Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) In Kaplan-Meier analysis, patients with tumors expressing high levels of AR (N = 39) had poor recurrence-free survival ( p = 0.0006 ). (B) In Kaplan-Meier analysis, there was a significant trend toward improved survival in cases showing high expression of CAMK2N1 (N = 19) as opposed to cases showing low expression of CAMK2N1 (N = 20) in recurrent patients with high AR expression ( p = 0.023 ). (C) IHC was also conducted to determine CAMK2N1 and AR expression in the prostate cancer specimens (N = 70). AR and CAMK2N1 were inversely correlated in human prostate cancers ( r = −0.384, p = 0.0027).

Article Snippet: 2 × 10 6 C4-2 cells knockdown CAMK2N1 were implanted subcutaneously into 4-6-week-old castrated mal nude mice purchased from Beijing HFK Bio-Technology.co., LTD. Tumor growth was measured using a digital caliper every 5 days for 4-5 weeks.

Techniques: Expressing

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A-B) LNCaP cells were treated with 1-100 nM R1881 for 6-10 hrs. CAMK2N1 mRNA levels were determined by qRT-PCR. (C-E) CAMK2N1, AR protein and mRNA levels were determined by Western blot and qRT-PCR in LNCaP cells with stably knockdown of AR. LNCaP cells were treated with 10 nM R1881 for 10 hrs. (F-H) CAMK2N1 promoter reporters (−39~−1011, −1112~−2006) were assessed in AR-positive LNCaP cells and AR-negative PC3 cells. R1881 repressed activity of CAMK2N1 gene reporters (−39~−1011) in the presence of AR.

Article Snippet: 2 × 10 6 C4-2 cells knockdown CAMK2N1 were implanted subcutaneously into 4-6-week-old castrated mal nude mice purchased from Beijing HFK Bio-Technology.co., LTD. Tumor growth was measured using a digital caliper every 5 days for 4-5 weeks.

Techniques: Quantitative RT-PCR, Western Blot, Stable Transfection, Activity Assay

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) CAMK2N1, AR and p21 protein levels were determined by Western blot in LNCaP cells with stably knockdown of CAMK2N1. LNCaP cells were treated with 10 nM R1881 for 10 hrs. (B-F) CAMK2N1, AR, PSA, TMPRSS2 and p21 mRNA levels were determined by qRT-PCR in LNCaP cells with stably knockdown of CAMK2N1. LNCaP cells were treated with 10 nM R1881 for 10 hrs. (G-J) Androgen-responsive luciferase reporter genes (PSA-Luc, MMTV-Luc) were assessed for AR activity. LNCaP cells with CAMK2N1 overexpression or knockdown were treated with R1881 for 24 hrs. (K) CHIP analysis of AR for PSA promoter region in LNCaP cells with stable knockdown of CAMK2N1. LNCaP cells were treated with R1881 or vehicle for 10 hrs. CHIP assay was performed using an anti-AR antibody.

Article Snippet: 2 × 10 6 C4-2 cells knockdown CAMK2N1 were implanted subcutaneously into 4-6-week-old castrated mal nude mice purchased from Beijing HFK Bio-Technology.co., LTD. Tumor growth was measured using a digital caliper every 5 days for 4-5 weeks.

Techniques: Western Blot, Stable Transfection, Quantitative RT-PCR, Luciferase, Activity Assay, Over Expression

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) Expression levels of pAKT ser473 , AKT, AR, Bcl-2, BAX, and p21 were determined by Western blot in LNCaP cells with stable CAMK2N1 knockdown. (B) Expression levels of pAKT ser473 , AKT and AR were determined by Western blot in LNCaP cells with stable CAMK2N1 overexpression. (C-D) CAMK2N1 knockdown cells treated with 10 nM R1881 and/or 20μM KN-93 inhibitor. PSA-Luc was assessed for AR activity. CAMKIIβ and AR protein levels were determined by Western blot. (E-F) LNCaP cells transiently transfected with CAMK2N1 and/or CAMKIIβ while treated with 10 nM R1881. PSA-Luc was assessed for AR activity. CAMK2N1 and CAMKIIβ protein levels were determined by Western blot. (G-H) CAMK2N1 knockdown cells treated with 10 nM R1881 and/or 20 μM AKT VIII. PSA-Luc was assessed for AR activity. pAKT ser473 , AKT and AR β protein levels were determined by Western blot. (I-J) LNCaP cells transiently transfected with CAMK2N1 and/or m-AKT. PSA-Luc was assessed for AR activity. CAMK2N1 and CAMKIIβ protein levels were determined by Western blot.

