Fig 1: Incubation with the CD44 antibody, 5F12, abolished the differences in cell viability or apoptosis induced by cells treated with IGF-1 and either IGFBP-3 or P-IGFBP-3 with or without cisplatin. Cells (0.2 × 105) were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media overnight, then treated for 72 h with cisplatin (10 μM when using A549 cells and 30 μM when using H1299 cells), IGF-1 (100 ng/mL), IGFBP-3 or P-IGFBP-3 (50 nM), the CD44 antibody 5F12 (5 μg/mL), added either separately or 2 h prior to addition of other treatments as indicated, or in combination. The media containing the specific components in the various treatments was replaced every 24 h. Viability and apoptosis of A549 (A,B) and H1299 (C,D) cells were then assessed as described in the Methods section. Data from five independent assays, each carried out in triplicate, were averaged, normalized, and expressed as fold change relative to untreated cells (control). The graphs summarize the results expressed as means ± SD (n = 5). Asterisks indicate a statistically significant difference from the control or single treatments of each cell line group while absence of asterisks indicates no significance using the GraphPad 9.4.1 software, Mann–Whitney test. Statistical differences between different groups were analyzed by an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test, * p < 0.05, ** p < 0.0l. Absence of asterisks indicates no significance (ns).
Fig 2: Phosphorylation of IGFBP-3 by CK2 affects its binding to HA but not to IGF-1. IGFBP-3 (50 nM) unphosphorylated or phosphorylated by CK2 (A) was bound to the wells, then increasing concentrations of biotin-HA (B) or IGF-1 (C) were added and processed as described in the Methods section. Optical densities (ODs, 450 nm) were normalized for the curves by expressing each point relative to the best fitted Emax value for IGFBP-3 (set to 100%). The data were then plotted as a function of increasing biotin-HA or IGF-1 concentrations and, using the GraphPad Prism 9.4.1 software, fit with a nonlinear regression curve fitting approach. Before analysis, the ODs from the data were corrected for non-specific binding by subtracting the mean background absorbance for the negative controls containing the same IGFBP-3 concentration but using water instead of biotin-HA or IGF-1. Data were expressed as the mean ± S.D. of three independent experiments, each carried out in triplicate.
Fig 3: Midkine knockdown inhibits the PI3K/Akt pathway in PDGF‐BB‐treated ASMCs. ASMCs were transfected with si‐midkine for 48 h and then incubated with the PI3K/Akt pathway activator IGF‐1 (100 ng/mL) and PDGF‐BB (20 ng/mL) for a further 24 h. (a) Representative gel blots depicting the levels of phosphorylated PI3K and phosphorylated Akt. (b, c) Quantification of p‐PI3K and p‐Akt protein band intensities. Data are presented as mean ± SD from at least three independent biological replicates, each performed in six duplicate wells, and analyzed using one‐way ANOVA, followed by Bonferroni post‐hoc test.
Supplier Page from Abcam for Recombinant human IGF1 protein (Active)