Journal: Neural Regeneration Research
Article Title: Reduced mesencephalic astrocyte–derived neurotrophic factor expression by mutant androgen receptor contributes to neurodegeneration in a model of spinal and bulbar muscular atrophy pathology
doi: 10.4103/NRR.NRR-D-23-01666
Figure Lengend Snippet: Mutant AR with polyQ expansion reduces MANF expression in N2a cells. (A) A schematic of AR0Q and AR48Q plasmids. AR containing 0Q and 48Q were linked with an N-terminal EGFP tag. Created with Microsoft PowerPoint (2312 Build 16.0.17126.20132). (B) Western blotting analysis showed that the AR0Q and AR48Q plasmids expressed the corresponding proteins in the transfected N2a cells. EGFP and AR antibodies were used to detect both AR0Q and AR48Q. 1C2 antibody preferentially reacts with AR48Q. β-tubulin was used as a loading control. (C) Subcellular fractionation was performed using N2a cells transfected with AR0Q and treated with different concentrations (0, 100 nM, 10 nM, or 1 nM) of R1881. Western blotting analysis showed that without R1881, AR0Q was predominantly localized in the cytoplasm, whereas with R1881, AR0Q was predominantly localized in the nucleus. AR antibody was used to detect AR0Q. Lamin B1 was used as a marker for the nucleus (N), and GAPDH was used as a marker for the cytoplasm (C). (D) Subcellular fractionation was performed using N2a cells transfected with AR0Q or AR48Q and treated with or without R1881. Western blotting analysis showed that in the presence of R1881, both AR0Q and AR48Q translocated into the nucleus. AR antibody was used to detect both AR0Q and AR48Q. Lamin B1 was used as a marker for the nucleus (N), and GAPDH was used as a marker for the cytoplasm (C). (E) Fluorescent microscopy images showing the localization of AR0Q and AR48Q in N2a cells treated with or without R1881. Without R1881, AR0Q and AR48Q were present in the cytoplasm and nucleus, whereas with R1881, AR0Q and AR48Q were predominantly localized in the nucleus. DAPI was used to stain nuclei. Scale bars: 20 μm. (F) Western blotting analysis of MANF expression in N2a cells transfected with AR0Q or AR48Q and treated with R1881. AR antibody was used to detect both AR0Q and AR48Q, and MANF antibody was used to detect MANF. β-Actin was used as a loading control. (G) Quantitative analysis of MANF expression ( n = 7, one-way analysis of variance with Tukey’s post hoc test). MANF expression was significantly lower in AR48Q-expressing cells compared with AR0Q-expressing cells. (H) Real-time polymerase chain reaction was performed to measure Manf mRNA levels in N2a cells transfected with AR0Q or AR48Q and treated with R1881 ( n = 4, one-way analysis of variance with Tukey’s post hoc test). The Manf mRNA level was significantly lower in AR48Q-expressing cells than in AR0Q-expressing cells. (I) MTT assay was performed using N2a cells transfected with AR0Q or AR48Q and treated with R1881 ( n = 4, one-way analysis of variance with Tukey’s post hoc test). The viability of AR48Q-expressing cells was significantly lower than that in AR0Q-expressing cells. * P < 0.05, *** P < 0.001, **** P < 0.0001. Data are presented as mean ± SEM. AR: Androgen receptor; DAPI: 4′,6-diamidino-2-phenylindole; EGFP: enhanced green fluorescent protein; MANF: mesencephalic astrocyte-derived neurotrophic factor; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; polyQ: polyglutamine.
Article Snippet: The AR containing no polyQ (AR0Q) and AR containing 48 polyQ repeats (AR48Q) plasmids were purchased from Addgene (Watertown, MA, USA, 86427 and 86428).
Techniques: Mutagenesis, Expressing, Western Blot, Transfection, Control, Fractionation, Marker, Microscopy, Staining, Real-time Polymerase Chain Reaction, MTT Assay, Derivative Assay
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![(A) Western blots (WB) of indicated proteins in LNCaP cells treated with increasing concentration R1881 under ADT (CSS) for 72 h. (B) Schematic representation of the experimental design for AR regulation of BCL2 expression. (C) WB of LNCaP cells either treated with 1 nM R1881 alone or in combination with increasing concentration (0.03–10 μM) of ENZA normalized to DMSO for 48 h. (D) WB of LAPC4 cells either treated with 1 nM R1881 alone or in combination with indicated concentration of ENZA normalized to DMSO for 48 h. (E) WB of LNCaP cells either treated with 1 nM R1881 alone or in combination with indicated concentration of apalutamide or darolutamide normalized to DMSO for 48 h. (F) WB of LNCaP cells either transfected with control siRNA or AR siRNA for 72 h. Post-transfected cells are cultures either in complete medium or under ADT (CSS) alone for 2 days or CSS supplemented with 1 nM R1881 for (first day in CSS, second day in CSS + R1881). (G) qPCR analysis of 2-month-old normal mouse prostate and castrated mouse prostate (15 days post castration) for bcl2 and bclxl mRNA expression. Error bar, SD. (H) qPCR analysis of indicated genes in the LNCaP cells transiently transfected (overexpression [OE]) for 72 h with full-length AR or <t>AR-V7</t> or GFP (control). Post-transfected cells were cultured in complete medium. Error bar, SD. (I) WB of LNCaP cells treated with indicated concentration of PU-h71 normalized to DMSO for 48 h. (J) WB of LNCaP-Abl cells treated with 1 nM R1881 alone or in combination with 0.10 μM ENZA normalized to DMSO for 48 h. (K) Schematic model of BCL2 expression regulation in CSPC vs. CRPC. WBs and qPCRs are representatives of n = 3 technical replicates.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6453/pmc12316453/pmc12316453__nihms-2092657-f0003.jpg)