Article Snippet: 2 × 10 6 C4-2 cells knockdown CAMK2N1 were implanted subcutaneously into 4-6-week-old castrated mal nude mice purchased from Beijing HFK Bio-Technology.co., LTD. Tumor growth was measured using a digital caliper every 5 days for 4-5 weeks.

Techniques: Expressing, Western Blot, Over Expression, Activity Assay, Transfection

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) LNCaP cells with stable knockdown of CAMK2N1 were treated with or without R1881. Cells were analyzed for cell proliferation by MTT assay. (B) LNCaP cells with stable knockdown of CAMK2N1 followed by treatment with or without Casodex (10 μM) were analyzed for cell proliferation by MTT assay. (C) LNCaP cells with stable knockdown of CAMK2N1 were treated with or without R1881. Cells were then analyzed for cell cycle by flow cytometry.

Article Snippet: 2 × 10 6 C4-2 cells knockdown CAMK2N1 were implanted subcutaneously into 4-6-week-old castrated mal nude mice purchased from Beijing HFK Bio-Technology.co., LTD. Tumor growth was measured using a digital caliper every 5 days for 4-5 weeks.

Techniques: MTT Assay, Flow Cytometry

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) Expression levels of pAKT ser473 , AKT, AR, Bcl-2, BAX, and p21 were determined by Western blot in C4-2 cells with stable CAMK2N1 knockdown. (B) Expression levels of pAKT ser473 , AR Bcl-2, BAX, and p21 were determined by Western blot in C4-2 cells with stable CAMK2N1 overexpression. (C) C4-2 cells with stable overexpression of CAMK2N1 and AR knockdown. Cells were analyzed for cell proliferation by MTT assay. CAMK2N1 and AR protein levels were determined by Western blot. (D) C4-2 cells with stable overexpression of CAMK2N1 followed by treatment with or without Casodex (10 μM) were analyzed for cell proliferation by MTT. (E) C4-2 cells with stable knockdown of CAMK2N1 were analyzed for cell cycle by flow cytometry. (F) C4-2 cells with stable overexpression of CAMK2N1 and AR knockdown. Cells were analyzed for cell apoptosis by Annexin V staining.

Article Snippet: 2 × 10 6 C4-2 cells knockdown CAMK2N1 were implanted subcutaneously into 4-6-week-old castrated mal nude mice purchased from Beijing HFK Bio-Technology.co., LTD. Tumor growth was measured using a digital caliper every 5 days for 4-5 weeks.

Techniques: Expressing, Western Blot, Over Expression, MTT Assay, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A-C) C4-2 tumors with stable CAMK2N1 knockdown were injected into nude mice. Tumor size was measured every 5 days. The data was shown as mean ± SEM for N > 6 separate tumors for each group. (A) Images of tumors dissected from the mice. (B) The tumor size (mm 3 ) versus days of post injection. (C) Tumor was weighted after resection at the end of experiment. (D) mRNA levels of AR, PSA, P21, BAX and BCL2 were determined by qRT-PCR in tumors. (E) IHC staining detected the protein expression of CAMK2N1, AR, p21, Ki67, pAKT ser473 and BCL2 in C4-2 tumor tissues derived from mice. Data for quantified IHC was shown as mean ± SEM for N = 4 tumors in each group.

Article Snippet: 2 × 10 6 C4-2 cells knockdown CAMK2N1 were implanted subcutaneously into 4-6-week-old castrated mal nude mice purchased from Beijing HFK Bio-Technology.co., LTD. Tumor growth was measured using a digital caliper every 5 days for 4-5 weeks.

