Journal: Research
Article Title: Trabecular-Like Scaffold Dictates Osteogenesis via Fluid Shear Stress-Induced Metabolic Reprogramming through the CAV1–HIF-1α Axis
doi: 10.34133/research.1307
Figure Lengend Snippet: Integrated transcriptomic and proteomic analyses and in vitro validation of mechano-metabolic remodeling in BMSCs under HFSS. (A) Schematic workflow of the integrated analysis. DEGs and DEPs between the HFSS and Ctrl groups were identified from RNA-seq and proteomic datasets, respectively, and concordantly regulated targets showing the same direction of change at both transcriptomic and proteomic levels were selected for downstream analyses. Created with BioRender.com . (B) Venn diagram illustrating the overlap between DEGs and DEPs. (C) Nine-quadrant plot comparing log₂ fold changes at the RNA and protein levels, highlighting concordant and discordant regulation patterns. (D) GO enrichment analysis of concordantly regulated genes and proteins. (E) KEGG pathway enrichment analysis of concordant targets. (F) GSEA plots for glycolysis, HIF-1 signaling, and PI3K–AKT signaling, showing positive enrichment of all 3 pathways in the HFSS group. (G) Heatmap of selected genes and proteins involved in glycolysis and mechanosignaling. (H) Phalloidin staining of the actin cytoskeleton in BMSCs cultured on Voronoi scaffolds under dynamic FSS. (I) Seahorse extracellular flux analysis of BMSCs cultured on V50, V60, and V70 scaffolds. (J) Representative ECAR curves and quantitative analysis of glycolysis rate, glycolytic reserve, and glycolytic capacity. (K) Western blot analysis of GLUT1, HIF1A, and CAV1, and (L) PI3K, p-PI3K, AKT, and p-AKT in BMSCs cultured on scaffolds, with corresponding quantitative analyses of Western blot protein bands. (M) Quantitative analysis of multiple glycolytic flux fractions in BMSCs seeded onto scaffolds. (N) Representative immunofluorescence images of CAV1 expression in BMSCs cultured on V50, V60, and V70 scaffolds. Scale bar, 100 μm. (O) qPCR analysis of Hif1a , Ldha , Slc2a1 , and Cav1 gene expression. Data are presented as mean ± SD ( n = 3 per group). Statistical significance was assessed using unpaired 2-tailed Welch’s t test and one-way ANOVA with Tukey’s HSD post hoc test.
Article Snippet: Transcriptomic, proteomic, and untargeted metabolomic analyses were performed by Hipro Life Sciences Co., Ltd. (China).
Techniques: In Vitro, Biomarker Discovery, RNA Sequencing, Staining, Cell Culture, Western Blot, Immunofluorescence, Expressing, Gene Expression