Fig 2: ITGB4 promotes migration, invasion, metastasis and EMT process of ccRCC cells. a Overexpression of ITGB4 in ACHN and Caki-1 cells respectively confirmed by western blot. b Wound-healing assay used to detect the alteration of cell migratory ability after ITGB4 overexpression. Bar scale = 200 µm. c, d Cell migration and cell invasiveness respectively detected by migration assay (c) and invasion assay (d). e Expression of EMT-related markers in ACHN and Caki-1 cells with or without ITGB4 overexpression determined by western blot. f Representative and HE-staining images of lungs excised from mice injected with ITGB4-overexpressing and negative control cells. Bar scale = 50 µm. Each experiment was carried out independently for three times. Bar graphs: means ± SDs. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig 3: High expression of ITGB4 in glioma patients was associated with glioma grades. a-c Representative images from immunohistochemical staining of ITGB4. The expression levels of ITGB4 between normal tissues (n = 8) and glioma tissues (n = 112) were compared. The association between ITGB4 and the grades were analysed. * p < 0.05 vs. control. d ITGB4 mRNA expression in normal tissues (n = 23) and the glioblastoma tissues (n = 81) were analysed. The data were extracted from the Oncomine database. e The mRNA levels of ITGB4 between normal tissues (n = 3) and glioblastoma tissues were analysed by q-RT-PCR

Fig 4: ITGB4 knockdown suppressed stem-like properties of glioma cells. a-d ITGB4+ and ITGB4- cells were isolated from LN229 and U251 cells by flow cytometry sorting. The expression levels of Oct4 and Nanog were analysed by western blotting and q-RT-PCR assays. Data represent the mean ± SD of three independent experiments. *** p < 0.001 vs. control. e-h GSCs were enriched from LN229 and U251 cells by sphere formation assay. We then knocked down ITGB4 expression using siRNA in the GSCs. The expression levels of ITGB4, Oct4, and Nanog were analysed by western blotting and q-RT-PCR assays. Data represent the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001 vs. control. i-n ITGB4 was knocked down in LN229 and U251 cells. The mammosphere-forming abilities, ALDH1-positive populations, and CD133-positive populations were analysed. Data represent the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001 vs. control

Fig 5: Overexpression of ITGB4 is associated with occurrence and poor prognosis of ccRCC. a, b The abundance of ITGB4 transcripts in ccRCC tissues and normal kidney tissues from TCGA database (a) and GSE53757 dataset (b). c, d Kaplan–Meier analysis of ccRCC patients’ overall survival (c) and disease-free survival (d) based on ITGB4 expression in TCGA database. e–h ITGB4 expression of ITGB4 in ccRCC tissues of different clinical stages (e), pathological grades (f), nodal metastasis status (g) and subtypes (h). i Analysis of ITGB4 protein level in ccRCC tissues and normal kidney tissues in CPTAC database. j The mRNA expression of ITGB4 in 50 paired ccRCC tissues and normal kidney tissues detected by qRT-PCR. k The protein expression of ITGB4 in 24 ccRCC paired tissues and normal kidney tissues determined by western blot assay. N, normal tissues; T, tumor tissue. l ITGB4 expression in metastatic ccRCC and non-metastasis ccTCC detected by immunohistochemistry assay. Bar scale = 20 µm. Bar graphs: means ± SDs. *p < 0.05; ****p < 0.0001