Techniques: Injection, Quantitative RT-PCR, Immunohistochemistry, Expressing, Derivative Assay

Journal: Nature communications

Article Title: YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer

doi: 10.1038/ncomms9126

Figure Lengend Snippet: (a) Genetic alterations of YAP1 gene in human prostate cancer compiled from the www.cbioportal.org online platform. DEL: Deletion, AMP: Amplification. (b) Immunohistochemical (IHC) analysis of YAP1 protein in human normal prostate (NP, n = 9) and prostate cancer (PC, n = 22) clinical samples. (c) Co-immunoprecipitation (co-IP) and western blot (WB) analysis of AR and YAP1 proteins in total lysates obtained from fresh-frozen non-cancerous prostate or benign prostatic hyperplasia (BPH) and PC tissues. Tissue lysate consisting of high levels of both YAP1 and AR proteins was used in IgG control for the representation of other samples subjected to the co-IP and WB. (d) Co-IP and WB analysis of AR and YAP1 proteins in total lysates obtained from castration-sensitive LNCaP and castration-resistant C4-2 cells grown in serum-fed conditions. Mixture (1:1 ratio) of lysates from LNCaP and C4-2 cells was used in IgG control (e) Co-IP and WB analysis of AR and YAP1 proteins in total lysates obtained from LNCaP and C4-2 cells treated with vehicle (EtOH) and androgen (10 nM, Dihydrotestosterone, DHT) in charcoal-striped serum (CSS) growth conditions for 24 h. Co-IP and WB experiments were performed with antibodies to corresponding proteins. IgG was used as negative control in co-IP/WB experiments. Data are representative of two independent experiments. f.c., fold change; Scale bar, 100 mm.

Article Snippet: Briefly, shRNA control or shRNA YAP1 C4-2 cells (1 × 10 6 cells per well) mixed with Matrigel (1:1 ratio in 100 μl volume) were injected subcutaneously in the right and left flanks of the hormonally intact, four-week old (approximately 25 g), immune deficient (SCID), male mice (Charles River, Boston, MA).

Techniques: Amplification, Immunohistochemical staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Control, Negative Control

Journal: Nature communications

Article Title: YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer

doi: 10.1038/ncomms9126

Figure Lengend Snippet: (a,b) Co-IP and WB analysis of AR and YAP1 proteins in cytoplasmic and nuclear fractions obtained from (a) LNCaP cells treated with DHT for 0, 4 or 24 h and (b) from C4-2 cells treated with EtOH or DHT for 24 h in CSS growth conditions. Co-IP and WB were probed with antibodies to corresponding proteins. Lamin A/C was used as a nuclear extraction control. (c,d) Co-immunofluorescence (co-IF) analysis of AR and YAP1 proteins in LNCaP (c) and C4-2 (d) cells that were treated with EtOH and DHT in CSS conditions for 24 h. Alexa Fluor 488 stained AR (green), Cy3 stained YAP1 (red) and DAPI stained cell nuclei (blue). Magnification: 40×. Micrographs are the representation of multiple confocal images. Data are from two independent experiments. C, Cytoplasm; N, Nuclei, Scale bar, 100 μm.

Article Snippet: Briefly, shRNA control or shRNA YAP1 C4-2 cells (1 × 10 6 cells per well) mixed with Matrigel (1:1 ratio in 100 μl volume) were injected subcutaneously in the right and left flanks of the hormonally intact, four-week old (approximately 25 g), immune deficient (SCID), male mice (Charles River, Boston, MA).

Techniques: Co-Immunoprecipitation Assay, Extraction, Control, Immunofluorescence, Staining

Journal: Nature communications

Article Title: YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer

doi: 10.1038/ncomms9126

Figure Lengend Snippet: (a,b) Co-IP and WB analysis of AR and YAP1 proteins in cytoplasmic and nuclear fractions obtained (a) from LNCaP cells that were treat with DMSO, enzalutamide (ENZ) plus or mice DHT or (b) from C4-2 cells that were treated with DMSO or enzalutamide without DHT exposure. (c) Analysis of LNCaP or C4-2 cell growth after treatment with DMSO or enzalutamide without DHT in vitro; *P<0.001. (d) Co-IP and WB analysis of AR and YAP1 proteins in cytoplasmic and nuclear fractions obtained from LNCaP cells with or without MST1 knockdown plus or minus DHT treatment. Co-IP and WB were probed with antibodies to corresponding proteins at 24 in experiment (a,b) and at 48 h in experiment ‘d’ post treatment. Lamin A/C was used as a nuclear extraction control. Blots are representative of two independent experiments. C, Cytoplasm; N, Nuclei. (e) Analysis of LNCaP cell growth with or without MST1 knockdown plus or minus DHT treatment in vitro; *P<0.002. To assess growth, cell viability was determined by MTS assay at 72 h post treatment. Data (±s.e.) in c and e are representation of two independent experiments in triplicates.

