Journal: EJNMMI Radiopharmacy and Chemistry

Article Title: Radiolabeled 15-mer peptide internalization is mediated by megalin (LRP2 receptor) in a CRISPR/Cas9-based LRP2 knockout human kidney cell model

doi: 10.1186/s41181-024-00262-2

Figure Lengend Snippet: Verification of LRP2 edits by flow cytometry analysis, gentamicin and FITC-albumin accumulation. A Morphological changes in parent HK2 and B LRP2 KO cells observed using the FLoid™ cell imaging station after treatment with cytotoxic doses of gentamicin (48 h, 6 mg/mL). C Cells were incubated with 0.05 mg/mL megalin polyclonal antibody for 2 h followed by CF™ 594 secondary antibody staining (1h, 5 µg/mL). The fluorescence intensity was quantified using a median based on fluorescent histograms of the cell populations. D Viability of edited and parent HK2 cells was evaluated after 48 h treatment with cytotoxic doses of gentamicin (6 mg/mL), with the graph representing the relative cellular viability. E FITC-albumin accumulation studies (24 µg/mL) were performed after 2 h incubation. One-way ANOVA with Dunnett’s post hoc test or t- test were implemented to compare differences in parent and edited HK2 cells, * P < 0.05, *** P ≤ 0.001

Article Snippet: The membrane was incubated with primary LRP2 Polyclonal Antibody (1:500, 1 h, 19700–1-AP, ThermoFisher) followed by the incubation with Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP (1:200 000, 1 h, 31460, ThermoFisher).

Techniques: Flow Cytometry, Imaging, Incubation, Staining, Fluorescence

Journal: EJNMMI Radiopharmacy and Chemistry

Article Title: Radiolabeled 15-mer peptide internalization is mediated by megalin (LRP2 receptor) in a CRISPR/Cas9-based LRP2 knockout human kidney cell model

doi: 10.1186/s41181-024-00262-2

Figure Lengend Snippet: Verification of LRP2 knockout by western blotting analysis and laser scanning confocal microscopy. A HK2 cells expressing LRP2 and LRP2 KO cells shown by confocal microscopy. Cells were incubated with megalin polyclonal antibody (red), Hoechst 33342 to visualize the nuclei of viable cells (blue) and ActinGreen™ 488 ReadyProbes was used for cytoskeleton imaging (green). B Western blot analysis of LRP2 knockout using the parent HK2, LRP2 KO cells, and negative controls without LRP2 expression, SK-OV-3 and U-87 MG cells

Article Snippet: The membrane was incubated with primary LRP2 Polyclonal Antibody (1:500, 1 h, 19700–1-AP, ThermoFisher) followed by the incubation with Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP (1:200 000, 1 h, 31460, ThermoFisher).

Techniques: Knock-Out, Western Blot, Confocal Microscopy, Expressing, Incubation, Imaging

Journal: EJNMMI Radiopharmacy and Chemistry

Article Title: Radiolabeled 15-mer peptide internalization is mediated by megalin (LRP2 receptor) in a CRISPR/Cas9-based LRP2 knockout human kidney cell model

doi: 10.1186/s41181-024-00262-2

Figure Lengend Snippet: Accumulation of radiolabeled 15-mer. The [ 68 Ga]Ga-NODAGA-15-mer and [ 99m Tc]Tc-KDC-15-mer internalization assays in HK2 parent, partial KO and LRP2 KO cells. The accumulation of radiolabeled 15-mers measured in parent HK2 cells was set as 100%. All the data were normalized to the total protein level. A The HK2 parent, partial KO and LRP2 KO cells were treated with [ 99m Tc]Tc-KDC-15-mer (2 h, 27 µg/mL, 37 °C). The experimental data were obtained from three independent experiments performed in biological triplicates. B The [ 68 Ga]Ga-NODAGA-15-mer (2 h, 20 µg/mL, 37 °C) treatment was performed in HK2 parent, LRP2 KO 1 and LRP2 KO 2 cells in biological triplicates to examine 68 Ga-radiolabeled 15-mer megalin-mediated accumulation. ANOVA with Dunnett’s post hoc test was used to compare KO models with control cells, * P ≤ 0.05, ** P ≤ 0.01

Article Snippet: The membrane was incubated with primary LRP2 Polyclonal Antibody (1:500, 1 h, 19700–1-AP, ThermoFisher) followed by the incubation with Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP (1:200 000, 1 h, 31460, ThermoFisher).

Techniques: Control