RARRES1 modulates angiogenesis.

<p>Human umbilical vein endothelial cells (HUVECs) were transfected with empty vector or RARRES1 expression vector. After 48 hours HUVECs were trypsinized and seeded on a matrigel at a density of 5×10³/well in triplicates. After 18h, tube-like structures were photographed. <b>A)</b> Representative photographs of tube-like structure. <b>B)</b> Quantitative analysis of total tube numbers; *p<0.01 vs. empty vector; #p <.05 vs. empty vector and PMA. <b>C)</b> PC3 cells were transfected with empty vector or RARRES1 expression vector. HUVECs were seeded on a matrigel at a density of 5×10³/well and treated with the conditioned media (CM) from PC3 transfected cells. Each group was in triplicate. After 18h, tube-like structures were photographed and quantitated. The quantitation results of total tube numbers are reported. <b>D)</b> Real time qPCR analysis of TSP1 and HIF1 expression on HUVECs treated with conditioned media from empty vector or RARRES1 expression vector transfected PC3 cells. Fold changes have been calculated by 2^^-(ΔΔCT) method, *p<0.05.</p

RARRES1 induces autophagy.

<p><b>A)</b> PC3 cells were plated in 8 well chamber slides and transfected with empty vector or RARRES1 expression vector. Cells were then fixed with parafomaldehyde and immunocytochemistry with antibodies, FLAG and LC3B was performed. Confocal images were taken at 20X magnification. <b>B)</b> Protein lysates from PC3 cells transfected with empty vector or RARRES1 expression vector were immunoblotted and immunoprobed with antibodies against autophagy markers, LC3B, ATG3, Beclin, FLAG and GAPDH. <b>C)</b> Prostate cancer cells C4-2 and PC3 cells were co-transfected with LC3B and empty vector or RARRES1 expression vector. Immunocytochemistry was performed using LC3B (green) and FLAG (red) antibodies and images were taken with 40X objective using Zeiss LSM 700 confocal microscope.</p

Mechanism of RARRES1 function.

<p>Based on our data and cited references, the outline of RARRES1’s role of tumor suppression in prostate cancer has been depicted. The green arrows indicate a positive induction and the red lines indicate repression. The black dashed arrows indicate the presence of intercellular signaling between the processes/molecules. The solid black arrows indicate the results we present in the paper to support our conclusion.</p

RARRES1 modulates ER stress and the expression of antioxidant enzymes.

<p><b>A)</b> PC3 cells were treated with tunicamycin after being transfected with empty vector or RARRES1 expression vectors. Immunoblot analysis was performed for ER stress with antibodies against GRP78, IRE-1, FLAG and GAPDH (loading control). <b>B)</b> Real time qPCR data of PC3 cells transfected with empty vector or RARRES1 expression vector. Fold changes have been calculated by 2^^-(ΔΔCT) method; *p<0.05. <b>C)</b> PC3 cells were transfected with empty vector or RARRES1 expression vector. Immunoblot analysis was performed for autophagy modulating signaling proteins, with antibodies against mTOR, SIRT1, RARRES1 and GAPDH (loading control). <b>D)</b> Immunoblot data with antibodies against acetylated and total forms of FOXO1 with protein lysates from cells transfected with empty vector or RARRES1 expression vector. <b>E)</b> Levels of antioxidant enzymes, catalase, Glutathione peroxidase (GPX), Manganese superoxide dismutase (MnSOD) after empty vector or RARRES1 expression vector transfection; *p<0.01.</p

Clinical-pathological characteristics of the prostate cancer patients.

<p>Clinical-pathological characteristics of the prostate cancer patients.</p