vegfa  (R&D Systems)

Journal: bioRxiv

Article Title: VEGFR2 blockade overcomes acquired KRAS G12D inhibitor resistance driven by PI3Kγ activation

doi: 10.1101/2025.09.16.676456

Figure Lengend Snippet: (A) Schematic diagram of generating MRTX1133-resistant cancer models and characterization of these models with a uniform manifold approximation and projection (UMAP) plot showing both parental and resistant models and the proportion of parental and resistant cells within each cluster. (B) GSVA signals of Angiogenesis, Hypoxia, Epithelial-Mesenchymal Transition, MYC Targets V2, E2F Targets, and MYC Targets V1 in the MSigDB for each cluster, with the proportions of parental and resistant cells displayed below. (C) Z-score normalized expression for nine representative genes involved in angiogenesis, hypoxia, and EMT pathways. Each gene was labeled with an abbreviation for its respective pathway: Angiogenesis (Angio), Hypoxia (Hyp), and EMT. The proportions of parental and resistant cells are shown beneath the heatmap. (D) VEGFA concentration in conditioned media (CM) derived from parental and MRTX1133-resistant patient-derived organoids (PDOs). The experiment was performed in triplicate. *P <□0.05, **P <□0.01, and ***P <□0.001. (E) Levels of p-VEGFR2, total VEGFR2, and vinculin in parental and MRTX1133-resistant models. The normalized values by vinculin were indicated. (F) Colony-forming assays were performed to assess the proliferative effect of KDR knockout in MRTX1133-resistant cells. TLCV2-sg KDR- resistant cells were treated with 1 μM of MRTX1133 and either VEH or doxycycline for 2 weeks. The colony images were captured using ChemiDoc Touch (Bio-Rad) and quantified using the ImageJ software (NIH). The Y-axis of the graph indicates the number of colonies. The experiment was performed in triplicate. ***P <□0.001. (G) The viability of VEGFR2-high and -low resistant cells sorted by FACS after MRTX1133 treatment. All experiments were repeated four times. Data represent the mean□±□SD. The IC 50 values were calculated using GraphPad Prism Software.

Article Snippet: Recombinant human VEGFA was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Labeling, Concentration Assay, Derivative Assay, Knock-Out, Software

Journal: bioRxiv

Article Title: VEGFR2 blockade overcomes acquired KRAS G12D inhibitor resistance driven by PI3Kγ activation

doi: 10.1101/2025.09.16.676456

Figure Lengend Snippet: (A) Relative luciferase reporter activity of two VEGFA promoter regions (1-500 bp and 500-1000 bp upstream of the transcription starting site) in parental and resistant cells. pGL basic was a negative control. The graph indicates the normalization of firefly luminescence by Renilla luminescence. All experiments were repeated three times. Data represent the mean□±□SD. **P <□0.01 and ***P <□0.001. (B) Levels of SP1, Lamin B, and α-tubulin in fractionated cytoplasmic and nuclear protein samples of PANC-1-P and PANC-1-R cells. SP1 expression was normalized by Lamin B expression. (C) Levels of SP1, p-VEGFR2, and VEGFR2 of resistant cells that were transfected with negative control (N/C) or SP1 siRNA. The values indicate the normalized band intensity by vinculin. (D) Deleting 5-bp in the predicted SP1 binding site of the VEGFA promoter region significantly decreased the VEGFA mRNA expression. VEGFA transcriptional levels in PANC-1-R cells transfected with wild-type and deletion vectors were plotted. The values are normalized with 18s rRNA. All experiments were performed in triplicate. The graph indicates the mean ± SD. ***P <□0.001. (E) Representative immunofluorescence images for p-VEGFR2 (green) and SP1 (red) in PANC-1-R cells after transfection with wild-type and deletion vectors. Nuclei were stained with DAPI (blue). Scale bar: 65.3 μm.

Article Snippet: Recombinant human VEGFA was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Luciferase, Activity Assay, Negative Control, Expressing, Transfection, Binding Assay, Immunofluorescence, Staining