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

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

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 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

Autophagy potentiates the effects of celastrol on inhibition of cell proliferation and induction of apoptosis.

<p>Cell viability was measured by MTT assay after treatment with celastrol. <b>A</b>, LNCaP cells were transfected with negative control (NC) or miR-101 mimic at indicated concentrations for 24 h, followed by additional 96 h treatment with DMSO or celastrol at 1.5 μM. **, <i>P</i><0.01 <i>versus</i> NC with celastrol treatment. <b>B</b>, LNCaP cells were treated with celastrol (1.5 μM) in the presence of miR-101 mimic or negative control (NC) for indicated time points. **, <i>P</i><0.01 <i>versus</i> NC. <b>C</b>, LNCaP cells were treated with celastrol (CEL, 1.5 μM) or DMSO in the presence or absence of bafilomycin A1 (BAF, 10 nM) for 24 h. #, <i>P</i> <0.05 <i>versus</i> CEL, **, <i>P</i><0.01 <i>versus</i> other two groups. <b>D</b>, LNCaP cells were treated with celastrol (CEL, 2.0 μM) in the presence or absence of bafilomycin A1 (BAF, 10 nM) or Z-VAD (20 μM) for up to 72 h. Protein extracts were immunoblotted with antibodies against PARP, LC3, p62 and GAPDH (loading control). <b>E</b>, Colony formation assay. LNCaP cells were treated with DMSO or celastrol (CEL, 1.5 μM) in the presence of miR-101 mimic or negative control (NC) or bafilomycin A1 (BAF, 10 nM) for 24 h. Colony formation was performed as described in the <b>Materials and Methods</b>. Colonies with over 50 cells were counted (<b>E</b>) and quantified in <b>F.</b> **, <i>P</i><0.01 <i>versus</i> NC with celastrol treatment.</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

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

Western blot and morphological analysis (concentration-dependent study).

<p><b>A</b>, Western blot analysis of breast cancer MDA-MB-231 cell extracts. Cells were treated with the complexes AuD6 and AuD8 at the indicated concentrations for 24 h. <b>B</b>, Western blot analysis of the p27 protein amount in MDA-MB-231 cell extract after treatment with the compound AuD8 at the indicated concentrations for 24 h. The solvent DMSO was used as a control while GAPDH as a loading control. <b>C</b>, Apoptotic morphological changes of MDA-MB-231 cells after treatment with AuD6 and AuD8 at the indicated concentrations for 24 h (phase contrast imaging, 100× magnification).</p

<i>In vitro</i> inhibition of proteasome.

<p>IC₅₀ values (µM±SD) obtained for the three proteasomal activities on the purified 20S proteasome and on MDA-MB-231 cell extract after 2 h incubation.</p

Table_1_Chemoresistance Transmission via Exosome-Transferred MMP14 in Pancreatic Cancer.docx

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies. Gemcitabine is the most commonly used chemotherapy for the treatment of PDAC, but the development of drug resistance still remains challenging. Recently, exosomes have emerged as important mediators for intercellular communication. Exosomes affect recipient cells’ behavior through the engulfed cargos, however the specific cargos responsible for gemcitabine resistance in PDAC are poorly understood. Here, we reported that exosomes could transfer gemcitabine resistance via a metalloproteinase 14 (MMP14)-dependent mechanism. MMP14 was identified as a major differentially secreted protein from the gemcitabine-resistant PDAC cells by comparative secretome. It was packaged into the exosomes and transmitted from the chemoresistant cells to the sensitive ones. The exosome-transferred MMP14 could enhance drug resistance and promotes the sphere-formation and migration abilities of the recipient sensitive PDAC cells. Mechanically, exosome-transferred MMP14 promotes the stability of CD44, the cancer stem cell marker in the recipient cells. Our results indicate that MMP14 is a key player for exosome-mediated transfer of gemcitabine resistance, thus targeting MMP14 in exosomes may represent a novel strategy to limit gemcitabine resistance in PDAC.</p