Fig 1: Expression of SNX9 in human colorectal cancer tissues. (a) The domain structure of SNX9 and western blot analysis of recombinant biotinylated (Bio‐) SNX9 proteins. The numbers in the domain structure represent the order of amino acids from the N‐terminus. The red line indicates the location of antigen for the anti‐SNX9 antibody used in this study. The anti‐SNX9 antibody failed to recognize Bio‐SNX9 (ΔLC) recombinant proteins because the antigen amino acid sequence is located in the LC domain. (b) Validation of the anti‐SNX9 antibody for immunohistochemistry in the human colorectal cancer tissues. The anti‐SNX9 antibody was preincubated with biotinylated full‐length SNX9 (Bio‐SNX9 [FL]), LC domain‐deleted SNX9 (Bio‐SNX9 [ΔLC]), or LC‐PX domain of SNX9 (Bio‐LC‐PX). The mixtures of the antibody and recombinant proteins were used as primary antibody. The neighboring sections of a human colorectal cancer tissue were subjected to H&E staining and immunohistological staining for SNX9. Bars, 500 µm. Inset: magnifications of the yellow squared areas. (c) Representative images of the H&E staining and immunohistological staining for SNX9 and CD31. The neighboring sections of the human normal colon and colorectal cancer tissues were subjected to H&E staining and immunohistochemistry. In normal colon, CD31+‐endothelial cells are SNX9‐negative (yellow arrows). In colorectal cancer tissues, CD31+‐endothelial cells are SNX9‐positive (yellow arrowheads). Bars, 500 µm (HE staining, left panels of SNX9 and CD31 staining) and 50 µm (right panels of SNX9 and CD31 staining). Magnifications of the squared areas are shown on the right. H & E: hematoxlyin–eosin; SNX9: sorting nexin 9 [Color figure can be viewed at wileyonlinelibrary.com]
Fig 2: SNX9 is essential for tube formation in HUVECs. (a) Western blot analysis of HUVEC lysates 72 hr posttransfection of siRNAs. (b) Confocal images and quantitation of tube formation. HUVECs seeded on collagen I gel were treated with control, or SNX9 siRNAs (siSNX9 #1–#3) and packed on collagen I followed by VEGF stimulation for 66 hr. HUVECs were visualized by the staining for F‐actin with rhodamine‐labeled phalloidin. Bars, 200 µm. A total of 30 of the tubes from three independent experiments were analyzed. Data shows the mean ± SEM. ***p < .001. (c) Confocal images and quantitation of cell spreading. HUVECs were seeded on the Matrigel followed by incubation for 1 hr. The cells were visualized by staining of F‐actin with rhodamine‐labeled phalloidin. Bars, 20 µm. The size of 50 cells from three independent experiments were analyzed. Data shown are the mean ± SEM. ***p < .001. (d) Confocal images and quantitation of network formation. HUVECs were seeded on the Matrigel followed by incubation for 12 hr. The cells were visualized by staining for F‐actin with rhodamine‐labeled phalloidin. Bars, 200 µm. A total of 30 of networks from three independent experiments were analyzed. Data shown are the mean ± SEM. ***p < .001. (e,f) Rescue experiments for SNX9 knockdown: (e) Western blot analysis of cell lysates of HUVECs infected with siRNA resistant‐Myc‐SNX9‐carrying lentivirus. (f) Confocal images and quantitation of network formation of HUVECs infected with siRNA resistant‐Myc‐SNX9‐carrying lentivirus (f). HUVECs were seeded on the Matrigel followed by incubation for 12 hr. Cells were visualized by the staining for F‐actin with rhodamine‐labeled phalloidin. The Myc‐SNX9 was labeled with anti‐Myc antibody. Bars, 200 µm. Magnifications of the squared areas are shown in the lower panels (bars, 20 µm). A total of 30 of the tubes from three independent experiments were analyzed. Data shown are the mean ± SEM. ***p < .001. HUVEC: human umbilical vein endothelial cells; SEM: standard error of mean; siRNA: small interfering RNA; SNX9: sorting nexin 9; VEGF: vascular endothelial growth factor [Color figure can be viewed at wileyonlinelibrary.com]
Fig 3: Snx9 knockout mice do not display pathological changesTissues of 2-month-old Snx9+/− and Snx9−/− mice were sectioned and stained with hematoxylin-eosin (HA). Shown are results from heart A, B. lung C, D. kidney E, F. and brain G, H.
Fig 4: Strategy for generation of Snx9 knockout miceA. Schematic drawing of the domain structures of Drosophila melanogaster SH3PX1 and Mus musculus SNX9, SNX18 and SNX33. The identity of different domains between SH3PX1 and its mouse homologs is indicated below each domain. B. Schematic drawing of targeting strategy for Snx9 gene disruption. The targeting vector has two loxP sites flanking exon 5 of Snx9 gene. After homologous recombination, the two loxP sites are placed flanking exon 5 of Snx9 gene in the mouse genome. Then exon 5 is excised with the help of Cre recombinase, resulting in disruption of Snx9 gene. The localizations of genotyping primers in the genome are indicated.
Fig 5: Survival analysis with SNX9 expression profiles in human colorectal cancers. The Kaplan–Meier plot with SNX9 expression profiles in GSE17536 and GSE14333 datasets using endpoint overall survival status (left) and endpoint disease‐free survival status (right). Shading along the curve showed 95% confidential interval. In human colorectal cancers, the high expression of SNX9 was correlated to poor prognosis for overall survival (high: n = 99; low: n = 78) and disease‐free survival (high: n = 25; low: n = 201). SNX9: sorting nexin 9 [Color figure can be viewed at wileyonlinelibrary.com]
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