Journal: Scientific reports

Article Title: Adiponectin deficiency contributes to the development and progression of benign prostatic hyperplasia in obesity.

doi: 10.1038/srep43771

Figure Lengend Snippet: Figure 1. Expression of adiponectin receptors in prostatic tissues and cells. (a) IHC staining for AdipoR1 and AdipoR2 on human postoperative BPH tissue samples. Scale bar, 100 μm or 200 μm. (b) Semi-quantitation based on the average of optical density (AOD) measured by Image J software (n = 5, Student’s t-test, **p < 0.01). (c) WB analysis of adiponectin receptors (AdipoR1 and AdipoR2) expression on RWPE1 and WPMY1 cells. Positive controls were skeletal muscle tissue extract for AdipoR1 and liver extract for AdipoR2. The results were adjusted by GAPDH expression and quantitated as described in the Methods section (n = 3, Student’s t-test, **p < 0.01). Full-length bolts are presented in Supplementary Fig. S3. (d) mRNA expression levels of AdipoR1 and AdipoR2 in RWPE1 and WPMY1 cells were determined by RT-PCR. The results were adjusted by β-actin and quantitated by the 2−ΔΔCt method (n = 3, Student’s t-test, **p < 0.01). (e) Immunofluorescence staining (green) for AdipoR1 and AdipoR2 in RWPE1 and WPMY1 cells, DAPI staining (blue) for nucleus. Magnification, ×400.

Article Snippet: Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R&D System.

Techniques: Expressing, Immunohistochemistry, Quantitation Assay, Software, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: Scientific reports

Article Title: Adiponectin deficiency contributes to the development and progression of benign prostatic hyperplasia in obesity.

doi: 10.1038/srep43771

Figure Lengend Snippet: Figure 2. Adiponectin inhibits growth factor-mediated proliferation of prostatic epithelial and stromal cells. (a,b) CCK-8 proliferation analysis of RWPE1 and WPMY1 cells cultured in regular growth medium, with 0, 1, 5, 10 or 20 μg/ml human recombinant adiponectin treatment. QD450 values were converted to cell numbers according to the standard curve. (one-way analysis of variance followed by Dunnett’s post-tests; n = 5; *p < 0.05 versus control, NS, not significant). (c,d) Cells were cultured with 10 ng/ml EGF or 10% FBS and treated with 10 μg/ml APN or the same volume of PBS. CCK-8 proliferation analysis was performed after 24 h of incubation. The results were expressed as the mean ± s.d. of three independent experiments (one-way analysis of variance followed by Bonferroni post-tests; ***p < 0.001, **p < 0.01, NS, not significant). (e,f) Cells were treated with 10 μg/ml adiponectin for 12 h or the same volume of PBS as a control after starvation. Then, constituents of the cell cycle were determined by flow cytometry (Student’s t-tests; n = 3; ***p < 0.001, **p < 0.01, *pp < 0.05, NS, not significant).

Article Snippet: Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R&D System.

Techniques: CCK-8 Assay, Cell Culture, Recombinant, Control, Incubation, Flow Cytometry

Journal: Scientific reports

Article Title: Adiponectin deficiency contributes to the development and progression of benign prostatic hyperplasia in obesity.

doi: 10.1038/srep43771

Figure Lengend Snippet: Figure 3. Adiponectin arrests prostatic cells in G0/G1 phase. (a) RWPE1 and WPMY1 cells were treated with the indicated conditions. Then, the cell cycle distribution was analysed with ModFit LT software (V4 1.7, ME). The results are expressed as the mean ± s.d. of three independent experiments (one-way analysis of variance followed by Bonferroni post-tests; ###p < 0.001, ##p < 0.01, ns, not significant, shAdipoR1 versus scramble; ***p < 0.001, **p < 0.01, *p < 0.05, NS, not significant). (b) Cellular extracts were analysed for the expression of cyclinD1 and PCNA after the indicated treatment. The experiment was performed twice with similar results. Full-length bolts are presented in Supplementary Fig. S3. Illustration: shAdipoR1, knockdown of AdipoR1; Scramble, control for shAdipoR1; Mock, untransfected cells; APN, treatment with 10 μg/ml adiponectin for 12 h; U0126, 10 μM pretreated for 2 h; EGF or FBS: supplement of 10 ng/ml EGF for RWPE1 or 10% FBS for WPMY1.

Article Snippet: Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R&D System.

