Fig. 5: A pathway of matrix stiffness-tuned exosomal let-7d-5p/HMGA2/E2F1 acetylation/GLUT1, PFKP, and HK2 in lung fibroblasts participates in modulating glucose enrichment.

a Predicted target proteins of let-7d-5p using three publicly available bioinformatic tools. b Western blot analysis of HMGA2 in lung fibroblasts with let-7d-5p overexpression or knockdown. c Schematic illustration of luciferase reporter plasmids for HMGA2 3’UTR (left panel) and relative luciferase activity determined after co-transfection of miRNA mimic and plasmids (right panel) (n = 3 biological replicates). d Effects of HMGA2 overexpression (HMGA2-OE) on GLUT1, PFKP, and HK2 expressions in lung fibroblasts with let-7d-5p overexpression. The samples derive from the same experiment but different gels for GLUT1 and PFKP, and another for HK2, HMGA2, and β-actin were processed in parallel. e Immunoprecipitation assay of interaction between HMGA2 and Rb in the nuclear protein of lung fibroblasts. f Immunoprecipitation assay of interaction between HMGA2 or HDAC1 and E2F1-Rb complex in the nuclear protein of lung fibroblasts with let-7d-5p overexpression or downregulation. g E2F1 acetylation level (Pan Ac-Lys levels of E2F1-captured proteins) and HDAC1 interacted with E2F1 in the nuclear protein of lung fibroblasts with let-7d-5p overexpression or downregulation determined by immunoprecipitation assay. h ChIP-qPCR analysis of E2F1 occupancy on SLC2A1, PFKP, and HK2 promotors in lung fibroblasts (relative to input) (n = 3 biological replicates). i Effects of TSA intervention on E2F1 acetylation levels in the nuclear protein (left panel) and GLUT1, PFKP, and HK2 expression in the total protein (right panel) of lung fibroblasts with let-7d-5p overexpression. The samples derive from the same experiment but different gels for GLUT1 and HK2, and another for PFKP and β-actin were processed in parallel. j, k Effects of E2F1 mutation (K117/120/125 R) on E2F1 acetylation levels in the nuclear protein (j) and GLUT1, PFKP, and HK2 expressions in the total protein (k) of lung fibroblasts with let-7d-5p downregulation. The samples derive from the same experiment but different gels for GLUT1 and PFKP, and another for HK2 and β-actin were processed in parallel. Representative blot was shown from 3 biologically independent experiments (b, d–g, i–k). Protein loading was 20 μg in each group and β-actin was used to normalize total protein quantification (b, d, i, k), and Histone H3 was used as a loading control in the nuclear protein (f, g, i, j). Data are presented as mean ± SD, and P-values were calculated using one-way ANOVA (b–d, i, k) or two-tailed unpaired Student’s t-test (h). Ac-Lys acetylated-Lysine, WT wild type, mut mutant, WCL whole cell lysate. Source data are provided as a Source Data file.