Fig 1.
Molecular schematic of peptide modification and molecular interaction.
The peptide cAmbly (shown in red) is modified with the following components: photo-leucine (purple), which enables covalent binding upon UV excitation; a PEG4 spacer (green) to minimize non-specific interactions; and biotin (blue), which serves as a tag for peptide-target complex pulldown. Agarose-streptavidin beads are employed to capture the peptide-target complex, which is subsequently purified for mass spectrometry analysis.
Fig 2.
Cytotoxicity evaluation using the MTT assay.
T98G cells were treated for 24 h or 48 h with varying concentrations of cAmbly (0.125 µ M to 2 µ M). DMEM and DMSO were used as negative controls. cAmbly did not interfere with cell viability at either time point.
Fig 3.
Quantification of cAmbly internalization.
T98G cells were treated with either N-terminal modified cAmbly (B-cAmbly) or C-terminal modified cAmbly (cAmbly-PL) for 30 min, 1 h, 3 h, or 6 h. Peptide presence was quantified as the percentage of cells containing internalized peptide. cAmbly-PL exhibited robust internalization, with approximately 70% of cells containing the peptide after 1 h and 6 h.
Fig 4.
Internalization of C-terminal modified cAmbly (cAmbly-PL).
T98G cells were treated with cAmbly-PL for 30 min, 1 h, 3 h, or 6 h, followed by crosslinking, PFA fixation, and staining for nuclei, actin, and peptide. A: Green arrows indicate cells with internalized peptides. B: Enlarged sections provide detailed views of peptide localization at each time point.
Fig 5.
Internalization of N-terminal modified cAmbly (B-cAmbly).
T98G cells were treated with B-cAmbly for 30 min, 1 h, 3 h, or 6 h, followed by crosslinking, PFA fixation, and staining for nuclei, actin, and peptide. A: Green arrows highlight cells containing internalized peptides. B: Enlarged sections show detailed peptide distribution within cells at each time point.
Fig 6.
Colocalization map of cAmbly and mitochondria.
T98G cells were treated with cAmbly-FITC (green), and mitochondria were labeled with MitoTracker Deep Red (red). 2D maximum projection (A), 3D isosurfaces (B) and colocalization quantification (C) were performed using Huygens software. Most cAmbly colocalized with mitochondria (M2), while only a fraction of mitochondria interacted with cAmbly (M1).
Fig 7.
Dose-response analysis of cAmbly targets.
T98G cells were treated with B-cAmbly and cAmbly-PL in the presence of increasing concentrations of native cAmbly as a competitor. The peptide-target complexes were crosslinked, purified with streptavidin beads, and identified via mass spectrometry. Specific targets exhibit the following pattern: background intensity with native cAmbly alone (first point), high intensity with modified cAmbly alone (second point), and decreasing intensity as the concentration of native cAmbly increases.
Table 1.
UniProt entry, gene name, protein name, and Gene Ontology of identified targets.