(A) Schematic representation of MMC-induced PVOD mouse model (top left) and immunoblot analysis of the indicated proteins in total lung lysates from vehicle (Veh)- or MMC-treated Control (Ctrl) and PKR-KO (KO) mice on day 5 (bottom left). The relative amounts of the indicated proteins, normalized to β-actin, are shown as mean ± SEM (right). n = 4 lung samples from Veh-treated Ctrl or KO mice and 6 lung samples from MMC-treated Ctrl or KO mice. (B) The levels of ATF4 target gene mRNAs, such as ATF3, ATF4, PKR, GADD34, GDF15, and VEGFA, in the lungs of Ctrl and KO mice treated with Veh or MMC on day 5 were analyzed by RT-qPCR. The results were normalized to β-actin levels and are presented as mean ± SEM. n = 9 independent samples. (C) ChIP assay was performed using the lungs harvested from Ctrl and KO mice treated with Veh or MMC employing an anti-ATF4 antibody, followed by PCR amplification corresponding to the genomic region of the PKR, ATF3, GADD34, and GDF15 genes spanning the ATF4 binding sequence. The PCR results are presented as fold enrichment over the input as mean ± SEM. n = 4 independent experiments. (D) In vivo puromycin incorporation assay. Ctrl and KO mice treated with Veh or MMC were administered puromycin on day 5, followed by lung lysate preparation. After normalizing the total protein content, samples were subjected to SDS-PAGE. Puromycin-labeled proteins were visualized by immunoblotting using anti-puromycin and anti–β-actin antibodies (left panel). The levels of puromycin-labeled proteins, normalized to β-actin as a loading control, are shown as mean ± SEM (right panel). n = 4 independent samples per group. Statistical analysis was performed using 2-way ANOVA with Tukey’s multiple-comparison test.