Fig. 2: Inhibiting the IGF2BP3-induced secretion of CSF3 suppresses NETosis and reduces glioma survival. | Nature Communications

Fig. 2: Inhibiting the IGF2BP3-induced secretion of CSF3 suppresses NETosis and reduces glioma survival.

From: Overcoming therapeutic resistance in oncolytic herpes virotherapy by targeting IGF2BP3-induced NETosis in malignant glioma

Fig. 2

a, b mRNA-seq analysis was conducted on U87MG cells with IGF2BP3 overexpression a or knockout b, together with the corresponding control groups. The top 20 pathways with the smallest P values were selected for GO enrichment analysis. n = 3 biological independent experiments. c The heatmap displays the top 10 genes that were affected by IGF2BP3 knockout, based on mRNA-seq results. n = 3 biological independent experiments. d–g RT-qPCR analysis revealed the relative levels of CSF3(Csf3) mRNA in U87MG and GL261 cells with IGF2BP3 knockdown or overexpression. n = 3 biological independent experiments. h, i ELISA analysis demonstrated the relative levels of CSF3 protein in U87MG and GL261 cells. n = 6 biological independent samples for panel h, n = 5 biological independent samples for panel i. j, n Representative immunofluorescence images j and quantification n of NET formation in the GL261 cell/TAN uncontacted transwell assay treated with CSF3 neutralizing antibody (Anti-CSF3 nAb) or recombinant protein (CSF3). “Control” indicates TANs unstimulated with glioma cells in vitro. Scale bars, 20 μm. Representative images of n = 5. k, m, o, p Representative immunofluorescence images k, m and quantification o, p of NET formation in the GL261 cell/neutrophil and U87MG cell/induced neutrophil (HL-60) uncontacted transwell assays treated with Anti-CSF3 nAb or CSF3 recombinant protein. “Control” indicates neutrophils unstimulated with glioma cells in vitro. “ND” as undetected. Scale bars, 20 μm. Representative images of n = 5. l Electron microscopic analysis of TANs co-cultured with GL261-Luc cells stably expressing vector or Igf2bp3 with the indicated treatment. Yellow arrows indicate NETs, characterized by membrane protrusions and fiber formation. Scale bars are 5 μm. Representative images of n = 3. q–t The effect of CSF3 neutralizing antibody and recombinant protein on NET-induced tumor cell survival in contacted co-culture assays was determined using a luciferase assay. n = 5 biological independent samples. Statistical significance was determined using two-tailed Student’s t-test in d–i, or one-way ANOVA in n–r, t. Data represent the mean ± SD. Source data are provided in the Source Data file.

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