Journal: bioRxiv

Article Title: Targeting nucleolin facilitates the development of blood-tumor barrier-penetrating and glioblastoma-specific PROTACs

doi: 10.1101/2025.07.22.666084

Figure Lengend Snippet: ( A ) Flow cytometric analysis of surface NCL expression on astrocytes and U87MG cells using an anti-NCL antibody. ( B ) Relative mRNA expression of NCL in bEnd.3 cells after coculture with astrocytes or U87MG cells at 0, 48, and 120 h. ( C ) Protein expression of NCL in U87MG cells stably transfected with plasmids encoding shCtrl or shNCL. Experiments were performed in triplicate and repeated three times. Graphs represented means ± SEM, and statistical significance was calculated by Student’s t -test (A) and one-way ANOVA (B). **** P < 0.0001. ns, no significance.

Article Snippet: Following blocking with 5% non-fat dry milk in TBST, the membrane was incubated overnight at 4°C with primary antibodies against VEGFR2 (1:1000, CST, USA), EGFR (1:1000, CST, USA), NCL (1:1000, Proteintech, CHN), MDM2 (1:1000, Proteintech, CHN), Na + /K + - ATPases (1:1000, Proteintech, CHN), and ACTIN (1:2000, Abclonal, CHN).

Techniques: Expressing, Stable Transfection, Transfection

Journal: bioRxiv

Article Title: Targeting nucleolin facilitates the development of blood-tumor barrier-penetrating and glioblastoma-specific PROTACs

doi: 10.1101/2025.07.22.666084

Figure Lengend Snippet: ( A ) Co-immunoprecipitation (IP) of NCL, MDM2, VEGFR2, and EGFR in U87MG cells using isotype-matched IgG or an anti-NCL antibody. ( B ) Pull-down assay of NCL, MDM2 VEGFR2, and EGFR in U87MG cells treated with 500 nM biotin-labeled NC (Bio-NC) or AS1411 (Bio-AS1411) for 6 h by streptavidin-coated magnetic beads. ( C ) Pull-down assay of NCL and MDM2 in siNCL-transfected U87MG cells after treatment with 500 nM Bio-NC or Bio-AS1411 for 6 h. ( D ) Structure of AS1411-cediranib (AS1411-Ced). ( E ) Structure of AS1411-gefitinib (AS1411-Gef). ( F ) Absorption spectra of N-methylmesoporphyrin IX (NMM) in the presence of 10 µM NC, AS1411, AS1411-Ced, and AS1411-Gef. The concentration of NMM was 1 µM. ( G ) Co-IP of NCL, MDM2, and VEGFR2 in U87MG cells treated with PBS or 500 nM AS1411-Ced for 6 h using an anti-VEGFR2 antibody in the presence of 10 µM MG132. ( H ) Co-IP of NCL, MDM2, and VEGFR2 in scrambled control siRNA (siCtrl)- or NCL siRNA (siNCL)- transfected U87MG cells after treatment with PBS or 500 nM AS1411-Ced for 6 h using an anti-VEGFR2 antibody in the presence of 10 µM MG132. ( I ) Co-IP of NCL, MDM2, and VEGFR2 in siCtrl or siMDM2- transfected U87MG cells after treatment with PBS or 500 nM AS1411-Ced for 6 h using an anti-VEGFR2 antibody in the presence of 10 µM MG132. ( J ) Co-IP of NCL, MDM2, and EGFR in U87MG cells treated with PBS or 500 nM AS1411-Gef for 6 h using an anti-EGFR antibody in the presence of 10 µM MG132. ( K ) Co-IP of NCL, MDM2, and EGFR in siCtrl- or siNCL-transfected U87MG cells after treatment with PBS or 500 nM AS1411-Gef for 6 h using an anti-EGFR antibody in the presence of 10 µM MG132. ( L ) Co-IP of NCL, MDM2, and EGFR in siCtrl- or siMDM2-transfected U87MG cells after treatment with PBS or 500 nM AS1411-Gef for 6 h using an anti-EGFR antibody in the presence of 10 µM MG132. Experiments were performed in triplicate and repeated three times.

