Treatment with streptozotocin reduces androgen receptor staining in prostate tumors in PAC120 mouse model.

<p>(<b>A</b>) HE staining of prostate tumor xenografts from control mice, (<b>D</b>) citrate treated mice, (<b>G</b>) STZ-treated before tumor implantation and (<b>J</b>) STZ-treated after tumor implantation. (<b>B</b>) AR staining of prostate tumor xenografts from non-treated mice, (<b>E</b>) citrate treated mice, (<b>H</b>) STZ-treated mice before tumor implantation and (<b>K</b>) STZ-treated mice after tumor implantation. (<b>C</b>) Cytokeratine staining of prostate tumor xenografts from control mice, (<b>F</b>) citrate treated mice, (<b>I</b>) STZ-treated mice before tumor implantation and (<b>L</b>) STZ-treated mice after tumor implantation.</p

Treatment with streptozotocin reduces prostate tumor growth in PAC120 mouse model.

<p>(<b>A</b>) Pictures of tumors developed in vehicle treated mice (n = 5) (i), STZ treated after or before tumor implantation (n = 7 for both) with reduced (ii) or no tumor growth (iii). (<b>B</b>) Comparison of tumor volume for non-treated (n = 5) and citrate treated (n = 5) mice. (<b>C</b>) Tumor volume of STZ-treated animals after tumor implantation compared with tumor volume of citrate treated mice. (<b>D</b>) Tumor volume of STZ-treated animals before tumor implantation compared with tumor volume of citrate treated mice. Data are mean ± SD. *P<0.05 and **P<0.01 compared with the control.</p

Analysis of the androgen receptor and p65 levels in tumor xenografts of control and STZ treated mice.

<p>(<b>A</b>) Relative AR mRNA levels for citrate and STZ-treated mice. Human 18 S mRNA was amplified as a control. Data points are shown as mean ± SD of triplicates. (<b>B</b>) Analysis of AR (left panel) and phospho-p-65 (right panel) protein levels from citrate and STZ-treated mice measured by Western blotting using PPIA as a housekeeping reference protein.</p

Synergic effects of hyperglycemia and TNFα on the androgen receptor downregulation in LNCaP cells.

<p>(<b>A</b>) Relative AR mRNA levels in LNCaP treated with an increasing concentration of D-glucose with or without TNFα. Human 18 S mRNA was amplified as a control. Data points are shown as mean ± SD of triplicates. *P<0.05 and **P<0.01 compared with 5 mM of D-glucose. (<b>B</b>) Analysis of AR protein levels in LNCaP cells treated as in <b>A</b> measured by Western blotting using PPIA as a housekeeping reference protein.</p

Hyperglycemia reduces androgen receptor levels via NF-κB activation in LNCaP cells.

<p>(<b>A</b>) Relative AR mRNA levels in LNCaP treated with 5 mM or 30 mM glucose. Human 18 S mRNA was amplified as a control. Data points are shown as mean ± SD of triplicates. *P<0.05 and **P<0.01 compared with 5 mM glucose. (<b>B</b>) Analysis of AR and phospho-p-65 protein levels in LNCaP cells treated as in <b>A</b> measured by Western blotting using PPIA as a housekeeping reference protein. (<b>C</b>) Relative AR mRNA levels in LNCaP treated with 5 mM or 30 mM glucose in the absence or presence of QNZ (25 nM). Human 18 S mRNA was amplified as a control. Data points are shown as mean ± SD of triplicates. *P<0.05 and **P<0.01 compared with 5 mM glucose. (<b>D</b>) Analysis of AR protein levels in LNCaP cells treated as in <b>C</b> measured by Western blotting using PPIA as a housekeeping reference protein.</p

Hyperglycemia downregulates androgen receptor levels in LNCaP cells.

<p>(<b>A</b>) Relative AR mRNA levels in the presence of different D-glucose concentrations are shown. Human 18S mRNA was amplified as a control. Data points are shown as mean ± SD of triplicates. (<b>B</b>) Analysis of AR protein levels in the presence of different D-glucose concentrations in LNCaP cells by western blot. PPIA has been used as a housekeeping reference protein.</p

List of specific primers for each exon used for RT-PCR.

<p>List of specific primers for each exon used for RT-PCR.</p

FAIM-S_2a and FAIM-L_2a are localized in the cytoplasm and nucleus.

<p><b>A:</b> Western blot analysis using anti-FLAG to detect the presence of FAIM-S, FAIM-L, FAIM-S_2a and FAIM-L_2a in the distinct cellular compartments. Anti-calnexin was used as a marker for the membrane fraction, anti-actin as a marker of the cytosolic fraction, and anti-Tri-Methyl-Histone H3 as a marker of the nucleus. <b>B:</b> Immunofluorescence in Vero cells 24 h after transfection with pcDNA3-GFP containing the extra-long isoforms. Anti-calnexin (reticular protein), Mitotracker (mitochondrial marker) and Hoechst (nuclei staining) were used to examine the co-localization of FAIM isoforms. Scale bars 10 <b>μ</b>m.</p

Isoforms expression in cell lines.

<p><b>A:</b> SH-SY5Y cells were transfected with the pCDNA3-FLAG-FAIM-S, pCDNA3-FLAG-FAIM-S_2a, pCDNA3-FLAG-FAIM-L or pCDNA3-FLAG-FAIM-L_2a vector. At a range of time points, cells were harvested and protein expression was assessed by western blot using an anti-FLAG antibody (dilution 1:20000). <b>B</b>: PC12 cells were transfected with the isoform vectors (above mentioned) and treated with MG-132 (2.5 μM). Cell extracts were then resolved by western blot analysis, and FAIM expression was measured using an anti-FLAG antibody (dilution 1:20000). <b>C:</b> HEK293T cells transfected with pcDNA3-FLAG-FAIM-L, pcDNA3-FLAG-FAIM-S, pcDNA3-FLAG-FAIM-L-2a or pcDNA3-FLAG-FAIM-S-2a vector were lysed, and protein extracts were analyzed by western blot. An anti-FAIM-L (anti-2b FAIM, specific for neuronal exon 2b) and anti-FAIM (that recognizes the common part of the isoforms) were used. Anti-tubulin was used as a loading control. Two different exposures of the film are shown in order to facilitate observation of the bands of all isoforms. DIV: days <i>in vitro</i> (n = 3).</p

Treatment with Actinomycin D (ActD).

<p>SH-SY5Y cells were treated with ActD for a range of times (3, 6, 9 and 12 h). <b>A:</b> The half-life of mRNA was measured by treating cells with ActD (5 μg/ml) and collecting total RNA at the times shown, whereupon the levels of <i>FAIM</i> mRNA and <i>18S mRNA</i> (a stable, housekeeping control mRNA) were measured by RT–qPCR analysis. mRNA half-life was calculated as the time needed to reduce transcript levels to half (50%, discontinuous line) of their initial abundance at time 0. <b>B:</b> Number of cycles needed to detect similar size product of FAIM-S, FAIM-L, FAIM-S_2a and FAIM-L_2a by qPCR (SybrGreen) using the following pairs of primers: (1bF2/3R for FAIM-S; 2bF/3R for FAIM-L; 2aF/2a3R for FAIM-S_2a and 2aF/2bR for FAIM-L_2a).</p