Fig 1: Actinification requires INF2 and F-actin depolymerization.a SMIFH2, not CK666, inhibits actinification. n = 2 independent experiments; two-way ANOVA [(-) NMDA vs (+) NMDA, F (1, 6) = 513.2; p = 0.0001; ctrl vs SMIFH2 vs CK666, F (2, 6) = 101, p = 0.0001], with Tukey’s post hoc multiple comparison analysis (p < 0.0001 NMDA vs NMDA w/ SMIFH2; n.s. p = 0.9168 NMDA vs NMDA w/ CK666). b INF2 immunoreactivity in neurons (NeuN). Arrows: distal (yellow) and proximal (blue) dendrites (MAP2), enlarged in lower panels. Scale bar, 12 µm (upper panels), 5 µm (lower panels). c INF2 immunoreactivity is higher in early-actinified than non-actinified neurons. Blue arrows: neuronal soma. Scale bar: 20 µm. n = 2 independent experiments; unpaired t-test,two-tailed (p = 0.0175). d Wildtype INF2 (INF2WT) enhances NMDA-induced actinification; constitutively active INF2 (INF2CA) actinifies without NMDA. n = 4 independent experiments; two-way ANOVA [(-) NMDA vs (+) NMDA, F (1, 18) = 1645; p = 0.0001; eGFP vs INF2WT vs INF2CA, F (2, 18) = 2394, p = 0.0001], with Tukey’s post hoc multiple comparison analysis (p = 0.0001 eGFP +/- NMDA; p = 0.0001 INF2WT +/- NMDA; n.s. p = 0.999 INF2CA +/- NMDA). e INF2 shRNA prevents actinification; rescue by shRNA-resistant INF2WT. n = 2 independent experiments; two-way ANOVA [(-) NMDA vs (+) NMDA, F (1, 6) = 481.1; p = 0.0001; empty v. vs INF2shRNA vs INF2shRNA + INF2WT, F (2, 6) = 100.6, p = 0.0001], with Tukey’s post hoc multiple comparison analysis (p = 0.0001 empty v. +/- NMDA; n.s. p = 0.7496 INF2shRNA +/- NMDA; p = 0.0001 INF2shRNA & INF2WT +/- NMDA). f Jasplakinolide (JASP) prevents NMDA-induced actinification (GFP-F-tractin). n = 2 independent experiments; two-way ANOVA [(-) NMDA vs (+) NMDA, F (1, 4) = 76.34; p = 0.0009; (-) JASP vs (+) JASP, F (1, 4) = 76.34; p = 0.0009]. Scale bar, 4 µm. g C646 formin-dependent actinification, despite NMDAR antagonists (APV + MK801). n = 3 independent experiments; two-way ANOVA [(-)C646 vs (+) C646, F (1, 12) = 144.4; p = 0.0001; vehicle vs NMDA antagonists vs SMIFH2, F (2, 12) = 21.2; p = 0.0001], with Tukey’s post hoc multiple comparison analysis (p < 0.0001 vehicle +/- C646; (p < 0.0001 NMDAR anta +/- C646; n.s. p = 0.5596 SMIFH2 +/- C646). Scale bar, 12 µm. All data are mean ± SEM. Source data are provided as source data file.