Fig. 4: Matrix stiffness-tuned exosomal let-7d-5p promotes glucose enrichment during lung pre-metastatic niche formation. | Nature Communications

Fig. 4: Matrix stiffness-tuned exosomal let-7d-5p promotes glucose enrichment during lung pre-metastatic niche formation.

From: A glucose-enriched lung pre-metastatic niche triggered by matrix stiffness-tuned exosomal miRNAs in hepatocellular carcinoma

Fig. 4

a Heatmap of the known differentially expressed miRNAs between MHCC97H-L-Exo and MHCC97H-H-Exo (n = 3 biological replicates). b miRNA qRT-PCR analysis of let-7d-5p, miR-365a-5p, miR-194-5p, and miR-125a-5p in MHCC97H/Hep3B-L/H-Exo (n = 3 biological replicates). Data were normalized to U6. c qRT-PCR analysis of glycolysis-related genes in lung fibroblasts transfected with indicated miRNA mimics or mimic NC (n = 4 biological replicates). Data were normalized to β-actin. d Western blot analysis of glucose transporters and glycolytic enzymes in lung fibroblasts with let-7d-5p overexpression or knockdown (n = 3 biological replicates). The samples derive from the same experiment but different gels for GLUT1 and PFKP, and another for PKM2, HK2, and β-actin were processed in parallel. e 2-NBDG uptake in lung fibroblasts with let-7d-5p overexpression or knockdown (n = 3 biological replicates). f Flow cytometry analysis of CD11b+CD45+ BMDCs recruitment in the pre-metastatic niche lung tissues induced by Hepa1-6-Mock/Exo or Hepa1-6-let-7d-5p-OE/Exo (n = 4 mice per group). g 2-NBDG uptake in lung fibroblasts (CD45-CD31-CD140a+ cells) in the pre-metastatic niche lung tissues induced by Hepa1-6-Mock/Exo or Hepa1-6-let-7d-5p-OE/Exo (n = 4 mice per group). h IHC staining of GLUT1, PFKP, and HK2 expressions in the pre-metastatic lung tissues induced by Hepa1-6-Mock/Exo or Hepa1-6-let-7d-5p-OE/Exo (n = 4 mice per group). Scale bars: black, 200 μm; red, 50 μm. i HE staining and quantification of lung metastasis lesions in lung tissues on day 61 (n = 5 mice per group). Scale bars: red, 2 mm; blue, 500 μm; black, 100 μm. Representative images are presented from indicated biologically independent experiments (e–i). Representative blot (20 μg protein per group) was shown from 3 biologically independent experiments (d), and β-actin was used to normalize protein quantification. Data are presented as mean ± SD, and P values were calculated using two-tailed unpaired Student’s t-test (b, c, f–i) or one-way ANOVA (d, e). L low-stiffness substrates, H high-stiffness substrates, MFI mean fluorescence intensity, Exo exosome, WT wild type, OE overexpression. Source data are provided as a Source Data file.

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