Fig 2: Surgery impaired spine structural plasticity and decreased the activity of Rac1 signaling pathways during learning. a Dorsal hippocampal profile image of Golgi staining. b Representative tracing images of dorsal hippocampal CA1 pyramidal neurons on postoperative day 14. Sholl analysis of apical and basal dendrites of CA1 pyramidal neurons from rats c, d without behavioral training (home cage) and e, f with behavioral training (behavior, n = 24 neurons from 4 rats per group). g Total number of dendrites and h total dendritic lengths of traced images of dorsal hippocampal CA1 pyramidal neurons were analyzed using FIJI software (n = 24 neurons from 4 rats per group). i Representative images of the secondary branch of apical dendrites of CA1 pyramidal neurons in the dHPC on postoperative day 14 captured with a 100 × objective. Quantitation of the total spine, mushroom-shaped spine, thin-shaped spine, filopodia-shaped spine, and stubby-shaped spine densities in rats j without behavioral training and k with behavioral training (n = 24 neurons from 4 rats per group). l Representative immunoblots and m densitometry analysis of Rac1-GTP (active) and total Rac1 proteins levels in the dHPC on postoperative day 14 (n = 6 rats per group). n Representative confocal images of immunohistochemical staining for Rac1-GTP+ cells (green) in the dHPC captured with a 10 × objective. o Representative immunoblots and densitometry analysis of p phospho-Cofilin, Cofilin, and q Arp3 proteins in the dHPC on postoperative day 14 (n = 6 rats per group). Control-H control-home cage, Surgery-H surgery-home cage, Control-B control-behavior, Surgery-B surgery-behavior. Data are presented as means ± SEM and statistical analyzed by a Student’s t test (c, d, e, f, j, and k), a one-way ANOVA followed by a Tukey’s test (g, h, m, p, and q). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with Control

Fig 3: EGF-driven microcircuitry steers a two-pronged pathway toward metastasis. (A) List of selected interacting partners of DIAPH1 shortlisted from BioID. (B) Western blot analysis on SCC12 lysates immunoprecipitated with anti-DIAPH1 and probed for Arpin (top). Input cell lysates were probed for Arpin (middle) and DIAPH1 (bottom). Representative image is from three independent experiments. (C) Western blot on SCC12 control or shDIAPH1 cell lysates immunoprecipitated with anti-Arpin and probed for Arp3 (top). Input cell lysates were probed for Arp3 (middle) and ß-actin (bottom). Representative image is from three independent experiments. IP, immunoprecipitation; WB, Western blotting. (D) Analysis of cell displacement upon DIAPH1 knockdown and rescue with concurrent knockdown of both DIAPH1 and Arpin. Representative image is from three independent experiments. (E) Heat map showing expression values of selected genes from microarray data of SCC12 cells transfected with control shRNA or shFSTL1. Expression values displayed in shades of red (high) or blue (low) relative to the individual mean value of the gene in linear scale. (F) Gelatin zymography and quantification of band intensities represented as a histogram (top). **, P < 0.001; error bars represent SD (n = 3). (G) Western blot on control or shFSTL1 SCC cell lysates from EGF-pretreated cells. Lysates were probed for phosphoERK or total ERK. (H) Western blot showing rescue of phosphoERK upon treatment with recombinant FSTL1 (n = 2). (I) Relative transcript abundance of MMP9 in control or shFSTL1 A253 cells treated with EGF. **, P < 0.001. Error bars represent SD (n = 5). (J) Rescue of MMP9 expression upon treatment with recombinant FSTL1. Error bars represent SD (n = 3). (K) A253 cell lysates were immunoprecipitated with IgG or anti-FSTL1 antibody and probed for Wnt7a by Western blot analysis. Representative image is from three independent experiments. (L) Western blot on lysates from shFSTL1 cells transfected with specific siRNA against Wnt7a and cells treated with EGF posttransfection. Lysates were probed for phosphoERK or total ERK (n = 3).

Fig 4: Co-IP’d peptides and proteins identified comparing aCSF vs. KCl in WT lysates. (A) Volcano plot showing peptides that were enriched and depleted in aCSF vs. KCl. Peptides that were identified as enriched in WT vs KO in the aCSF condition are color-coded as shown in the key. (B) The same volcano lot from (A) is shown with the scale changed to highlight significantly depleted peptides, and all peptides that meet significance criteria color-coded by protein identity. Key shows color-coding of peptides by protein identify. (C) Volcano plot showing proteins that were depleted in aCSF vs. KCl. Depleted proteins are labeled. (D) StringDB node-edge diagram of the identified peptides from (B), highlighting the known interactions. (E) IP-western blots confirm that ARP3, PSD95 (DLG4) and SynGAP co-associate with Homer1, but not with nonspecific IgG control.