Techniques: Software, Expressing, Knockdown, Control

Journal: Scientific reports

Article Title: Adiponectin deficiency contributes to the development and progression of benign prostatic hyperplasia in obesity.

doi: 10.1038/srep43771

Figure Lengend Snippet: Figure 4. Adiponectin promotes apoptosis of prostatic epithelial and stromal cells by regulating the activation of caspase3. (a,b) Caspase3 activity assay of RWPE1 and WPMY1 cultured in regular growth medium (control), with 0, 5, 10 or 20 μg/ml human recombinant adiponectin treatment for 12 h, the lentivirus-transfected cells (RWPE1-scramble, RWPE1-shAdipoR1, WPMY1-scramble and WPMY1-shAdipoR1) were treated with 10 μg/ml adiponectin for 12 h. Caspase3 activity is the active unit of caspase3 per unit weight (U/g). The results are presented as the means ± s.d. of three independent experiments (One-way analysis of variance followed by Bonferroni post-tests; ###p < 0.001 versus control, ***p < 0.001, NS, not significant). (c,d) Transfected cells were treated with the indicated conditions before the caspase 3 activity analysis. The results are presented as the means ± s.d. of three independent experiments (one-way analysis of variance followed by Bonferroni post-tests; ***p < 0.001, **p < 0.01, *p < 0.05, NS, not significant). (e) Cellular extracts were analysed for the expression of caspase 3 and cleaved-caspase 3 after the indicated treatment by immunoblotting. The experiments were performed twice with similar results. Full-length bolts are presented in Supplementary Fig. S3.

Article Snippet: Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R&D System.

Techniques: Activation Assay, Caspase-3 Activity Assay, Cell Culture, Control, Recombinant, Transfection, Activity Assay, Expressing, Western Blot

Journal: Scientific reports

Article Title: Adiponectin deficiency contributes to the development and progression of benign prostatic hyperplasia in obesity.

doi: 10.1038/srep43771

Figure Lengend Snippet: Figure 5. Adiponectin negatively regulates the MEK-ERK-p90RSK axis. (a) RWPE1 cells were cultured in growth medium and treated with or without 10 μg/ml human recombinant adiponectin for the indicated times, then the cellular extracts were analysed by immunoblotting for expression of the indicated proteins. (b) Cells were starved for 24 h and incubated for 2, 4 or 6 min with supplement of 10 ng/ml EGF for RWPE1 or 10% FBS for WPMY1. NC, negative control treated with PBS. Then, immunoblotting was performed to determine the expression of phospho-ERK1/2. (c) RWPE1 and WPMY1 cells were treated with the indicated conditions for 2 h, followed by immunoblotting for the indicated proteins. All immunoblotting experiments were performed twice with similar results. Full-length bolts are presented in Supplementary Fig. S3. Illustration: shAdipoR1, knockdown of AdipoR1; Scramble, control for shAdipoR1; Mock, untransfected cells; APN, treatment with 10 μg/ml adiponectin for 2 h; U0126, treatment with 10 μM U0126 for 2 h; EGF or FBS: supplement of 10 ng/ml EGF for RWPE1 or 10% FBS for WPMY1.

Article Snippet: Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R&D System.

Techniques: Cell Culture, Recombinant, Western Blot, Expressing, Incubation, Negative Control, Knockdown, Control

Journal: Scientific reports

Article Title: Adiponectin deficiency contributes to the development and progression of benign prostatic hyperplasia in obesity.

doi: 10.1038/srep43771

Figure Lengend Snippet: Figure 6. HFD resulted in obesity and metabolic disorders in mice. C57BL/6 mice were fed a low-fat control diet (CD) or a high-fat diet (HFD) for 14 weeks with or without mouse recombinant adiponectin treatment (APN) as described in the Methods section. (a) Weight was monitored for the indicated time points. (b) Photograph of peri-testis adipose tissues. (c) H&E staining for mouse adipose tissues ( ×100 magnification). (d) Oil Red O staining for mouse liver tissues to determine the lipid levels in hepatocytes (×200 magnification). (e) Abdominal adipose was collected and weighed at the end of 14 weeks. The adiposity index was calculated as the total adipose tissue weight (g) divided by the body weight (g). (One-way analysis of variance followed by Bonferroni post-tests, n = 5, ***p < 0.001, **p < 0.01). (f) Monitoring results of blood glucose. (g) Serum adiponectin levels were measured by an ELISA (Student’s t-test, n = 5, ***p < 0.001). (h) Blood lipid levels were determined by ADVIA 2400 Biochemistry Analyzer (Siemens). The results are shown as the mean ± s.d. (Kruskal-Wallis nonparametric test, n = 5, **p < 0.01, *p < 0.05). Abbreviations: TC, total cholesterol; TG, triglyceride; HDL, high density lipoprotein; LDL, low density lipoprotein; FFA, free fatty acid.

