Journal: Nature Communications

Article Title: Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

doi: 10.1038/s41467-019-14184-0

Figure Lengend Snippet: 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.

Article Snippet: The pcDNA3.1(+) SOX2 was purchased from GenScript, pcDNA3.1(+)-SPINK1-2xV5 was synthesized from GeneArt and pcDNA3.1(+) empty vector was kindly gifted by Dr. Arun K. Shukla.

Techniques: Expressing, Western Blot, Immunostaining, Cell Culture, Microarray

Journal: Nature Communications

Article Title: Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

doi: 10.1038/s41467-019-14184-0

Figure Lengend Snippet: a Schematic showing SOX2 binding elements ( S1 , S2, and S3 ) on the SPINK1 promoter (top). ChIP-qPCR data for SOX2 occupancy on the SPINK1 promoter in wildtype LNCaP and LNCaP-AI cells (androgen-deprived for 15 days) (bottom). b Same as in a , except 22RV1 cells. c ChIP-qPCR data for RNA Pol-II binding on the SPINK1 promoter using cells as a . d Same as c , except 22RV1 cells. e Immunoblot for SOX2 and SPINK1 in siRNA mediated SOX2- silenced LNCaP-AI and control cells. f Same as e , except 22RV1 cells. g QPCR data showing relative expression of SOX2 and SPINK1 upon transient SOX2 overexpression in LNCaP cells (left). Immunoblot for SOX2 and SPINK1 expression(right). h Luciferase reporter activity of the SPINK1 distal-promoter (SPINK1-DP) using same cells as g . i REST binding motif obtained from JASPAR (top). Genomic location for AR and REST binding on the SPINK1 promoter (bottom). j ChIP-qPCR data showing AR and REST occupancy on the SPINK1 promoter in R1881 stimulated (10 nM) LNCaP cells. k Immunoblot for the REST and SPINK1 levels in 22RV1 cells treated with Casein Kinase 1 inhibitor (iCK1) as indicated (top). QPCR data for relative SPINK1 and SYP expression (bottom). l Cell proliferation assay using 22RV1 cells treated with different concentrations of iCK1. m Foci formation assay using 22RV1 cells treated with iCK1 (20 µM). Inset showing representative images depicting foci (scale bar: 500 µm). n Representative phase contrast microscopic images of 3D tumor spheroids using same cells as m (left). Bar plots depict mean area and efficiency of the sphere formation. Scale bar represents 1000μm. Experiments were performed with n = 3 biologically independent samples; data represents mean ± SEM. For panels a – d , h , j , m – n two-tailed unpaired Student’s t -test; g two-way ANOVA, Sidak’s multiple-comparisons test; k two-way ANOVA, Dunnett’s multiple-comparisons test were applied. ∗ P ≤ 0.05 and ∗∗ P ≤ 0.001. Source data for e , f , g , k are provided as a Source Data file.

Article Snippet: The pcDNA3.1(+) SOX2 was purchased from GenScript, pcDNA3.1(+)-SPINK1-2xV5 was synthesized from GeneArt and pcDNA3.1(+) empty vector was kindly gifted by Dr. Arun K. Shukla.

Techniques: Binding Assay, ChIP-qPCR, Western Blot, Control, Expressing, Over Expression, Luciferase, Activity Assay, Proliferation Assay, Tube Formation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

doi: 10.1038/s41467-019-14184-0

Figure Lengend Snippet: Schematic showing androgen deprivation therapy (ADT) induced progression of prostate adenocarcinoma (ADPC) to neuroendocrine prostate cancer (NEPC) (top panel). ADT using AR-antagonists relieve AR-REST mediated transcriptional repression of SPINK1 , and subsequently, SOX2 gets recruited on the SPINK1 promoter resulting in its upregulation (left box). In addition, Casein kinase 1 inhibitor (iCK1) restores REST levels causing downregulation of SPINK1, leading to reduced stemness and cellular plasticity (right box).

Article Snippet: The pcDNA3.1(+) SOX2 was purchased from GenScript, pcDNA3.1(+)-SPINK1-2xV5 was synthesized from GeneArt and pcDNA3.1(+) empty vector was kindly gifted by Dr. Arun K. Shukla.

Techniques:

Journal: Molecular Oncology

Article Title: Isoform‐specific effects of transcription factor TCFL5 on the pluripotency‐related genes SOX2 and KLF4 in colorectal cancer development

doi: 10.1002/1878-0261.13085

Figure Lengend Snippet: CHA and TCFL5_E8 directly control SOX2 and KLF4 promoters. (A) CHA and TCFL5_E8 binding to the SOX2 promoter. Above, scheme of SOX2 promoter and region studied by chromatin immunoprecipitation (ChIP). Below, CHA (red bars) and TCFL5_E8 (blue bars) binding to the control, SRR1 and CORE regions of SOX2 promoter determined by qPCR analysis of immunoprecipitated DNA. (B) CHA and TCFL5_E8 binding to the KLF4 promoter. Above, scheme of KLF4 promoter and region studied by ChIP. Below, CHA (red bars) and TCFL5_E8 (blue bars) binding to the negative and positive regions of the KLF4 promoter determined by qPCR analysis of immunoprecipitated DNA. (C) Luciferase activity of CORE region of SOX2 promoter in WT, KO23 and KO34 HCT116 cell lines with control (grey bars), CHA (red bars), TCFL5_E8 (blue bars) or pEP4 (white bars) transient expression. (D) Luciferase activity of SRR1 region of SOX2 promoter in WT, KO23 and KO34 HCT116 cell lines with control (grey bars), CHA (red bars), TCFL5_E8 (blue bars) or pEP4 (white bars) transient expression. (E) KLF4 activity by KLF4‐dependent reporter luciferase assay in WT, KO23 and KO34 HCT116 cell lines with control (grey bars), CHA (red bars), TCFL5_E8 (blue bars) or pEP4 (white grey bars) transient expression. (F) KLF4 activity determined by KLF4‐dependent reporter luciferase assay in HCT116 cells transfected with EV, CHA, TCFL5_E8, and an equal amount of CHA and TCFL5_E8. (G) Luciferase activity of 4× E‐box minimal promoter in HCT116 cells transfected with EV, CHA, TCFL5_E8, and different amounts of CHA and TCFL5_E8. Results are expressed as means ± SEM of three independent experiments ( t ‐test; * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Cells were grown in 24‐well plates and transfected with pCDNA3‐CHA‐Flag, pCDNA3‐TCFL5‐Flag or pEP4 E02S CK2M EN2L, expression vector carrying the Oct4 and Sox2; Klf4 and Myc; Nanog and Lin28 genes (a gift from James Thomson, Addgene Plasmid#20924) plasmids in combination with pGL4‐SOX2‐CORE, pGL4‐SOX2‐SRR1, pGL4‐KLF4‐RE, pGL4‐4x‐E‐box reporter plasmids and SV40‐Renilla control plasmid using Metafectene Pro (Biontex) according to manufacturer’s instructions.

Techniques: Control, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Luciferase, Activity Assay, Expressing, Transfection