Article Snippet: Briefly, shRNA control or shRNA YAP1 C4-2 cells (1 × 10 6 cells per well) mixed with Matrigel (1:1 ratio in 100 μl volume) were injected subcutaneously in the right and left flanks of the hormonally intact, four-week old (approximately 25 g), immune deficient (SCID), male mice (Charles River, Boston, MA).

Techniques: Co-Immunoprecipitation Assay, In Vitro, Knockdown, Extraction, Control, MTS Assay

Journal: Nature communications

Article Title: YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer

doi: 10.1038/ncomms9126

Figure Lengend Snippet: (a) Co-IP and WB analysis of YAP1 and AR proteins in C4-2 cells with or without MST1 induction by doxycycline (Dox) followed by DHT exposure. (b) Analysis of YAP1 in cytoplasm and nuclear fraction in C4-2 cells transiently transfected with vector or HA-MST1. (c) IF analysis of YAP1 and HA-MST1 in C4-2 cells that were transiently transfected to express HA-MST1 in serum-fed conditions. Experiments in (a, b, c) were performed at 48 h post MST1 induction. (d) Co-IP and WB analysis of endogenous MST1 and YAP1 interactions in LNCaP cells. (e) In vitro kinase assay with GST–YAP1 (2–150) peptide and the MST1 immune complex precipitated from LNCaP cells. (f) GST–pull-down and in vitro kinase assays with the recombinant, purified MST1 kinase and GST–YAP1 (2–150) peptide. GST only peptide was used as a negative control in kinase and binding assays in (e) and (f). Experiments related to co-IP, WB and IF were performed with antibodies to corresponding proteins. Data are representative of two independent experiments. C, Cytoplasm; N, Nuclei; and Scale bar, 100 μm.

Article Snippet: Briefly, shRNA control or shRNA YAP1 C4-2 cells (1 × 10 6 cells per well) mixed with Matrigel (1:1 ratio in 100 μl volume) were injected subcutaneously in the right and left flanks of the hormonally intact, four-week old (approximately 25 g), immune deficient (SCID), male mice (Charles River, Boston, MA).

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, In Vitro, Kinase Assay, Recombinant, Purification, Negative Control, Binding Assay

Journal: Nature communications

Article Title: YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer

doi: 10.1038/ncomms9126

Figure Lengend Snippet: (a) Schematic representation of the functional domains of YAP1. (b) GST–pull-down with GST–YAP1 fusion peptides. The binding of AR protein to GST–YAP1 peptide was probed with WB using the AR specific antibody. Total cell lysate from LNCaP was used as a source of AR in GST–pull-down experiment. M, Marker. (c) Co-IP and WB analysis of endogenous YAP1 interacting with the exogenous HA–AR full-length (FL), HA–AR–NTD or AR–DBD–LBD deletion mutant in C4-2 cells. (d) AR-responsive PSA promoter reporter activity; *P<5.6E–06. LNCaP cells were transiently transfected with PSA-Luc and Vector control, YAP1–WT, YAP1-ΔN (58-504 residues), or YAP1-ΔC (2–290 residues) construct, followed by EtOH vehicle or androgen (10 nM DHT) treatment for 48 h. (e) Quantitative PCR analysis of YAP1 and well-characterized AR responsive genes KLK3, PSMA, FKBP5 and TMPRSS2 in LNCaP cells that were transiently transfected with control shRNA or two different YAP1 shRNAs, followed by DHT treatment in CSS growth condition; *P<0.002. Quantitative RT–PCR with total RNA was performed at 24 h post DHT treatment or at 72 h post transfection. Data (±s.e.) are from two independent experiments in triplicate.

Article Snippet: Briefly, shRNA control or shRNA YAP1 C4-2 cells (1 × 10 6 cells per well) mixed with Matrigel (1:1 ratio in 100 μl volume) were injected subcutaneously in the right and left flanks of the hormonally intact, four-week old (approximately 25 g), immune deficient (SCID), male mice (Charles River, Boston, MA).