Article Snippet: Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R&D System.

Techniques: Control, Recombinant, Staining, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: Adiponectin deficiency contributes to the development and progression of benign prostatic hyperplasia in obesity.

doi: 10.1038/srep43771

Figure Lengend Snippet: Figure 7. Adiponectin supplement protects prostate from microscopic BPH induced by HFD. C57BL/6 mice were fed a low-fat control diet (CD) or a high-fat diet (HFD) for 14 weeks with or without mouse recombinant adiponectin treatment (APN) as described in the Methods section. (a) Histopathological analysis with H&E staining showing characteristics of prostatic glands and stroma (×200 and ×400 magnification). (b,d,f,h) TUNEL assay and immunohistochemical staining with antibodies against PCNA, p-P90RSK and AdipoR1 ( ×200 and ×400 magnification). (c,e,g,i) The results were presented as the average percentage of positive cells over the total counted cells. Expression of AdipoR1 was presented as the average of optical density (AOD) (Kruskal-Wallis nonparametric test, n = 5, ***p < 0.001, **p < 0.01, *p < 0.05).

Article Snippet: Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R&D System.

Techniques: Control, Recombinant, Staining, TUNEL Assay, Immunohistochemical staining, Expressing

Journal: Scientific reports

Article Title: Adiponectin deficiency contributes to the development and progression of benign prostatic hyperplasia in obesity.

doi: 10.1038/srep43771

Figure Lengend Snippet: Figure 8. A model for adiponectin deficiency-induced facilitation of the MEK-ERK-p90RSK axis and aggravation of cell growth imbalance. Abbreviations: AdipoR1, adiponectin receptor 1; GPCR, G-protein- coupled receptors; RTK, receptor tyrosine kinase.

Article Snippet: Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R&D System.

Techniques:

Journal: Nucleic Acids Research

Article Title: DeepCLIP: predicting the effect of mutations on protein–RNA binding with deep learning

doi: 10.1093/nar/gkaa530

Figure Lengend Snippet: DeepCLIP predicts increased TDP-43 binding as mechanism behind ACADM exon 6 skipping. ( A ) DeepCLIP TDP-43 profile across the 5′ss of ACADM exon 6 with wt indicated in black and patient mutation indicated in red. Along the first axis the sequence is shown and along the second axis the DeepCLIP BLSTM values are shown. SPRi oligo location and SSO locations are indicated in blue and red bars above and below the sequence, respectively. ( B ) Splicing of wt and mutant minigenes with either TDP-43 targeting siRNA or non-targeting siRNA determined by RT-PCR. ( C ) Western blot of TDP-43 and HPRT from siRNA and minigene transfected samples. ( D ) Splicing of wt and mutant minigenes treated with either a control SSO (Ctrl-SSO), SSO1, or SSO2 determined by RT-PCR. ( E ) DeepCLIP profile of short RNA oligos used in SPRi measurement, reference in black and +7A>G variant in red. ( F ) The difference in DeepCLIP binding profiles in (E) between reference and variant. Positive score indicates higher score in variant. ( G ) SPRi measurements of TDP-43 binding to the wt oligo in (E). ( H ) SPRi measurements of TDP-43 binding to the variant oligo in (E). In both (G) and (H), the black line indicates the fitted binding model.

Article Snippet: Surface plasmon resonance imaging (SPRi) by IBIS MX-96 was used to measure the kinetics of recombinant hnRNP A1 (ab224866, Abcam), SRSF1 (GenScript, Piscataway, NJ, USA) and TDP-43 (R&Dsystems, AP-190) binding to the immobilized RNA oligonucleotides.

Techniques: Binding Assay, Mutagenesis, Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Variant Assay

Journal: Nucleic Acids Research

Article Title: DeepCLIP: predicting the effect of mutations on protein–RNA binding with deep learning

doi: 10.1093/nar/gkaa530

Figure Lengend Snippet: DeepCLIP analysis of TDP-43 repressed pseudoexons indicate position-dependent tissue-specificity. ( A ) The average DeepCLIP TDP-43 profile scores of 58 neuron-specific and 79 muscle-specific pseudoexons activated in TDP-43-null mice in the areas covering the 25 first and last nucleotides of the pseudoexon, and the 50 nt spanning intronic regions. 95%-confidence intervals are indicated by shaded areas.

Article Snippet: Surface plasmon resonance imaging (SPRi) by IBIS MX-96 was used to measure the kinetics of recombinant hnRNP A1 (ab224866, Abcam), SRSF1 (GenScript, Piscataway, NJ, USA) and TDP-43 (R&Dsystems, AP-190) binding to the immobilized RNA oligonucleotides.

Techniques: