Fig 2: Overexpression of CD133 in PC cell lines. (A) Western blot analysis of CD133 expression in LNCaPVec (Vec) and LNCaPCD133+ (CD133) cells. 293T cells were transiently transfected with pcDNA3.1/N-terminal green fluorescent protein (NT-GFP) or pcDNA3.1/NTGFPCD133, and 293TVec (Vec)/293TCD133+ (CD133) cells were used as a positive control. (B) Confocal microscopy analysis of GFP-CD133 expression by stably or transiently transfected LNCaP/293T-GFP-CD133 cells. Nuclei were stained with DAPI, blue. Magnification, ×630; scale bar, 10 µm. (C) Western blot analysis of stemness-related protein expression. GAPDH was used as a loading control. Similar results were obtained in three separate experiments. (D) Stable overexpression of CD133 led to a significant increase in colony-forming ability. Magnification, ×100; scale bar, 100 µm. Bar graphs represent the mean ± standard deviation. *P<0.05 vs. VEC. PC; prostate cancer; NT-GFP, N-terminal green fluorescent protein.

Fig 3: In vitro and in vivo expression of MIF in LNCaPVec and LNCaPCD133+ cells. (A) Expression of OPN in LNCaPVec (Vec) and LNCaPCD133+ (CD133+) cells was measured by western blotting. GAPDH was used as a loading control. (B) MIF mRNA expression was characterized in LNCaPVec (Vec) and LNCaPCD133+ (CD133+) cells via reverse transcription-quantitative PCR. (C) Confocal microscopy of MIF expression in LNCaP cells. Nuclei were stained with DAPI (blue). Magnification, ×630; scale bar, 20 µm. (D) Immunohistochemical analysis of MIF in mouse tissue sections at the end of the study period. Metastases in representative histological sections of spine tissues are shown (magnification, ×200; scale bar, 20 µm) and black arrows indicate strong MIF positive cells near bone tissue. *P<0.05 vs. VEC. MIF, macrophage migration inhibitory factor, B, bone; T, tumor mass; C, spinal cord; OPN, osteopontin; MIF, macrophage migration inhibitory factor.

Fig 4: Comparison of mouse tumors. (A) Luciferase activities between LNCaPVec (Vec) and LNCaPCD133+ (CD133+) cells. (B) Tumor growth and multiple metastases were monitored over time via in vivo bioluminescence imaging. Data per representative mouse are shown. A large hot-spot of bioluminescence was observed in vivo following inoculation of mice with LNCaPVec (Vec) cells. Multiple localized and distant metastases were induced in vivo after injection of LNCaPCD133+ (CD133+) cells into the hearts of nude mice. Colored bars indicate the bioluminescence signal intensity range (photon·-1s·-1cm·-2steradian-1). (C) Hematoxylin and eosin staining of mouse tissue sections at the end of the study period. Metastases in representative histological sections of spine tissues are shown (magnification, ×20; scale bar, 200 µm). (D) Cytokine profiling of LNCaPVec (Vec)/LNCaPCD133+ (CD133+) cells. Culture supernatant from each cell line and serum from each mouse were harvested after 24 h and assayed using a cytokine profile array kit. Bar represents the mean ratio ± SD from two experiments. *P<0.05 vs. respective supernatant. B, bone; T, tumor mass; C, spinal cord; IL-8, interleukin-8; MIF, macrophage migration inhibitory factor.