Techniques: Functional Assay, Binding Assay, Marker, Co-Immunoprecipitation Assay, Mutagenesis, Activity Assay, Transfection, Plasmid Preparation, Control, Construct, Real-time Polymerase Chain Reaction, shRNA, Quantitative RT-PCR

Journal: Nature communications

Article Title: YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer

doi: 10.1038/ncomms9126

Figure Lengend Snippet: (a) Quantitative PCR and WB analysis of YAP1 mRNA and protein, respectively, in stable shControl or shYAP1 C4-2 cells; *P<0.002. (b) Time-dependent analysis of cell growth. Cell growth was assessed by MTS assay after 24, 48, 72 and 96 h post cell seeding in serum-fed conditions; *P<0.001. (c) Sphere formation in 3D Matrigel. Equal numbers of shControl and shYAP1 C4-2 cells were grown for 14 days in presence of either EtOH (vehicle) or 10 nM DHT in CSS conditions, and spheres were counted manually and presented in a graph; *,**P<0.001. (d) Time-dependent analysis of cell invasion through Matrigel-coated Transwell chamber; *,**P<0.001. Equal numbers of shControl and shYAP1 C4-2 cells were grown in CSS conditions (upper chamber) and serum-fed growth condition (lower chamber). Cells invading through the chamber were visualized by crystal violet staining (micrographs) and counted manually at 16-, 24- and 48-h post cell seeding. The graph is the quantification of invaded cells at indicated times. Data (±s.e.) are from two independent experiments in triplicates. Scale bar, 100 μm.

Article Snippet: Briefly, shRNA control or shRNA YAP1 C4-2 cells (1 × 10 6 cells per well) mixed with Matrigel (1:1 ratio in 100 μl volume) were injected subcutaneously in the right and left flanks of the hormonally intact, four-week old (approximately 25 g), immune deficient (SCID), male mice (Charles River, Boston, MA).

Techniques: Real-time Polymerase Chain Reaction, MTS Assay, Staining

Journal: Nature communications

Article Title: YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer

doi: 10.1038/ncomms9126

Figure Lengend Snippet: (a,b) Assessing the impact of Verteporfin (VP) on cell growth. C4-2 cells were treated with increasing doses of VP for 48 h or DMSO (vehicle) or 1.25 μM VP for 24, 48, 72 and 96 h. Cell growth was assessed by MTS assay post treatment; *P<0.0001. (c) Apoptosis assay. C4-2 cells were treated with increasing doses (0, 1.25 or 5 μM) of VP for 48 h. Propidium iodide and Annexin V stained cells were analysed by flow cytometry to assess apoptosis; *P<0.001. (d) Sphere formation in 3D Matrigel assay. C4-2 cells grown in Matrigel under serum-fed conditions were treated with DMSO or 1.25 μM VP for 10 days. Spheres in Matrigel (micrographs) were counted manually and then plotted (graph); *P<0.001. (e) Cell invasion in Matrigel-coated Transwell. C4-2 cells were treated with DMSO (vehicle) or 1.25 μM VP. Invaded cells that were stained with crystal violet (micrographs) were counted and then plotted (graph); *P<0.001. (f) IF staining of C4-2 cells that were treated with DMSO or 1.25 μM VP for 24 h in serum-fed growth conditions. YAP1 protein (green) with Alexa Fluor 488 and nuclei (blue) with DAPI were visualized by confocal microscopy. Data (±s.e.) are from two independent experiments in triplicate; *P<0.001. (g) Co-IP and WB analysis of YAP1 and AR proteins in total lysate from C4-2 cells that were treated with DMSO or 1.25 μM VP. Co-IP and WB were probed with antibodies to corresponding proteins. f.c.: fold change. Scale bar, 100 μm.

Article Snippet: Briefly, shRNA control or shRNA YAP1 C4-2 cells (1 × 10 6 cells per well) mixed with Matrigel (1:1 ratio in 100 μl volume) were injected subcutaneously in the right and left flanks of the hormonally intact, four-week old (approximately 25 g), immune deficient (SCID), male mice (Charles River, Boston, MA).

