Journal: Bioactive Materials
Article Title: Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment
doi: 10.1016/j.bioactmat.2026.01.002
Figure Lengend Snippet: Generation of PKM2-overexpressing cells and generation of Tet-PKM2-enriched LEVs. ( A ) Schematic illustration depicting the overexpression of PKM2 in HEK293T cells by lentiviral transduction and subsequent allosteric activation of Tet-PKM2 via TEPP-46 treatment. Cells transfected with ov-NC or ov-PKM2 were named the NC or OV, respectively. ( B ) Relative mRNA expression levels of PKM2 in the NC and OV groups (qRT‒PCR, n = 4). ( C ) Representative immunoblot bands of PKM2 in the NC and OV groups. ( D ) Semiquantitative analysis of the expression levels of PKM2 in the NC and OV groups ( n = 6). ( E ) PK activity in PKM2-overexpressing HEK293T cells in response to treatment with various concentrations of TEPP-46 (0, 10, 20, 40, and 70 μM) ( n = 6). ( F ) Representative immunoblot bands of PKM2 conformational states in PKM2-overexpressing cells after treatment with TEPP-46 (40 μM) for 0, 8, and 24 h (DSS cross-linking assay). ( G ) Semiquantitative analysis of the expression levels of Tet-PKM2 in PKM2-overexpressing cells after treatment with TEPP-46 (40 μM) for 0, 8, and 24 h ( n = 4). ( H ) Schematic illustration of the isolation of LEVs and SEVs by differential velocity centrifugation. ( I ) Representative TEM images showing the morphology of SEVs and LEVs. ( J ) Size distributions of SEVs and LEVs (NTA). ( K ) Particle counts of SEVs and LEVs (NTA). ( L ) Particle-to-protein ratios of SEVs and LEVs. ( M ) Cellular uptake of SEVs and LEVs (labeled with PKH67; green) by macrophages (immunofluorescence assay); cell skeletons were stained with phalloidin (red), and nuclei were stained with DAPI (blue). ( N ) Expression of CD63, HSP70, TSG101, calnexin, and GM130 in whole-cell lysates (Cells), SEVs, and LEVs (Western blot). ( O ) Representative immunoblot bands of PKM2 conformational states in LEVs and SEVs. ( P ) Semiquantitative analysis of the expression levels of Tet-PKM2 in LEVs and SEVs ( n = 3) ( Q ) Conformational states of PKM2 in LEVs Tet−PKM2 in response to TEPP-46 treatment. ( R ) Semiquantitative analysis of the expression levels of Tet-PKM2 in LEVs following TEPP-46 treatment ( n = 3). The data are expressed as the mean ± SEM. Statistical analysis was performed by one-way ANOVA ( E and G ) and Student's t -test ( B , D , K , L , P , and R ). ns indicates no significant difference between the indicated columns; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 indicate significant differences between the indicated columns.
Article Snippet: PKH67-labeled SEVs and LEVs resuspended in complete medium were used to treat RAW 264.7 cells for 24 h. The cells were then fixed with 4 % PFA (Coolaber), permeabilized, and stained for cytoskeletal visualization using fluorescein phalloidin (1:1000 dilution; MCE) for 30 min.
Techniques: Over Expression, Transduction, Activation Assay, Transfection, Expressing, Western Blot, Activity Assay, Isolation, Centrifugation, Labeling, Immunofluorescence, Staining
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