyzed using western blot analysis with specific antibodies. g, h Total mRNA and protein levels of ISR target genes were quantified using RT-qPCR (g) and western blot analysis (h), respectively. Data are presented as mean ± s.d (n = 3) with statistical significance (t-tests; ****P < 0.0001). i The mRNA levels of specific genes were assessed using RT-qPCR. Data are presented as mean ± s.d (n = 3). Statistical analysis was performed using unpaired two-sided t-tests; *P < 0.05, **P < 0.01. j Western blot analyses were performed to assess the activation of the GCN2-mediated ISR pathway. k, l The mRNA levels of proinflammatory genes were measured in THP-1-derived macrophages exposed to typhoid toxin under ISRIB treatment (30 μM) (k) or in ATF3-deficient cells and their parental cell line (l). RT-qPCR was used for gene expression analysis. Data are presented as mean ± s.d (n = 3) (t-tests; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.). m Activation of GCN2 in STING-deficient THP-1-derived macrophages and their parental cell line exposed to typhoid toxin was determined by western blot analysis. The western blots shown in (c), (f), (h), (j), (m) are representative of 3 independent experiments. Source data are provided as a Source data file.