Fig 2: NMDA receptor-dependent Kalirin-7 expression decreases at puberty.(a,c,e,g) Representative images, scale, 2.5 µm. Arrows, spines. (a) Phalloidin (Phal), Kalirin-7 (Kal7) and merged images from pre-pub and pub CA1 hippocampus. (b) Mean pixel intensity, *t-test, t(26)=29.2, p<0.0001*, power=1; n=14 neurons (6 mice)/group. (c) Pfn2, Kal7 and merged images from pub WT and a4 KO CA1. (d) Mean pixel intensity, *t-test, t(26)=12.0, p<0.0001*, power=1; n=14 neurons (4 mice)/group. (e) Phal, Kal7 and merged images from pub CA1 hippocampus following in vivo treatment with vehicle or MK801 to increase NMDAR expression (Gao and Tamminga, 1995). (f) Mean pixel intensity, *t-test, t(26)=6.25, p<0.0001*, power=1; n=14 neurons (5 mice)/group. (g) Phal, Kal7 and merged images from post-pub CA1 hippocampus following in vivo treatment with vehicle or memantine (MEM), an NMDAR blocker. (h) Mean pixel intensity, *t-test, t(26)=6.5, p<0.0001*, power=1; n=14 neurons (5 mice)/group. Original uncropped images of Kal7 immunohistochemistry are shown in Figure 4—figure supplement 1. (Figure 4—source data 1) (i) Representative z-stack images, Pub, post-Pub Kal7 KO. Scale, 10 µm. (j) Averaged data, spine density. Proximal: t(32)=0.06, p=0.95, n=17 neurons (6 mice)/group; Distal: t(32)=0, p=1, n=17 neurons (6 mice)/group. (Figure 4—source data 2)DOI: http://dx.doi.org/10.7554/eLife.15106.02310.7554/eLife.15106.024Figure 4—source data 1.Figure 4b,d,f,h: Measurements of Kalirin-7 (Kal7) luminescence taken from CA1 hippocampal pyramidal cells for Pre-pub and Pub WT (4b), Pub, WT and a4 KO (4d), Pub WT-treated with MK-801 or vehicle (VEH) (4f) and Post-pub WT-treated with memantine or VEH (4h).DOI: http://dx.doi.org/10.7554/eLife.15106.02410.7554/eLife.15106.025Figure 4—source data 2.Figure 4j: Spine counts/20 µm on dendrites of CA1 hippocampal pyramidal cells – proximal (left) and distal (right) for pubertal (Pub) and post-pubertal (Post-pub) Kal7 KO mice.DOI: http://dx.doi.org/10.7554/eLife.15106.025

Fig 3: Cytoskeletal remodeling may be involved in PFN2-regulated EMT. (A and B) p-MLC expression was measured by western blotting in (A) normal colon, non-metastatic CRC and metastatic CRC tissues, and (B) in untransfected, vector- and PFN2-OE-transfected SW620 cells. (C) Protein expression levels of PFN2, p-MLC, E-cadherin and vimentin were examined by western blotting in PFN2-OE-transfected SW620 cells with or without co-treatment with Y27632; ß-tubulin served as the internal reference. (D) Wound-healing assays were performed to determine the migratory ability of PFN2-OE-transfected SW620 with or without co-treatment with Y27632. Data are presented as the mean ± sandard error of the mean of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001. CRC, colorectal cancer; EMT, epithelial-mesenchymal transition; MLC, myosin light chain; OE, overexpression; p, phosphorylated; PFN2, profilin 2; Y, Rho-associated kinase inhibitor Y27632.

Fig 4: PFN2 overexpression results in decreased EMT in colorectal cancer. PFN2 was transiently overexpressed in SW620 cells using a pQCXIH-PFN2 vector. (A) Western blotting was performed to confirm the overexpression efficiency in PFN2-OE transfected SW620 cells; untransfected and empty vector-transfected cells were used as controls. (B) Protein expression levels of EMT markers and regulators were analyzed by western blotting; GAPDH served as the internal reference. (C and D) Migratory abilities of the transfected SW620 cells were examined by (C) wound-healing and (D) Transwell migration assays, respectively. (E and F) Vector-transfected control SW620 cells or PFN2-OE-transfectd SW620 cells were injected into nude mice (E) via the spleen to induce liver metastasis or (F) via the tail vein to induce lung metastasis. The yellow arrows indicate the metastatic tumor nodules and hematoxylin and eosin staining was performed to confirm the tumor characteristics. (G) The volumes of the largest metastatic tumor nodules were calculated. Data are presented as the mean ± sandard error of the mean of three independent experiments. *P<0.05 and **P<0.01. Ctrl, control; EMT, epithelial-mesenchymal transition; OE, overexpression; PFN2, profilin 2.

Fig 5: PFN2 expression in normal colon mucosa and CRC tissues. (A) Representative examples of PFN2 immunohistochemical staining analysis in human CRC and corresponding para-CRC normal adjacent. (B and C) PFN2 expression levels in freshly collected human normal colorectal, non-metastatic CRC and metastatic CRC tissues was analyzed by (B) reverse transcription-quantitative polymerase chain reaction and (C) western blotting; GAPDH served as the internal reference. Data are presented as the mean ± standard error of the mean of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001. CRC, colorectal cancer; para-CRC, paracancerous; PFN2, profilin 2.