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. 2020 Jan 20;11:384. doi: 10.1038/s41467-019-14184-0

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Bar graph showing SPINK1 expression (top) and heatmap of AR-signaling associated genes including SPINK1 in long-term androgen deprived (AD) LNCaP cells (GSE8702). b Representative phase-contrast images of androgen-deprived LNCaP cells (LNCaP-AI). Red arrow-heads indicate neurite outgrowth. c QPCR data showing relative expression of SPINK1 and KLK3 using same cells as b. d Immunoblot assay for SPINK1, PSA, SYP, ENO2, SOX2 and REST using same cells as in b. e Immunostaining for SPINK1 using same cells as in b. f Schematic representation of NE-transdifferentiation (NE-td) using doxycycline (dox)-inducible AR-V7 overexpressing LNCaP cells subjected to androgen deprivation (AD) with or without induction (40 ng/ml) at day 10 and cultured upto 30 days. g QPCR data showing relative expression of SPINK1 and KLK3 using same cells as f. h Immunoblot assay for AR, AR-V7, SPINK1, and PSA using same cells as f. i QPCR data showing relative expression of SYP, NCAM1, ENO2 and CHGA using the same cells as f. j Schema describing generation of LNCaP-AI-shSPINK1 and LNCaP-AI-shSCRM cells by subjecting stable LNCaP-shSPINK1 and LNCaP-shSCRM cells to androgen deprivation (AD) for 30 days. k Representative images for the neurite outgrowths in LNCaP-AI-shSCRM cells and LNCaP-AI-shSPINK1 as j (top). QPCR data showing relative expression of SPINK1 (bottom, left) and measurement of neurite outgrowth (bottom, right). l Immunoblot analysis for SPINK1, E-Cad, VIM, and N-Cad expression using same cells as j. m Same as in l, except phospho (p) and total (t) AKT, SYP and ENO2 expression. n Representative IHC images for SPINK1 in SPINK1-negative (SPINK1−, top) and SPINK1-positive (SPINK1+, bottom) PCa tumor cores of the VPC tissue microarray (scale bar = 200 µm). Bar plot showing percentage cases of SPINK1 in untreated (n = 33) and neoadjuvant-hormone therapy (NHT) treated patients (n = 55). Experiments were performed with n = 3 biologically independent samples; data represents mean ± SEM. For panels b, e, k scale bar represents 20 µm. For panel c two-way ANOVA, Dunnett’s multiple-comparisons; g, i two-way ANOVA, Sidak’s multiple-comparisons test; k one-way ANOVA, Dunnett’s multiple-comparisons test were applied. ^∗P ≤ 0.05 and ^∗∗P ≤ 0.001. Source data for d, h, l, m are provided as a Source Data file.