bottom chamber contained serum free rpmi sdf 1 cxcl12  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Adenovirus E4ORF1 activates isoform-specific phosphatidylinositol 3-kinase signaling in human endothelial cells

doi: 10.1016/j.jbc.2025.110947

Figure Lengend Snippet: Human Ad5E4ORF1 associates with scaffold proteins IQGAP1, DLG1, CASK, and LIN7C but only DLG1 promotes E4ORF1-mediated AKT activation . A , western blot analysis of Ad5E4ORF1 proteins and phosphorylated AKT and ERK1/2 in HUVECs. ( Top panel ) Cells infected with vector control or Ad5E4ORF1 viruses were starved for 4 h prior to cell lysis in LDS loading buffer. Tagged and untagged Ad5E4ORF1 proteins were probed with Ad5E4ORF1-, Flag-, or HA-specific antibodies. VCN ( bottom ), genomic lentiviral vector copy numbers. ( Lower panels ) Densitometry quantification of immunoblots shown in the top panel . Phosphorylated AKT and ERK signals were normalized to their respective total protein levels and expressed relative to the vector control, which was set to 1. Ad5E4ORF1 protein levels, normalized to GAPDH, were quantified from anti-Ad5E4ORF1 immunoblots and expressed relative to native (untagged) E4ORF1. Data are presented as mean ± SD from 3 to 5 independent HUVEC lines. Statistical analysis was performed using one-way ANOVA (two-sided), followed by Tukey’s post hoc test. ns, nonsignificant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001 ( p value notation used throughout all figures). B , Ad5E4ORF1-enabled cell survival under starvation. Indicated transduced HUVECs were kept in minimal X-Vivo 20 medium for 7 days and cell survival rates (percentage of live cells in starvation relative to those in growth medium) presented as mean ± SD. One-way ANOVA with Tukey’s test (n = 3 independent HUVEC lines). C , identification of Ad5E4ORF1-interacting proteins in HUVECs. Cells expressing GFP (−) or Flag-Ad5E4ORF1 (+) were subjected to cross-linking anti-Flag immunoprecipitation. Flag peptide-eluted proteins were resolved on SDS-PAGE and gel slices analyzed by mass spectrometric protein ID. Shown is an SDS-PAGE silver stain with major identified candidate proteins marked on the right. The band corresponding to LIN7C was identified based on increased signal intensity over a comigrating background protein, denoted by an asterisk (∗). D , confirmation of identified interactions by protein immunoprecipitation (IP) in HUVECs expressing HA-tagged wild type (WT) or mutant Ad5E4ORF1. ΔC3, deletion of C terminal 3 amino acids (part of PDZ-binding motif). Whole cell lysates were prepared in IGEPAL CA-630 buffer without cell cross-linking. Bead-bound proteins, along with 15% of the corresponding input lysates, were analyzed by western blot. Data shown represent experiments using three HUVEC lines. E , interdependence of Ad5E4ORF1 scaffold interactions. DLG1, CASK, or LIN7C gene-specific shRNAs or a nontargeting (NT) shRNA were stably expressed under doxycycline induction for 4 days in control and HA-Ad5E4ORF1-transduced HUVECs. ( Left panel ) Immunoprecipitation blot performed as in ( D ). ( Right panels ) Densitometry quantification of DLG1, CASK, and LIN7C IP efficiency (IP/Input) in gene knockdown (KD) HUVECs relative to NT control. One-way ANOVA with Tukey’s test (mean ± SD; n = 3 HUVEC lines). F , schematic model of Ad5E4ORF1 interactions with scaffold proteins. G and H , effect of scaffold protein knockdowns on AKT activation. Indicated gene-specific shRNAs were doxycycline-induced for 4 days in HUVECs, which were then starved for 4 h and subjected to western blot analysis. Data shown represent experiments using three HUVEC lines. ( H , lower panel ) Quantification of AKT phosphorylation (average of two shRNAs) from upper panel blots. One-way ANOVA with Tukey’s test (mean ± SD; n = 3 HUVEC lines). I , effect of DLG1 knockdown on receptor-mediated AKT activation. DLG1-specific (+) and nontargeting (NT) shRNAs were doxycycline-induced in HUVECs for 4 days. After 4 h of starvation, cells were stimulated for 5 min with the indicated cytokines (20 ng/ml FGF2, HGF, SDF1; or 100 nM S1P) and lysed directly into LDS loading buffer for western blot. ( Lower panels ) Quantification of AKT and ERK phosphorylation from upper panel blots. Paired t test (mean ± SD; n = 3 HUVEC lines). Ad5E4ORF1, human adenovirus serotype 5 early gene E4ORF1; AKT, protein kinase B; CASK, calcium/calmodulin-dependent serine protein kinase; DLG1, discs large homolg 1; ERK, extracellular signal-regulated kinase; FGF2, fibroblast growth factor 2; HGF, hepatocyte growth factor; HUVEC, human umbilical vein endothelial cells; IQGAP1, IQ motif containing GTPase activating protein 1; LDS, lithium dodecyl sulfate; LIN7C, Lin-7 Cell Polarity Scaffold C; SDF1, stromal cell-derived factor-1; VCN, viral copy number.

Article Snippet: Other cytokines: HGF (PeproTech 100–39H); SDF1 (R&D Systems 350-NS); and S1P (Sigma-Aldrich 73914).

Techniques: Activation Assay, Western Blot, Infection, Plasmid Preparation, Control, Lysis, Expressing, Immunoprecipitation, SDS Page, Silver Staining, Mutagenesis, Binding Assay, shRNA, Stable Transfection, Knockdown, Phospho-proteomics, Derivative Assay