Article Snippet: Following blocking with 5% non-fat dry milk in TBST, the membrane was incubated overnight at 4°C with primary antibodies against VEGFR2 (1:1000, CST, USA), EGFR (1:1000, CST, USA), NCL (1:1000, Proteintech, CHN), MDM2 (1:1000, Proteintech, CHN), Na + /K + - ATPases (1:1000, Proteintech, CHN), and ACTIN (1:2000, Abclonal, CHN).

Techniques: Immunoprecipitation, Pull Down Assay, Labeling, Magnetic Beads, Transfection, Concentration Assay, Co-Immunoprecipitation Assay, Control

Journal: bioRxiv

Article Title: Targeting nucleolin facilitates the development of blood-tumor barrier-penetrating and glioblastoma-specific PROTACs

doi: 10.1101/2025.07.22.666084

Figure Lengend Snippet: ( A ) Co-localization of Cy5-labeled NC or AS1411 (red) with NCL (yellow) in brain sections from orthotopic GBM mice inoculated with U87MG-GFP-Luc cells (green) after intravenous injections of 0.2 µmol/kg Cy5-labeled NC or AS1411 for 1 h. Scale bars, 100 μm. ( B ) Co-localization of Cy5-labeled NC or AS1411 (red) with MDM2 (purple) in brain sections from orthotopic GBM mice inoculated with U87MG-GFP-Luc cells (green) after intravenous injections of 0.2 µmol/kg Cy5-labeled NC or AS1411 for 1 h. Nuclei were stained with DAPI (blue). N, non-tumor region; T, tumor region. Scale bars, 100 μm. n = 3 for each group.

Article Snippet: Following blocking with 5% non-fat dry milk in TBST, the membrane was incubated overnight at 4°C with primary antibodies against VEGFR2 (1:1000, CST, USA), EGFR (1:1000, CST, USA), NCL (1:1000, Proteintech, CHN), MDM2 (1:1000, Proteintech, CHN), Na + /K + - ATPases (1:1000, Proteintech, CHN), and ACTIN (1:2000, Abclonal, CHN).

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Targeting nucleolin facilitates the development of blood-tumor barrier-penetrating and glioblastoma-specific PROTACs

doi: 10.1101/2025.07.22.666084

Figure Lengend Snippet: ( A ) No transfer of NCL from astrocytes to the surface of brain capillary endothelial cells via EVs, maintaining the normal function of BBB. ( B ) EVs-mediated transfer of NCL from GBM cells to the surface of brain capillary endothelial cells, facilitating the transformation of BBB into BTB. ( C ) Transcytosis of AS1411 across the BTB in an NCL-dependent manner, enabling the targeting of GBM cells. ( D ) Development of BTB-penetrating and GBM-targeting PROTACs via conjugating AS1411 with ligands of POIs in GBM cells. Briefly, the AS1411-based PROTACs can transverse the BTB, selectively enter GBM cells, and induce the spatial proximity of POIs with the NCL-MDM2 complex. This will facilitate the ubiquitination and proteasomal degradation of POIs, leading to targeted therapy of GBM.

Article Snippet: Following blocking with 5% non-fat dry milk in TBST, the membrane was incubated overnight at 4°C with primary antibodies against VEGFR2 (1:1000, CST, USA), EGFR (1:1000, CST, USA), NCL (1:1000, Proteintech, CHN), MDM2 (1:1000, Proteintech, CHN), Na + /K + - ATPases (1:1000, Proteintech, CHN), and ACTIN (1:2000, Abclonal, CHN).

