Airs of samples, 54 CRC tissues (56.3 ) and 28 normal tissues (29.2 ) have been identified to be constructive for AF1q expression (p 0.05), with the AF1q protein becoming mostly located within the cell cytoplasm. Representative AF1q immunostaining images for standard, CRC, and metastatic liver tissues are shown in Figure 1C,D. AF1q upregulation was significantly connected with lymph node metastasis (p = 0.0159) and late tumor, lymph node, metastasis (TNM) stage (p = 0.018; Table 1).Table 1. The connection in between AF1q expression level and clinicopathologic variables in 96 CRC individuals.Clinicopathologic Parameters Gender Male Female Age (years) 50 50 T T1, T2 T3, T4 Tumor grade G1, G2 G3, G4 Lymph node metastasis Damaging Positive Distant metastasis Damaging Positive TNM Stage I, II III, IV 17 (17.7) 25 (26.0) ten (ten.4) 44 (45.eight) 0.018 40 (41.7) 2 (2.1) 45 (46.9) 9 (9.4) 0.135 20 (32.3) 22 (20.eight) 13 (13.5) 41 (42.7) 0.0159 34 (35.4) eight (8.three) 40 (41.7) 14 (14.six) 0.426 9 (9.4) 33 (34.4) 11 (11.5) 43 (44.eight) 0.899 6 (six.three) 36 (37.five) 7 (7.3) 47 (48.9) 0.851 22 (22.9) 20 (20.8) 31 (32.three) 23 (24.0) 0.623 AF1q Staining Low (n = 42) Higher (n = 54) pValueBioinformatic evaluation was performed to examine the relationship between AF1q and CRC patient survival outcomes. For this analysis, level 3 HiSeq RNASeq data for 380 cancer samples have been downloaded from the Cancer Genome Atlas (TCGA) web site [18,19]. Then, the R package `survival’Int. J. Mol. Sci. 2017, 18,three ofwas used to analyze the relationship in between AF1q expression and survival, utilizing data from the TCGA along with other CRC cohorts. The results showed that AF1q upregulation was indicative of each poor general survival (Figure 1E) and poor diseasefree survival (Figure 1F). Following this result, AF1q expression was evaluated in five CRC cell lines (SW480, SW48, KM12, SW620, and LoVo) and Int. J. Mol. Sci. 2017, 18, 987 3 of 14 1 normal intestinal epithelial cell line (NCM460). As shown in Figure 1G,H, AF1q expression at both the mRNA as well as the protein level was higher in the five CRC cell Figurethan inAF1q expression at one typical intestinal epithelial cell line (NCM460). As shown in lines 1G,H, NCM460 cells. These each the mRNA and is protein level was CRC, inside the five CRC cell lines than in NCM460 cells. benefits suggest that AF1qthe overexpressed inhigher and that this enhanced expression is predictive of these outcomes suggest that AF1q is overexpressed in CRC, and that this increased expression is poor Khellin Technical Information prognosis in CRC patients.predictive of poor prognosis in CRC individuals.Figure 1. Expression AF1q mRNA AF1q in colorectal38 CRC tissues and paired regular tissues; (B)lines. qPCR analyses of analyses of expression level in cancer (CRC) tissue specimens and cell (A) qPCR analyses of AF1q mRNA expression level in 38 CRC tissues and paired standard tissues; Histogram displaying the typical AF1q mRNA expression level in 38 CRC and paired standard tissues; (B) Histogram displaying the typical AF1q mRNA expression level in 38 CRC and paired regular tissues; (C,D) Representative immunostaining images displaying AF1q expression in standard tissues, CRC (C,D)tissues, and liverimmunostaining photos MLS1547 manufacturer showing100. Scale bars: 200 regular tissues, CRC tissues, Representative metastasis tissues (magnification: AF1q expression in m; (E,F) KaplanMeier overall (OS) and illness no cost (DFS) survival evaluation for bars: 200 ; (E,F) KaplanMeier general and liver metastasis tissues (magnification: 100. Scale CRC patients classified as obtaining higher or low (OS).