Fig 2: PLAC8 is decreased in BLM-induced lungs in mice.a, b Hematoxylin and eosin (HE) and Masson’s trichrome staining of lung tissues on day 14 post-BLM administration. c Immunofluorescence analysis of the Surfactant protein C (SPC, green) and TUNEL (red) in lung tissues. The white arrows were pointing to the double positive cells. d The percentages of double TUNEL + SPC+ positive cells in the SPC+ positive cells were determined. e, f Immunoblotting and semi-quantitative analysis of SPC, PLAC8, cleaved caspase-3, LC3, and p62 protein levels in lungs on indicated days after BLM treatment. g Immunofluorescence analysis of the PLAC8 (green) and E-cadherin (red) in lung tissues on day 3 after BLM treatment. h The percentages of double PLAC8 + E-cadherin + positive cells in the E-cadherin+ positive cells were determined. Data represent the mean ± SD (6 mice per group; representative of three independent experiments). Unpaired t-test applied for (d and h), and Tukey’s multiple comparison test after the one-way ANOVA was conducted for (e, f). *P < 0.05. NS non-significant.

Fig 3: Overexpression of PLAC8 inhibits BLM-induced apoptosis of pmATII cells and A549 cells.a–d The pmATII cells and A549 cells were treated with 50 μM BLM for 24 h. RT-qPCR analysis and immunoblotting analysis of PLAC8 levels. e, f The mRNA and protein levels of PLAC8 in pmATII cells after infection of adenovirus containing the mouse PLAC8 overexpression vector (oe-PLAC8) or negative control (oe-NC). g, h The mRNA and protein levels of PLAC8 in A549 cells after transfection of PLAC8 overexpression plasmid (OE-PLAC8) or negative control (OE-NC). Immunoblotting analysis of cleaved caspase-3 protein levels in PLAC8-overexpressing pmATII cells (i) and A549 cells (j) after BLM treatment. Cell apoptosis was detected by TUNEL staining of pmATII cells (k) and A549 cells (l). Data represent the mean ± SD (n = 3 per group; representative of three independent experiments. Unpaired t-test applied for (a and c), and Tukey’s multiple comparison test after the one-way ANOVA was conducted for (e–j). *P < 0.05.

Fig 4: Overexpression of PLAC8 alleviates BLM-induced lung fibrosis in mice.a Schematic diagram of the construction of alveolar epithelial type II (ATII)-specific overexpression of PLAC8 under the Surfactant protein B (SPB) promoter and BLM-induced mouse lung fibrosis in mice. (The schematic pictures were downloaded from sciDraw, mouse: doi.org/10.5281/zenodo.3925901; syringe: 10.5281/zenodo.4152947). b, c Hematoxylin and eosin (HE) staining of lung tissues on day 14 post-BLM administration and quantification of lung fibrosis by the Ashcroft score. d, e Masson’s trichrome staining of lung tissues and quantification of lung fibrosis. f Lung/body coefficient was calculated. g Collagen measurement by the hydroxyproline assay in lungs. Immunoblotting analysis of PLAC8 and α-SMA (h), collagen I and fibronectin (i), p53 (j), LC3, and p62 (k) in lung tissues of mice. Data represent the mean ± SD (6 mice per group; representative of three independent experiments). Tukey’s multiple comparison test after the one-way ANOVA was conducted for (c–k). *P < 0.05.

Fig 5: PLAC8 regulates p53 expression and apoptosis through interacting with VCP-UFD1-NPLOC4 complex.a Coimmunoprecipitation (COIP) verified the interaction among PLAC8, p53, VCP, UFD1, and NPLOC4 in BLM-treated cells. b Protein-protein interaction (PPI) network analysis of PLAC8, p53, VCP, UFD1, and NPLOC4 in ATII cells based on STRING database, COIP/MS analysis, and COIP experimentally determined. c A549 cells were transduced with OE-PLAC8 and incubated with MG132 and BLM, the ubiquitinated p53 was detected. d The p53 degradation was measured in PLAC8 overexpressing cells after incubation with CHX and BLM. e Cells were transfected with OE-PLAC8 and siRNA against VCP (siVCP) or UFD1 (siUFD1) and then treated with MG132 and BLM. The accumulation of ubiquitinated p53 was detected. f, g Immunoblotting analysis of the p53 expression and degradation in OE-PLAC8 and siVCP transfected cells. h Immunoblotting analysis of LC3 and p62 in A549 cells. i Cell apoptosis was detected by TUNEL staining. Data represent the mean ± SD (n = 3 per group; representative of three independent experiments). Tukey’s multiple comparison test after the one-way ANOVA was conducted for (f and h), Sidak’s test multiple comparison test two-way ANOVA was conducted for (d and g). *P < 0.05. NS non-significant.