Techniques: MTS Assay, Apoptosis Assay, Staining, Flow Cytometry, Matrigel Assay, Confocal Microscopy, Co-Immunoprecipitation Assay

Journal: Nature communications

Article Title: YAP1 and AR interactions contribute to the switch from androgen-dependent to castration-resistant growth in prostate cancer

doi: 10.1038/ncomms9126

Figure Lengend Snippet: (a) Table shows the number of tumours produced by shControl or shYAP1 C4-2 cells in immune deficient male mice (n = 10 per group). Representative tumour tissues are shown above the images. Micrographs show luciferase imaging of tumours in live animals. Tumours at the end of the fifth week post cell inoculation were dissected out at necropsy and their volumes were calculated and the data were plotted in a graph; *P<0.001. (b) Graph shows the quantification of photons from the luciferase imaging at each week; *P<0.001. (c) IHC analysis of YAP1, AR, c-Cas3 (cleaved-caspase 3), and Ki-67 proteins in xenografts tissue sections. Tissue sections were stained with YAP1, AR, Ki-67 or c-Cas3. Magnification: 40×. Micrographs are representative of multiple images. (d) Quantitative RT–PCR analysis of well-characterized AR-target genes KLK3 (PSA), PSMA, FKBP5 and TMPRSS2 in shControl and shYAP1 tumour xenografts. The qPCR data (±s.e.) are from two independent experiments in duplicates; *P<0.01. (e) Model summarizes the finding. Scale bar, 100 μm.

Article Snippet: Briefly, shRNA control or shRNA YAP1 C4-2 cells (1 × 10 6 cells per well) mixed with Matrigel (1:1 ratio in 100 μl volume) were injected subcutaneously in the right and left flanks of the hormonally intact, four-week old (approximately 25 g), immune deficient (SCID), male mice (Charles River, Boston, MA).

Techniques: Produced, Luciferase, Imaging, Staining, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: Enzalutamide-Induced Upregulation of PCAT6 Promotes Prostate Cancer Neuroendocrine Differentiation by Regulating miR-326/HNRNPA2B1 Axis

doi: 10.3389/fonc.2021.650054

Figure Lengend Snippet: PCAT6 expression was increased in NEPC cells and tissues. (A) qPCR analysis of the top 10 lncRNAs (PCAT6, LncRNA7816-1, LncRNA2323-2, LINC00319, lncRNA-p21, Malat1, PCA3, LncRNA8802, LncRNA1698-1 and LncRNA2303) mRNA expression in NCI-H660 cells and LNCaP cells. (B) qPCR analysis of PCAT6 mRNA expression in NE-like cells (PC3, DU145, and NCI-H660) and LNCaP and C4-2 cells. (C, D) The mRNA expression of PCAT6 was determined in LNCaP cells treated with different Enza concentration (2 μM, 4 μM, 8 μM, 10 μM) for 72 h or 10 μM for different times (0 h, 12 h, 24 h, 48 h, 72 h). (E) Fluorescent in situ hybridization (FISH) assay of the PCAT6 expression in LNCaP and NCI-H660 cells. (F) Fluorescent in situ hybridization (FISH) assay of the PCAT6 expression in LNCaP cells with 10 μM Enza treatment for 72 h. (G) qPCR analysis of PCAT6 expression in Pca tissues and adjacent normal tissues. (H) qPCR analysis of PCAT6 expression in NEPC (n=9) and CRPC (n=18) samples. * p <0.05. ** p <0.01.

Article Snippet: NE-like cells (PC3, DU145, and NCI-H660), LNCaP, C4-2 cells were purchased from ScienCell (Santiago, California, USA).

Techniques: Expressing, Concentration Assay, In Situ Hybridization

Journal: Frontiers in Oncology

Article Title: Enzalutamide-Induced Upregulation of PCAT6 Promotes Prostate Cancer Neuroendocrine Differentiation by Regulating miR-326/HNRNPA2B1 Axis

doi: 10.3389/fonc.2021.650054

Figure Lengend Snippet: PCAT6 promoted NED, proliferation, and invasion of PCa cells in vitro . (A) qPCR analysis of NE markers (NSE, SYP, and ChgA) mRNA expression in LNCaP cells after PCAT6 overexpression. (B, C) Western blot analysis for NSE, SYP, and ChgA protein level in LNCaP cells after PCAT6 overexpression. (D) qPCR analysis of NSE, SYP, and ChgA mRNA expression in NCI-H660 cells after PCAT6 knockdown. (E) LNCaP cell proliferation was assayed using CCK-8 after PCAT6 overexpression. (F) NCI-H660 cell proliferation was assayed using CCK-8 after PCAT6 knockdown. (G, H) LNCaP cell invasion was assessed using transwell invasion assay after PCAT6 overexpression. (I, J) NCI-H660 cell invasion was assessed using transwell invasion assay after PCAT6 knockdown. * p <0.05. ** p <0.01.

