Fig 2: RNA-seq revealed that KIAA1199 promoted complement activation.A–L Mice received an intraperitoneal injection of LPS (10 mg/kg) to induce sepsis-induced ALI, and samples were harvested after 12 h. A Volcano plot of differentially expressed genes (DEGs) in AT2 from LPS-treated KIAA1199fl/fl and KIAA1199Spccre mice. n = 3 per group. B The enriched Gene Ontology (GO) analysis of the DEGs. C The enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEGs. D–F The expression of CFH was detected by western blot, immunofluorescence and qPCR in lung tissues of mice. n = 6 per group. G, H CFH concentration in serum and BALF were detected by ELISA. n = 6 per group. I–K The markers of complement activation in the BALF of mice were detected by ELISA, including C3a, C3b, and membrane attack complex(C5b-9). n = 6 per group. L C5b-9 in the lung tissues of mice was detected by immunofluorescence. All the experiments were performed in triplicate. The data were presented as mean ± SD. One-way ANOVA (D, F–K) followed by the Tukey test was used as a parametric test. *P < 0.05, **P < 0.01, ***P < 0.001. ns, no significance.

Fig 3: KIAA1199 deficiency alleviated lung injury by upregulating CFH.A The efficiency of CFH knockdown was detected by Western blot in the lung tissues of mice. B Effect of CFH knockdown on survival rates in septic mice (n = 10 per group). C–I Mice received an intraperitoneal injection of LPS (10 mg/kg) to induce sepsis-induced ALI, and samples were harvested after 12 h. n = 6 per group. C The degree of pulmonary edema was detected by the lung wet-to-dry weight ratio. D The total proteins of BALF were detected using the BCA kit. E The MPO activity of lung tissues. F The lung injury in mice was detected by H&E staining. G–J Levels of cytokines in the BALF of mice were detected by ELISA, including IL-6, IL-1β, TNF-α, and IL-10. K The cell apoptosis of lung tissue was detected by TUNEL staining. L The expression of ZO-1, Occludin, and Claudin-4 was detected by Western blot of lung tissues. All the experiments were performed in triplicate. The data were presented as mean ± SD. Data were analyzed using one-way ANOVA (A, C–L) with Tukey’s post hoc test and log-rank test (B). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, no significance.

Fig 4: KIAA1199 as a potential prognostic biomarker for sepsis patients.A The levels of serum KIAA1199 between healthy controls and septic patients were detected by ELISA. B The levels of serum KIAA1199 between septic patients with or without ARDS. C The levels of serum KIAA1199 between septic patients who did or did not die within 28 days after admission. D Correlation of the level of serum KIAA1199 with the Murray Lung Injury Score of septic patients with ARDS were evaluated by Pearson correlation coefficient. E Correlation of the level of serum KIAA1199 with the SOFA score of all patients were evaluated by Pearson correlation coefficient. F Receiver operating characteristic (ROC) curves of serum KIAA1199 at admission in predicting 28-day mortality of septic patients. AUC was 0.80 (95% CI, 0.67–0.93) for serum KIAA1199 at admission in the cohort of septic patients. AUC = area under the curve. Unpaired t-test (A–C) was used as a parametric test. **P < 0.01, ***P < 0.001.

Fig 5: KIAA1199 downregulated CFH expression by promoting ubiquitination of p53.A The Gene Set Enrichment Analysis (GSEA) enrichment plot of the p53 pathway. B, C The expression of p53 was detected by qPCR and western blot. D Schematic representation of truncated fragments of CFH promoter. E Double luciferase reporter gene assay of CFH promoter fragments. Cells were transfected with pGL3-basic plasmids containing CFH promoters of different lengths and the reference pRL-TK plasmid, and harvested for luciferase activity analysis at 48 h post-transfection. F The sequence of p53 binding motif in the promoter of CFH was predicted by JASPAR. G Double luciferase reporter gene assay of wildtype (WT) and mutant (MUT) CFH promoter. Cells were transfected with pGL3-basic plasmids containing wildtype/mutant CFH promoters and the reference pRL-TK plasmid, and harvested for luciferase activity analysis at 48 h post-transfection. H Electrophoretic mobility shift assay (EMSA) was used to determine the binding of CFH probes to nuclear extract protein of A549 cells. I CHIP-qPCR assay of p53 binding to the promoter of CFH in A549 cells. J The protein expression of p53 was analyzed by western blot in A549 cells with KIAA1199 overexpression. After transfection for 48 h, cells were treated with DMSO or the proteasome inhibitor, MG132 (10 μM), for 6 h. K The ubiquitination of p53 was detected by western blot in A549 cells with KIAA1199 overexpression. After transfection for 48 h, cells were treated with MG132 (10 μM) for 6 h. L The protein stability of p53 was analyzed by western blot in A549 cells with KIAA1199 overexpression. After transfection for 48 h, cells were treated with cycloheximide (CHX, 50 μg/ml) at the indicated time points. Statistical significance was determined by two-way ANOVA of repeated measurements. M The interaction between p53 and MIB1 was analyzed by Co-IP and western blot in A549 cells at 48 h post-transfection. N The interaction between p53 and MIB1 was analyzed by Co-IP and western blot in A549 cells with KIAA1199 overexpression at 48 h post-transfection. O The ubiquitination of p53 was detected by western blot in A549 cells with MIB1 knockdown. After lentiviral infection for 48 h, cells were treated with MG132 (10 μM) for 6 h. P The protein stability of p53 was analyzed by western blot in A549 cells with MIB1 knockdown. After lentiviral infection for 48 h, cells were treated with CHX (50 μg/ml) at the indicated time points. Statistical significance was determined by two-way ANOVA of repeated measurements. All the experiments were performed in triplicate. The data were presented as mean ± SD. Unpaired t-test (B, C, G, I, J) and one-way ANOVA (E) followed by Dunnett test were used as parametric tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, no significance.