Skip to main content
Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: TMPRSS4 promotes cancer stem–like properties in prostate cancer cells through upregulation of SOX2 by SLUG and TWIST1

Fig. 3

TMPRSS4 promotes tumorsphere formation and upregulates stemness-related factors. A Tumorsphere formation assay. Transfected PC3 cells were dissociated to single cells and seeded at a density of 200 cells/ml in 96-well plates under suspension culture conditions in the presence of 20 ng/ml EGF, 10 ng/ml bFGF, and 2% B27 supplement. The cells were incubated for 7 days. Spheroids with diameter > 75 μm were counted. Bar, 50 μm. B ALDH assay. Transfected PC3 cells were incubated with ALDH substrate for 30 min, and then analyzed using flow cytometry. Cells were quenched with DEAB, an ALDH inhibitor, as a negative control. C PC3 and HEK293E cells transfected with TMPRSS4 expression vector were lysed for immunoblot analysis. Anti-myc antibody was used to detect myc-tagged TMPRSS4. D Transfected cells were lysed and subjected to real-time qPCR analysis. E Cells were co-transfected with a TMPRSS4 expression vector and a SOX2 promoter (− 2546/+ 544) reporter construct in the pGL3 vector. Firefly luciferase activity, representing SOX2 promoter activity, was measured after 48 h and normalized against Renilla luciferase activity. F–I 22Rv1 and LNCaP clone FGC cells were transfected with TMPRSS4-specific shRNA vectors for 48 h. Transfected cells were subjected to ALDH assay (F, H) or lysed for immunoblot analysis (G, I). Densitometric quantification was performed on the immunoblots using GAPDH as a loading control. The mean relative density from three independent experiments is shown under the immunoblots (C, G, I). Values represent mean ± SD. ***P < 0.001

Back to article page