AR suppression mediates miR-101 reduction after celastrol treatment.

<p><b>A</b>, miR-101 and AR expressions after celastrol (CEL) treatment. **, <i>P</i><0.01, compared with DMSO. <b>B</b>, Schematic depiction of luciferase reporter constructs as detailed in " <b>Materials and Methods</b>". Wild type and mutant AR binding sites were indicated by italic dash and cross, respectively. <b>C</b>, DU145 cells were transfected with indicated reporter constructs in the presence of pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) plasmid for 24 h, and then luciferase activity was detected. pRLSV40 plasmid was co-transfected for normalization. **, <i>P</i><0.01, between EGFP-AR and EGFP-V transfections. <b>D</b>, ChIP assay. Cell extracts from DU145 or LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) were immunoprecipitated with AR antibody or normal mouse IgG. Input was 1/100 of the sonicated chromatin prior to immunoprecipitation. PCR was performed as described in the "<b>Materials and Methods</b>". <b>E</b>, LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) were treated with celastrol (CEL, 2.0 μM) or DMSO for 3 h, pri-miR-101 and mature miR-101 levels were determined by qPCR. Asterisks denote significance between EGFP-AR and EGFP-V transfections. *, <i>P</i> <0.05; **, <i>P</i> <0.01.</p

Celastrol triggers autophagy in prostate cancer cells.

<p>Parental LNCaP cells (<b>A</b>, <b>D</b>) or the cells transfected with GFP-LC3 (<b>B</b>, <b>C</b>) were treated with celastrol at 2.0 μM for indicated times. mRNA expressions of autophagy related genes were measured by qPCR (<b>A</b>). RQ, relative quantity. GFP-LC3 transfected cells were stained by DAPI after treatments. GFP-LC3 puncta were observed under confocal microscope (<b>B</b>) and quantified in <b>C</b>. The cells that contained over 5 puncta were selected and fifty cells were analysed for each treatment. <b>D</b>, Protein extracts were immunoblotted with antibodies against LC3, p62 and GAPDH (loading control). Asterisks denote significance compared with control (0 h). *, <i>P</i> <0.05; **, <i>P</i> <0.01.</p

AR inhibition on celastrol-induced autophagy is related with miR-101 transactivation.

<p>To see whether miR-101 could affect AR suppression on celastrol-induced autophagy, AR positive LNCaP cells were transfected with miR-101 mimic or negative control (NC) for 24 h (<b>A</b>), followed by additional 24 h treatment with celastrol (2.0 μM, CEL). MiR-101 levels were determined by RT-PCR. #, <i>P</i><0.05 <i>versus</i> NC transfection without celastrol treament. **, <i>P</i><0.01 between miR-101 and NC transfections with celastrol treatment. The protein levels of LC3 and p62 as well as AR were determined by Western blotting using GAPDH as a loading control. <b>B</b>, AR negative DU145 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) in the presence of miR-101 inhibitor (miR-101 Inh) or negative control (NC). Cells were incubated in celastrol (2.0 μM, CEL) for an additional 24 h. MiR-101 levels were determined by RT-PCR. #, <i>P</i><0.05 <i>versus</i> EGFP-V plus NC transfections. *, <i>P</i><0.05 between EGFP-V and EGFP-AR in the presence of miR-101 inhibitor. The protein levels of AR, LC3and p62 were detected by Western blotting. <b>C</b>, DU145 cells were transfected with pGL3-B-miR-101-W (with wild type AR binding site) or pGL3-B-miR-101-M (with mutant AR binding site), along with AR expression vector (EGFP-AR) or empty vector (EGFP-V) for 24 h, then treated with DMSO or celastrol (CEL, 2.0 μM) for additional 24 h. MiR-101 levels were determined by RT-PCR. #, <i>P</i><0.05 <i>versus</i> EGFP-V plus pGL3-B-miR-101-M transfections. *, <i>P</i><0.05 between pGL3-B-miR-101-M and pGL3-B-miR-101-W transfections. The protein levels of AR, LC3 and p62 were detected by Western blotting using GAPDH as a loading control.</p

miR-101 expression in prostate cancer cells.

<p><b>A</b>, Expressions of pri-miR-101 and mature miR-101 were determined in AR positive or negative cell lines by qPCR. **, <i>P</i><0.01 between two compared groups. <b>B</b>, LNCaP or 22Rv1 cells were transfected with AR siRNA or control siRNA (ctrl siRNA). AR knockdown effects were verified by Western blotting. Expressions of pri-miR-101 and mature miR-101 were determined by qPCR (<b>C).</b> *, <i>P</i><0.05 <i>versus</i> control siRNA. <b>D</b>, DU145 or PC-3 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) and treated with DMSO or celastrol (CEL, 2 μM) for 24 h. Expressions of mature miR-101 were determined by qPCR. *, <i>P</i><0.05 between EGFP-AR and EGFP-V transfections. <b>E</b>, LNCaP cells were treated with R1881 (1 nM) for 24 h after androgen starvation, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140745#pone.0140745.g002" target="_blank">Fig 2</a><b>C</b>. AR protein levels were determined by Western blotting using GAPDH as a loading control. MiR-101 expressions were determined by qPCR. **, p<0.01 <i>versus</i> DMSO.</p

AR suppression on celastrol induced autophagy.

