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

Journal: The Journal of Cell Biology

Article Title: Energy stress activates AMPK to arrest mitochondria via phosphorylation of TRAK1

doi: 10.1083/jcb.202501023

Figure Lengend Snippet: Controls for rapalog motility experiments and perimitochondrial actin recruitment. (A) Representative example neurites showing rapalog recruits mCherry-FRB to mitochondria in the presence of vehicle or AntA. Neurons were transfected with mito-GFP-FKBP and mCherry-FRB and pretreated for 1 h with either 4 nM AntA or vehicle before addition of 1 µM rapalog as in . (B) Representative images of hippocampal neurons that were fixed and stained with Alexa Fluor 488 phalloidin conjugate and Hoechst. Either control neurons or neurons pretreated with 4 nM AntA were exposed to 5 µM LatA. Note the persistence of a LatA-resistant pool of actin after pretreatment with AntA. (C) Quantification of filamentous actin from the average Alexa Fluor 488 phalloidin intensity of 60× imaging fields of neurons treated as in B. (D) Quantification of mitochondrial motility in hippocampal neurons pretreated for 1 h with 4 nM AntA and then 30 min with both 5 uM LatA and 4 nM AntA. Once arrested by treatment with AntA, LatA could not reverse the arrest. (E) Time-lapse series of fibroblasts treated for 1 h with 40 nM AntA and imaged as in . The arrowhead indicates a mitochondrion that appears to lack perimitochondrial actin and moves, while nearby mitochondria do not. (F) Representative images of rat embryonic fibroblasts transduced with GFP-F-tractin and mito-mRaspberry from the quantification in . Cells cultured in galactose were treated with 40 nM AntA or vehicle. One hour after drug treatment, cells either had glucose or galactose added to a concentration of 25 mM, or no further addition. Cells were imaged after 30 min. P values for C were calculated by two-tailed, unpaired t tests with Welch’s correction. A blocked one-way ANOVA was performed with Tukey’s multiple comparison correction for D. Select comparisons are shown for D. For supplemental images, 3A scale bars = 10 µm. The scale bar in 3E is 5 µm. For supplemental images, 3B and 3F scale bars = 20 μm.

Article Snippet: The phalloidin buffer was washed off, and the sample was incubated for 30 min with AF488-conjugated phalloidin (sc-36379; Santa Cruz) dissolved in 1:1,000 in phalloidin buffer.

Techniques: Transfection, Staining, Control, Imaging, Transduction, Cell Culture, Concentration Assay, Two Tailed Test, Comparison