Journal: Advanced Science

Article Title: Metabolic Interplay in Acute Lung Injury: PARK7 Integrates FADS1/2‐Dependent PUFA Metabolism and H3K14 Lactylation to Attenuate Endothelial Ferroptosis and Dysfunction

doi: 10.1002/advs.202508725

Figure Lengend Snippet: PARK7 regulates FADS1/2 expression by upregulating bone morphogenetic protein (BMP)‐SMAD1/5/9 signaling. A) Quantitative real‐time PCR (qPCR) analysis of the expression of FADS1/2 precursor mRNA (n = 3). B) Gene set enrichment analysis (GSEA) of signaling pathways from the Pathway Interaction Database (PID). C) GSEA analysis of BMP signaling. D) Heatmap of expression levels for key BMP signaling molecules in human umbilical vein endothelial cells (HUVECs). E,F) qPCR (E) and Western blot (WB) (F) analysis of BMP2, BMP4, BMPR1A, and BMPR1B expression changes after PARK7 knockdown in HUVECs (n = 3). G) Immunofluorescence detection of phosphorylated SMAD1/5/9 (p‐SMAD1/5/9) expression in HUVECs with PARK7 overexpression or knockdown. H) WB analysis of expression levels of FADS1 and FADS2, the phosphorylation levels of SMAD1/5/9 in whole‐cell lysates, and the content of p‐SMAD1/5/9 in nuclear proteins were detected by WB in HUVECs overexpressing PARK7 , knocking down PARK7 , and treated with the BMP agonist sb4. I) WB quantification of the p‐SMAD1/5/9 to total SMAD5 ratio in HUVEC whole cell lysates (n = 4). J) WB quantification of nuclear p‐SMAD1/5/9 in HUVECs (n = 4). K) WB quantification of FADS1 and FADS2 in whole cell lysates of HUVECs (n = 4). L) ChIP‐qPCR validation of p‐SMAD1/5/9 binding to FADS1/2 gene promoters (n = 4). M) qPCR analysis of the expression of BMP2/4 and BMPR1A/B precursor mRNA (n = 4). N) WB analysis of NRF2 and KEAP1 after PARK7 knockdown. O) CoIP analysis of NRF2‐ubiquitin conjugation status following PARK7 knockdown. P) qPCR analysis of the expression of BMP2/4 after PARK7 knockdown and NEF2L2 overexpression (n = 4). Q) ChIP‐qPCR validation of NRF2 binding to BMP2/4 gene promoters (n = 3). R) Changes in BMPR1A/B mRNA levels over time after cycloheximide (CHX) administration (n = 3). S) qPCR analysis of the expression of BMPR1A/B after PARK7 knockdown and CMLD‐2 administration (n = 4). T) Western blot analysis of HuR expression changes following tBHP stimulation or PARK7 knockout, and the restorative effect of NAC. U) RIP assay validating changes in the binding capacity of HuR to BMPR1A/B mRNA (n = 5). V) Schematic diagram illustrating the dual pathways through which PARK7 regulates BMP/BMPR expression. CHX, cycloheximide; NAC, N‐Acetyl‐cysteine; tBHP, t‐Butyl hydroperoxide; ChIP, chromatin immunoprecipitation; CoIP, Co‐Immunoprecipitation; RIP, RNA immunoprecipitation; OE, overexpression; NC, negative control; Mock, transfection with vector plasmids and addition of corresponding solvents. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Main reagents used in this study included LPS (L9143; Sigma–Aldrich, USA), nigericin (HY‐127019; MCE, China), ferrostatin‐1 (T6500; TargetMol, China), SC26196 ( T12856 ; TargetMol, China), DHA (T5369; TargetMol, China), erastin (HY‐15763; MCE, China), ML385 (T4360; TargetMol, China), ALA (T3P2904; TargetMol, China), CMLD‐2 ( T36493 ; TargetMol, China), sodium lactate (HY‐B2227B; MCE, China), 2‐DG(HY‐13966; MCE, China), BMP agonist sb4 (HY‐12469; MCE, China), medium chain triglycerides (MCT, S25953 ; Yuanye, China), puromycin (HY‐K1057; MCE, China), cycloheximide (HY‐12320; MCE, China) and ROS assay reagent DCFH‐DA (HY‐D0940; MCE, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Protein-Protein interactions, Western Blot, Knockdown, Immunofluorescence, Over Expression, Phospho-proteomics, ChIP-qPCR, Biomarker Discovery, Binding Assay, Ubiquitin Proteomics, Conjugation Assay, Knock-Out, Chromatin Immunoprecipitation, Immunoprecipitation, RNA Immunoprecipitation, Negative Control, Transfection, Plasmid Preparation