Article Snippet: NE-like cells (PC3, DU145, and NCI-H660), LNCaP, C4-2 cells were purchased from ScienCell (Santiago, California, USA).

Techniques: In Vitro, Expressing, Over Expression, Western Blot, CCK-8 Assay, Transwell Invasion Assay

Journal: Frontiers in Oncology

Article Title: Enzalutamide-Induced Upregulation of PCAT6 Promotes Prostate Cancer Neuroendocrine Differentiation by Regulating miR-326/HNRNPA2B1 Axis

doi: 10.3389/fonc.2021.650054

Figure Lengend Snippet: PCAT6 functioned as a ceRNA and sponged miR-326. (A) Starbase v2.0 database was used to predict potential miRNA binding sites in PCAT6. (B) qPCR analysis of miR-326 mRNA expression in NE-like cells (PC3, DU145, and NCI-H660) and LNCaP and C4-2 cells. (C) Schematic representation of the miR-326 site in PCAT6-3’UTR. (D) Luciferase activity was assayed in LNCaP cells co-transfected with miR-326 and luciferase reporters containing PCAT6-WT or PCAT6-Mut. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. (E) Luciferase activity was assayed in LNCaP cells co-transfected with luciferase reporters containing PCAT6-WT and miR-326 or miR-326-Mut. (F) The direct binding of PCAT6 to miR-326 was affirmed using luciferase reporters in LNCaP cells treated with miR-326 or miR-326-mut. (G) qPCR analysis of miR-326 level in CRPC tissues (n=17) and NEPC tissues (n=9). ** p <0.01.

Article Snippet: NE-like cells (PC3, DU145, and NCI-H660), LNCaP, C4-2 cells were purchased from ScienCell (Santiago, California, USA).

Techniques: Binding Assay, Expressing, Luciferase, Activity Assay, Transfection

Journal: Cancer letters

Article Title: Histone deacetylase inhibitor valproic acid suppresses the growth and increases the androgen sensitivity of prostate cancer cells

doi: 10.1016/j.canlet.2011.07.015

Figure Lengend Snippet: Effect of NaB on cell growth and PAcP expression. LNCaP C-33 and C-81 cells were treated with different concentrations of NaB for 3 days. Cells were harvested for (A) cell growth analysis and (B) total RNA preparation. The expression of PAcP mRNA was analyzed by northern blot analyses. The same membrane was hybridized with a GAPDH probe as a loading control. (C) C-81 cells were transfected with the CAT reporter gene drived by PAcP promoters in the presence of different concentrations of NaB. Cells were harvested and the CAT activity was determined. The values represent the means ± SD of three independent experiments (n=3×3). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: LNCaP C4-2 cells were purchased from DIANON Company (Oklahoma City, OK).

Techniques: Expressing, Northern Blot, Membrane, Control, Transfection, Activity Assay

Journal: Cancer letters

Article Title: Histone deacetylase inhibitor valproic acid suppresses the growth and increases the androgen sensitivity of prostate cancer cells

doi: 10.1016/j.canlet.2011.07.015

Figure Lengend Snippet: Dosage and kinetic effects of HDAC inhibitors on cPAcP protein expression in LNCaP C-81 cells. LNCaP C-81 cells that were seeded at a density of 5×105 cells/T25 for 2 days in regular medium were treated with different concentrations of (A) NaB or (B) VPA for 48 hr, or treated with (C) 1 mM NaB or (D) 1 mM VPA for different time periods. The total protein was harvested and subjected to western blot analyses of cPAcP protein expression. (D) The acetylation and the methylation levels of H3 and H4 proteins were also analyzed in VPA-treated C-81 cells for different time periods. β-Actin was analyzed and used as a loading control in each experiment. Similar results were obtained from five sets of independent experiments.

Article Snippet: LNCaP C4-2 cells were purchased from DIANON Company (Oklahoma City, OK).

