GCN5L1 gene KO impaired surfactant production in MLE-12 cells. A Phospholipid secretion in WT and GCN5L1-KO MLE-12 cells. B ELISAs of secreted SPs in the culture supernatant of WT and GCN5L1-KO MLE-12 cells. C Reconstruction of GCN5L1 expression repaired phospholipid secretion in GCN5L1 mutant cells. D Reconstruction of GCN5L1 expression repaired SP-B and SP-C secretion in GCN5L1 mutant cells. The results are expressed as the mean ± SD of three independent experiments; **P < 0.01; t-test

Disruption of GCN5L1 altered the expression of Cebpα and surfactant-related genes. A Q-PCR verification of the surfactant-related DEGs in GCN5L1 mutant clones. B Q-PCR detection of Cebpα mRNA expression in WT and GCN5L1-KO MLE-12 cells. C Representative immunoblot image of Cebpα protein expression levels in WT and GCN5L1-KO MLE-12 cells. D Representative immunoblot images of Cebpα protein levels in WT cells, M2 cells, M2 cells infected with lentivirus expressing exogenous Cebpα (M2 + Cebpα), and M2 cells infected with lentivirus expressing exogenous GCN5L1 (M2 + GCN5L1). E Q-PCR assays of the RNA levels of surfactant-related genes in WT cells, M2 cells, and M2 cells infected with lentivirus expressing exogenous Cebpα (M2 + Cebpα), and M2 cells infected with lentivirus expressing exogenous GCN5L1 (M2 + GCN5L1). The results are expressed as the mean ± SD of three independent experiments; ns, not significant;*P < 0.05, **P < 0.01; t-test

Disruption of GCN5L1 altered the activity of the ROS–ERK–FOXO1 axis in MLE-12 cells. A Q-PCR detection of Foxo1 mRNA expression in WT, GCN5L1-KO MLE-12 cells. B Representative immunoblot image of Foxo1 protein expression levels in WT, GCN5L1-KO MLE-12 cells and M2 cells infected with lentivirus expressing exogenous GCN5L1 (M2 + GCN5L1). C Representative immunoblot images of ERK and p-ERK protein expression levels in WT, GCN5L1-KO MLE-12 cells and M2 cells infected with lentivirus expressing exogenous GCN5L1 (M2 + GCN5L1). D Cellular ROS levels of WT, GCN5L1-KO MLE-12 cells, and M2 cells infected with lentivirus expressing exogenous GCN5L1 (M2 + GCN5L1). Cells were incubated with CM-H2DCFDA and measured by FACS; mean fluorescence intensity was normalized to WT group. E Representative immunoblot images of ERK, p-ERK, Foxo1, and Cebpα protein expression levels in WT cells, M2 cells treated with DPI (M2 + DPI) or SCH772984 (M2 + SCH772984), and M2 cells infected with lentivirus expressing exogenous Foxo1 (M2 + Foxo1). F Q-PCR assays of the RNA levels of surfactant-related genes in WT cells, M2 cells treated with DPI (M2 + DPI) or SCH772984 (M2 + SCH772984), and M2 cells infected with lentivirus expressing exogenous Foxo1 (M2 + Foxo1). The results are expressed as the mean ± SD of three independent experiments; ns, not significant; *P < 0.05,**P < 0.01; t-test

Mitochondria-localized GCN5L1 is responsible for the regulation of surfactant-related genes. A Colocalization of GCN5L1–FLAG with TOM20 in MLE-12 cells. Cells were cotransfected with TOM20- and GCN5L1–FLAG-expressing plasmids before staining with anti-TOM20 and anti-FLAG antibodies. B The colocalization scatter plot of anti-TOM20 and anti-GCN5L1–FLAG signals. Pearson correlation coefficient is 0.78, Manders’ overlap coefficient is 0.82. C Q-PCR assays of the RNA levels of surfactant related genes in WT cells, M2 cells, and M2 cells infected with lentivirus expressing mito-GCN5L1 (M2 + mito-GCN5L1).The results are expressed as the mean ± SD of three independent experiments; *P < 0.05, **P < 0.01; t-test

Impairment of LB positioning/trafficking in GCN5L1-KO cells. A Lysotracker Red staining of WT, GCN5L1-KO clones (M2 and M29), and GCN5L1 reconstructed mutant cells (M2 + GCN5L1). B WT and M2 cells were metabolically labeled with BODIPY phosphatidylcholine. C WT and M2 cells were immunostained with Lamp1 antibody. D–F WT and M2 cells were transfected with h-ABCA3–EGFP or ABCA3–mCherry before staining with Lysotracker Red (D), BODIPY phosphatidylcholine (E), and Lamp1 antibody (F)

Abnormal size and number of LB-like organelles in GCN5L1-KO cells. A Representative images showing Lysotracker-stained organelles in WT, M2, M2 + GCN5L1, and M2 + mito-GCN5L1 cells. B, C The numbers and size (area) in WT, M2, M2 + GCN5L1, and M2 + mito-GCN5L1 cells; **P < 0.01 (Kolmogorov–Smirnov test). D Transmission electron micrographs of WT and M2 cells. Many LB-like organelles are present within the cytoplasm; arrows point to lamellar-like materials in the organelles. E, F The diameters and numbers of LBs in the TEM images were measured and counted with Image J. The results are expressed as the mean ± SD of four independently measured data; *P < 0.05; t-test

Localization of GCN5L1 in LBs. Cells were transfected with GCN5L1–FLAG-expressing plasmid and stained with anti-Lamp1 and anti-FLAG antibodies

Accumulation of SP proteins in GCN5L1-KO cells. A Immunoblot image of WT, M2, and GCN5L1 reconstructed M2 + GCN5L1 cells with an SP-B antibody (Santa Cruz). B Immunoblot image of WT, M2, and GCN5L1 reconstructed M2 + GCN5L1 cells with another SP-B antibody (Abcam). C WT and M2 cells were cotransfected with h-ABCA3–mCherry and m-Sftpb expressing plasmids and immunostained with an SP-B antibody (Abcam). D WT and M2 cells were cotransfected with h-ABCA3–EGFP- and m-Sftpc-expressing plasmids and immunostained with an SP-C antibody (Seven Hills Bioreagents). **P < 0.01; t-test

A hypothetical illustration of GCN5L1 functions in the regulation of pulmonary surfactant production