Techniques: Transformation Assay, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Targeting nucleolin facilitates the development of blood-tumor barrier-penetrating and glioblastoma-specific PROTACs

doi: 10.1101/2025.07.22.666084

Figure Lengend Snippet: ( A ) Flow cytometric analysis of surface NCL expression on astrocytes and U87MG cells using an anti-NCL antibody. ( B ) Relative mRNA expression of NCL in bEnd.3 cells after coculture with astrocytes or U87MG cells at 0, 48, and 120 h. ( C ) Protein expression of NCL in U87MG cells stably transfected with plasmids encoding shCtrl or shNCL. Experiments were performed in triplicate and repeated three times. Graphs represented means ± SEM, and statistical significance was calculated by Student’s t -test (A) and one-way ANOVA (B). **** P < 0.0001. ns, no significance.

Article Snippet: The cells were then incubated with 488-conjugated NCL antibody (1:200, Proteintech, CHN) for 1 h at room temperature.

Techniques: Expressing, Stable Transfection, Transfection

Journal: bioRxiv

Article Title: Targeting nucleolin facilitates the development of blood-tumor barrier-penetrating and glioblastoma-specific PROTACs

doi: 10.1101/2025.07.22.666084

Figure Lengend Snippet: ( A ) Co-immunoprecipitation (IP) of NCL, MDM2, VEGFR2, and EGFR in U87MG cells using isotype-matched IgG or an anti-NCL antibody. ( B ) Pull-down assay of NCL, MDM2 VEGFR2, and EGFR in U87MG cells treated with 500 nM biotin-labeled NC (Bio-NC) or AS1411 (Bio-AS1411) for 6 h by streptavidin-coated magnetic beads. ( C ) Pull-down assay of NCL and MDM2 in siNCL-transfected U87MG cells after treatment with 500 nM Bio-NC or Bio-AS1411 for 6 h. ( D ) Structure of AS1411-cediranib (AS1411-Ced). ( E ) Structure of AS1411-gefitinib (AS1411-Gef). ( F ) Absorption spectra of N-methylmesoporphyrin IX (NMM) in the presence of 10 µM NC, AS1411, AS1411-Ced, and AS1411-Gef. The concentration of NMM was 1 µM. ( G ) Co-IP of NCL, MDM2, and VEGFR2 in U87MG cells treated with PBS or 500 nM AS1411-Ced for 6 h using an anti-VEGFR2 antibody in the presence of 10 µM MG132. ( H ) Co-IP of NCL, MDM2, and VEGFR2 in scrambled control siRNA (siCtrl)- or NCL siRNA (siNCL)- transfected U87MG cells after treatment with PBS or 500 nM AS1411-Ced for 6 h using an anti-VEGFR2 antibody in the presence of 10 µM MG132. ( I ) Co-IP of NCL, MDM2, and VEGFR2 in siCtrl or siMDM2- transfected U87MG cells after treatment with PBS or 500 nM AS1411-Ced for 6 h using an anti-VEGFR2 antibody in the presence of 10 µM MG132. ( J ) Co-IP of NCL, MDM2, and EGFR in U87MG cells treated with PBS or 500 nM AS1411-Gef for 6 h using an anti-EGFR antibody in the presence of 10 µM MG132. ( K ) Co-IP of NCL, MDM2, and EGFR in siCtrl- or siNCL-transfected U87MG cells after treatment with PBS or 500 nM AS1411-Gef for 6 h using an anti-EGFR antibody in the presence of 10 µM MG132. ( L ) Co-IP of NCL, MDM2, and EGFR in siCtrl- or siMDM2-transfected U87MG cells after treatment with PBS or 500 nM AS1411-Gef for 6 h using an anti-EGFR antibody in the presence of 10 µM MG132. Experiments were performed in triplicate and repeated three times.

Article Snippet: The cells were then incubated with 488-conjugated NCL antibody (1:200, Proteintech, CHN) for 1 h at room temperature.

Techniques: Immunoprecipitation, Pull Down Assay, Labeling, Magnetic Beads, Transfection, Concentration Assay, Co-Immunoprecipitation Assay, Control