Techniques: Expressing, Western Blot, Methylation, Control

Journal: Cancer letters

Article Title: Histone deacetylase inhibitor valproic acid suppresses the growth and increases the androgen sensitivity of prostate cancer cells

doi: 10.1016/j.canlet.2011.07.015

Figure Lengend Snippet: Effect of cPAcP expression on VPA growth suppression and VPA effect on androgen sensitivity of PCa cell lines. (A) LNCaP C-81 cells were plated at a density of 1×105 cells/well in 6-well plates for 72 hr and then transfected with PAcP shRNA-126 plasmids. Control cells were transfected with the vector containing the scramble DNA. Five hours after transfection, the cells were fed with RPMI medium containing 10% FBS for 24 hr. The cells were then treated with 1mM VPA for 48 hr. The cell numbers were counted. The ratio of cell growth was calculated by normalizing the cell number to that of the control cells transfected with the control vector and treated with the solvent alone. The result shown is the average from two sets of independent experiments in triplicates (left panel, n= 3×2). Total cell lysate proteins from 3-day VPA treatments were analyzed for cPAcP protein expression. β-Actin was used as a loading control (right panel). (B) LNCaP C-81; (C) C4-2; (D) MDA PCa2b-AI cells were plated at a density of 3×104, 3×104 and 1×105 cells/well, respectively, in 6-well plates for 72 hr and then treated with 1 mM VPA. Control cells were treated with solvent alone. After 48 hr VPA treatment, cells were maintained in a steroid-reduced medium in the absence or presence of 10 nM DHT for 48 hr. The cell numbers were counted. The ratio of cell growth was calculated by normalizing the cell number to that of the control cells without any treatment (column #1). The result shown is the average from three sets of independent experiments in triplicates (n= 3×3). Total cell lysate proteins from 3-day DHT treatment were analyzed for cPAcP, PSA, AR protein expression. The AR protein in Fig. 3B was obtained after 3-hr hybridization with anti-AR Ab followed by an exposure time period of less than 1 min; while the AR proteins in Fig. 3C & 3D were obtained after overnight hybridization with anti-AR Ab and with an exposure time period of 1 hr. β-Actin was used as a loading control and it was obtained with the same reaction time periods.

Article Snippet: LNCaP C4-2 cells were purchased from DIANON Company (Oklahoma City, OK).

Techniques: Expressing, Transfection, shRNA, Control, Plasmid Preparation, Solvent, Hybridization

Journal: Cancer letters

Article Title: Histone deacetylase inhibitor valproic acid suppresses the growth and increases the androgen sensitivity of prostate cancer cells

doi: 10.1016/j.canlet.2011.07.015

Figure Lengend Snippet: Effects of VPA on cPAcP protein expression and ErbB-2 tyrosyl phosphorylation. LNCaP C-81 cells were plated in three cell dinsities (0.3, 0.5 and 1×106 cells/T25) in regular medium for 2 days and then treated with 1 mM VPA for 48 hr. The cells were harvested and the total protein was subjected to western blot analyses of functional proteins expression. VPA effects on cPAcP protein expression, ErbB-2 phosphorylation at Tyr1221/2 and Tyr1248, histone H3 and H4 acetylation and methylation, JNK and p38 phosphorylation, Bcl-2 and Bax protein levels. β-Actin was detected as a loading control. The data shown is a representative from three sets of independent experiments.

Article Snippet: LNCaP C4-2 cells were purchased from DIANON Company (Oklahoma City, OK).

Techniques: Expressing, Phospho-proteomics, Western Blot, Functional Assay, Methylation, Control

Journal: Cancer letters

Article Title: Histone deacetylase inhibitor valproic acid suppresses the growth and increases the androgen sensitivity of prostate cancer cells

doi: 10.1016/j.canlet.2011.07.015

Figure Lengend Snippet: Effects of HDAC inhibitors on cell cycle distribution, apoptosis and cell cycle protein expression in LNCaP C-81 cells. LNCaP C-81 cells were plated in regular medium for 48 hr and treated with 1 mM NaB, 1 mM VPA or 300 nM TSA for 48 hr. All cells were harvested and subjected to cell cycle analyses using flow cytometry and western blot analyses of indicated proteins. (A) Cell cycle analyses. The data shown is the mean of three sets of independent experiments. (B) Western blot analyses. The total cellular lysate proteins were analyzed for cPAcP protein, ErbB-2 phosphorylation, histone H3/H4 acetylation and methylation, AR, cPSA, cyclin B1 and D1, PCNA and p21 protein levels. β-Actin was analyzed as a loading control in each experiment. The data shown is a representative of three sets of independent experiments.

Article Snippet: LNCaP C4-2 cells were purchased from DIANON Company (Oklahoma City, OK).

Techniques: Expressing, Flow Cytometry, Western Blot, Phospho-proteomics, Methylation, Control