<p><b>A</b>, LNCaP cells were treated with celastrol at 2.0 μM for indicated times, the protein level of AR was revealed by Western blotting using GAPDH as a loading control. <b>B</b>, LNCaP cells were transfected with AR siRNA or control siRNA for 24 h, the effects on AR knockdown and autophagy induction were verified by Western blotting using AR, LC3, p62 and GAPDH (loading control) antibodies. <b>C</b>, LNCaP cells were subjected to androgen starvation as described in "<b>Materials and Methods</b>", followed by 1 nM of R1881 treatment for 24 h. AR and its target PSA, as well as autophagic makers LC3 and p62 were detected by Western blotting using GAPDH as a loading control. LNCaP (<b>D</b>) or DU145 (<b>E</b>) cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V). <b>D</b>, After transfection, LNCaP cells were cultured in the medium containing R1881 (1 nM) and treated with or without celastrol (CEL) at 2.0 μM for 24 h (<b>D</b>). <b>E</b>, DU145 stably transfected cells were pretreated with R1881 (1 nM) for 24 h before celastrol treatment as <b>D</b>. LC3 was detected by Western blotting using GAPDH as a loading control.</p

AR modulates miR-101 levels without affecting cell death.

<p><b>A</b> and <b>B</b>, LNCaP cells were treated with MDV3100 at 5 μM for indicated times. Protein extracts were immunoblotted with antibodies against PARP, AR, LC3, p62 and GAPDH (loading control) (<b>A</b>). Mature miR-101 levels were determined by qPCR (<b>B</b>). <b>C</b> and <b>D</b>, LNCaP cells were subjected to androgen starvation as described in "<b>Materials and Methods</b>", followed by 1 nM of R1881 treatment for indicated times. PARP, AR, LC3, p62 and GAPDH (loading control) were detected by Western blotting (<b>C</b>). Mature miR-101 levels were determined by qPCR (<b>D</b>). Asterisks denote significance compared with control (0 h). *, <i>P</i> <0.05.</p

Population characteristics of Parkinson Cases and controls.

#<p>Chi-Square tests for categorical variables, t-tests for continuous variables *number of participants that provided data.</p

Effect of Everolimus on Treg Generation in MLR (n = 7).

<p><b><i>A</i>:</b> Parallel to the experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156535#pone.0156535.g001" target="_blank">Fig 1</a>, 5x10⁵ CFSE-labeled PBMC responders (*A) were stimulated with 5x10⁵ PKH-26 labeled irradiated stimulators from HLA-2DR-matched (Bx) or completely HLA-mismatched (Ix) volunteers in the presence of indicated doses of Everolimus. Five color flow cytometric analysis was performed on days 0, 5, 7 and 9 (Day 0 = baseline; data from day 7 as the peak are shown here). Viable lymphocytes were gated followed by gating for CFSE bright and dim cells which were negative for both CD127-PE and PKH26 (thus gating out CD127⁺ responders and any residual stimulators). This was followed by gating for CD4⁺ cells. CFSE dilution in these CD4⁺ responders assessed the extent of proliferation, i.e., non-proliferating (CFSE high) or proliferating (CFSE low) cells. Then the percentage of CD4⁺CD127^-CD25⁺FOXP3⁺ total Tregs or CD4⁺CD127^-CD25^HIGHFOXP3⁺ natural Tregs (nTregs) were estimated in the non-proliferating and proliferating responders. Shown here is a representative experiment to demonstrate the analysis scheme. <b>B:</b> On the left, the percentage of total Tregs (top) and nTregs (bottom) are depicted. On the right, the raw percentages for the Tregs obtained in each experiment are converted to percentage of medium controls (in which no Everolimus was added) to minimize the variations. Note that Everolimus enhanced the generation of both total Treg and nTregs in the proliferating fraction depicted here, without discernible effect on the non-proliferating fraction (not shown). This was more definitively seen in the clinically applicable drug concentrations tested (p<0.05).</p

Effect of Everolimus plus Tacrolimus on allogeneic Lymphoproliferation and the generation of FOXP3⁺ Tregs in the Treg-MLR.

<p><b>A.</b> Lymphoproliferation assays (left) and CFSE Treg-MLRs (right) as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156535#pone.0156535.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156535#pone.0156535.g002" target="_blank">2</a> respectively were performed, but in the presence of indicated concentrations of TAC (instead of EVL). TAC inhibited the proliferative responses and the generation of new Tregs in the responding cells (unlike other agents) in a dose dependent manner (n = 7). <b>B.</b> EVL at indicated concentrations without or with 1 and 5 ng/ml TAC were tested in standard 7-day ³H-TdR assays lymphoproliferation assays (left) and in the CFSE Treg-MLRs (right). As in other experiments, the data were calculated as percentage of medium control (i.e. 100%) (n = 4). (Left): Note that both EVL and TAC inhibited the MLRs in a dose dependent manner and that their combinations had an additive inhibitory effect on lymphoproliferation. Statistical significance was observed below the horizontal dashed line at 67% (p<0.05). (Right): TAC inhibited the generation of new Tregs (comparing the 3 lines at 0 EVL) confirming results in Fig 4A, and unlike by EVL (solid line). When combinations of EVL + TAC were tested, EVL failed to abrogate the inhibition mediated by TAC.</p

Effect of Everolimus on Lymphoproliferation in MLR (n = 7).

<p>1x10⁵ responding PBMC from healthy volunteer A were cultured with 1x10⁵ irradiated stimulator cells from an HLA-2DR-matched volunteer (Bx) or HLA-mismatched laboratory third party indifferent volunteer (Ix), in the presence of the indicated concentrations of Everolimus. Standard ³H-TdR assays were performed on day 7 and the data were calculated as percentage of medium control (i.e. 100%). The dotted line indicates the level below which statistical significance at p≤0.05 was obtained. Note that Everolimus inhibited the MLRs in a dose dependent manner and that the degree of inhibition was equivalent against both DR-identical and HLA-mismatched stimulators.</p