Fig 2: Characterization and cardiac-specific cellular uptake of C-sEVs without cytotoxicity in vitro(A) Schematic representation of sEVs targeting cardiac cells. (B) Schematic representation of targeting via CTP-expressing sEVs. (C–E) TEM images (scale bar, 100 nm) (C), size distribution (D), and zeta potential (E) of sEVs. (F) Western blot analysis of FLAG and GAPDH from stable cell lines as well as FLAG, LAMP2, Alix, and CD81 in sEVs. CTP expression levels were indirectly quantified via the detection of FLAG expression. (G) Cell viability was measured through an MTS assay. (H) Expression of FLAG-LAMP2b in H9C2 cells. (I) Fluorescence microscopy images of PKH26-labeled (red) sEVs in H9C2 cells. Values are expressed as mean ± SEM. ∗p < 0.05 (compared to N-sEVs).

Fig 3: Characterization of EVs produced by Renca cells cultured under normoxia or hypoxia by ExoView. Names of the capture chips are shown in the upper left corners, total number of detected EVs in a sample (1 µg total protein) are shown by grey bars, number of EVs expressing CD63, CD81, and CD9 depicted by red, green, and blue bars, correspondingly.

Fig 4: Exosome-mediated delivery of CRISPR/Cas9 disrupts oncogenic KrasG12D in vitro and inhibits proliferation. (A) Representative size distribution and concentration of HEK293T exosomes measured by nanoparticle tracking analysis. (B) Representative Western blot for exosome markers Alix and CD81 and exclusion markers calnexin and β-actin. Cell, HEK293T cell lysate; Exo, HEK293T exosomes. (C, D, E) Quantitative PCR was used to evaluate mRNA expression levels of cas9 (C), KrasG12D (D), and WT Kras (E) of KPC689 cells treated with HEK293T exosomes (2,500 exosomes/cell) containing plasmid DNA (10 μg DNA/109 exosomes) every day for 3 d. (C) Data in (C) are normalized to 18s. (D, E) Data in (D, E) are normalized to 18s and untransfected control. (C, D) One-way ANOVA with Tukey’s multiple comparisons test was used to evaluate mean differences among groups based on ΔCT values for (C, D). (E) Brown–Forsythe ANOVA with Dunnett’s T3 multiple comparisons test was used to evaluate mean differences among groups based on ΔCT values for (E). (F) Western blot for KrasG12D, pERK1/2, total ERK1/2, and β-actin (left panel) of KPC689 cells following treatment with exosomes containing CRISPR/Cas9 plasmid DNA. Quantification of KrasG12D (normalized to β-actin) as assessed via Western blot (right panel). Data are normalized to untreated. One sample t test performed comparing each group to untreated. Full length blots are presented in Fig S3A. (G) Quantification of phospho-ERK1/2 (normalized to total ERK1/2 and β-actin) as assessed via Western blot (right panel). Data are normalized to untreated. One sample t test performed comparing each group to untreated. Full length blots are presented in Fig S3A. (H) T7/Surveyor assay was used to evaluate editing in genomic DNA of KPC689 cells. (I) MTT assay was used to evaluate cell viability/proliferation rates in KPC689 over the course of 72 h following treatment with exosomes loaded with CRISPR/Cas9 plasmid DNA. Two-way ANOVA with Tukey’s multiple comparison test was performed. Data are expressed as mean ± standard deviation. ***P < 0.001, ****P < 0.0001, ns, not significant.Source data are available for this figure.

Fig 5: Mutant KRAS cells treated with ethanol display a higher level of transcriptional and translational coordination. (A) Multiple Co-inertia analysis of RNA-Seq and proteomics data. Length of the line connecting the proteomics and RNA-Seq data represents their dissimilarity; RNA-Seq n = 1, proteomics n = 3. (B) Venn diagrams showing differentially expressed protein coding genes (RNA-Seq, ±2-fold change) and proteins (Proteomics, ±1.25-fold change) common in the RNA-Seq and proteomics data. (C) Heatmaps of all conditions showing expression of commonly detected up-(33) and down-(11) regulated protein-coding transcripts (left) and proteins (right) found in HPNE-KRAS EtOH cells compared to HPNE-KRAS cells. (D–I). Validation of omics data by qRT-PCR and Western blot for CD81 (D), RHOG (E), ZPR1 (F), RUVBL1 (G), PRPF19 (H), and NT5E (I); indicated in (C) by *. The qRT-PCR was performed to validate RNA-Seq expression with GAPDH used for normalization. Mean ± SEM; n = 4. Western blots were used to validate proteomics expression using HSC70 or GAPDH as loading control. Cluster of Differentiation 81 (CD81); Ras Homolog Family Member G (RHOG); ZPR1 zinc finger (ZPR1); RuvB like AAA ATPase 1 (RUVBL1); Pre-MRNA Processing Factor 19 (PRPF19); 5′-Nucleotidase (NT5E). The original blots of Figure 5 could be found in Figure S5.