Journal: International Journal of Molecular Sciences

Article Title: Radiotherapy Metastatic Prostate Cancer Cell Lines Treated with Gold Nanorods Modulate miRNA Signatures

doi: 10.3390/ijms25052754

Figure Lengend Snippet: Graphical representation of miRNA expression levels in the HPrEpiC cell line isolated from normal human prostate tissue. MiRNA expression was analyzed using RT-qPCR and compared to a control group that was not treated. The RNU6 housekeeping gene was used as a reference for normalization and relative expression was calculated using the ΔCT expression/ΔCT control ratio. The cells were irradiated in three doses of 2.5 Gy, with the first dose being 1 × 2.5 Gy, the second dose being 2 × 2.5 Gy, and the third dose being 3 × 2.5 Gy. N = 2.

Article Snippet: PC3, DU145, and LNCaP cell lines were provided by the Cancer Biology and Epigenetics Group at the Portuguese Oncology Institute of Porto, and human prostate Epithelial cells (HPrEpiC) were acquired from Innoprot (Innovative Technologies in Biological Systems, Derio, Spain).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Irradiation

Journal: International Journal of Molecular Sciences

Article Title: Radiotherapy Metastatic Prostate Cancer Cell Lines Treated with Gold Nanorods Modulate miRNA Signatures

doi: 10.3390/ijms25052754

Figure Lengend Snippet: MiRNA expression in radiation response in prostate cancer cell lines. With regard to the data presented from our study, the results of the 3rd day of the study, i.e., 3 × 2.5 Gy, were taken into account.

Article Snippet: PC3, DU145, and LNCaP cell lines were provided by the Cancer Biology and Epigenetics Group at the Portuguese Oncology Institute of Porto, and human prostate Epithelial cells (HPrEpiC) were acquired from Innoprot (Innovative Technologies in Biological Systems, Derio, Spain).

Techniques: Expressing, Irradiation, Functional Assay

Journal: Oncotarget

Article Title: The metastasis suppressor CD82/KAI1 inhibits fibronectin adhesion-induced epithelial-to-mesenchymal transition in prostate cancer cells by repressing the associated integrin signaling

doi: 10.18632/oncotarget.13767

Figure Lengend Snippet: A. Human prostate epithelial (PZ-HPV-7) and cancer (DU145, LNCaP, and PC3) cell lines and stably CD82-transfected DU145 and antisense CD82 fragment-transfected LNCaP cell clones were assessed for CD82 expression levels through immunoblotting analysis. B. Stably CD82-transfected DU145 and antisense CD82 fragment-transfected LNCaP cell clones grown on fibronectin (FN) were viewed under a phase-contrast microscope. Scale bar, 10 μm. C. The cells were seeded onto plates precoated with poly-L(+)-lysine (p-Lys) or FN and cultured for the indicated time periods. Expression of E-cadherin and mesenchymal marker proteins was examined through immunoblotting analysis using antibodies specific to each protein.

Article Snippet: PZ-HPV-7 (ATCC), a human prostate epithelial cell line transformed by human papillomavirus-18, was cultured in K-SFM medium (Invitrogen, Carlsbad, CA) supplemented with 0.05 mg/ml bovine pituitary extract and 5 ng/ml EGF.

Techniques: Stable Transfection, Transfection, Clone Assay, Expressing, Western Blot, Microscopy, Cell Culture, Marker

Journal: Oncotarget

Article Title: The metastasis suppressor CD82/KAI1 inhibits fibronectin adhesion-induced epithelial-to-mesenchymal transition in prostate cancer cells by repressing the associated integrin signaling

doi: 10.18632/oncotarget.13767

Figure Lengend Snippet: A. PZ-HPV-7 prostate epithelial cells were lysed with Brij 97 detergent, and immunoprecipitation (IP) was performed with normal mouse IgG or anti-CD82 antibody. The immunoprecipitates were analyzed by immnublotting using anti-integrin β 1 , α 3 , α 5 , or α 6 antibody. B. CD82 mutant cDNA, which encodes CD82 with a large extracellular loop (LEL) substituted with that of TM4SF2 as illustrated, was generated by PCR and subcloned into the pAdEasy-1 adenoviral vector to produce recombinant adenovirus. C. CD82-deficient PC3 prostate cancer cells grown on fibronectin (FN) were infected with adenovirus containing a wild-type (wt) or mutant (mt) CD82 expression construct, and Brij 97 detergent lysates were subjected to immunoprecipitation with an anti-β 1 integrin antibody followed by immunoblotting analysis using antibodies that recognize the C-terminus or LEL of CD82 and the LEL of TM4SF2. D. PC3 cells grown on poly-L(+)-lysine (p-Lys) or FN were infected with adenovirus containing a wt- or mt-CD82 expression construct and then assessed for the protein levels of E-cadherin and Snail. E. PC3 cells grown on FN were infected with wt-CD82 construct-containing adenovirus either alone or together with mt-CD82 construct-containing adenovirus and examined for E-cadherin and Snail expression. Numbers in parentheses represent the MOI values of adenovirus.

Article Snippet: PZ-HPV-7 (ATCC), a human prostate epithelial cell line transformed by human papillomavirus-18, was cultured in K-SFM medium (Invitrogen, Carlsbad, CA) supplemented with 0.05 mg/ml bovine pituitary extract and 5 ng/ml EGF.

Techniques: Immunoprecipitation, Mutagenesis, Generated, Plasmid Preparation, Recombinant, Infection, Expressing, Construct, Western Blot

Journal: Cell Death Discovery

Article Title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m 6 A demethylation

doi: 10.1038/s41420-024-02060-w

Figure Lengend Snippet: a The genes involved in m 6 A modification are shown in the table as candidate targets of ZFHX3 and were analyzed by Chip-Enrich using the ZFHX3 Chip-Seq results. b ZFHX3 correlated with WTAP , FTO , and ALKBH5 when using a cluster of PCa patients containing mRNA expression of these genes. c The m 6 A level was detected by immunofluorescence in RWPE1 cells after knocking down ZFHX3, and the arrows indicated m 6 A translocating from the nucleus to the cytoplasm. n = 3. d The expression of ZFHX3, FTO, and ALKBH5 in human prostate epithelial cell lines, was determined by western blot. The efficiency of ZFHX3 knockdown was detected by western blot, and the global m 6 A levels were reduced by ZFHX3 knockdown and detected by RNA dot blot in RWPE1 ( e ) and LNCaP ( f ). n = 3. MB (methylene blue) represents the loading control of the RNA samples. *** P < 0.001.

Article Snippet: Human immortalized prostate epithelial cell line RWPE1 and other human PCa cell lines were purchased from KeyGEN Biotech (Jiangsu, China).

Techniques: Modification, ChIP-sequencing, Expressing, Immunofluorescence, Western Blot, Knockdown, Dot Blot, Control

Journal: Cell Death Discovery

Article Title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m 6 A demethylation

doi: 10.1038/s41420-024-02060-w

Figure Lengend Snippet: a , b Knockdown of ZFHX3 upregulated FTO expression in RWPE1 and LNCaP cells at the protein and mRNA levels, as detected separately by western blot and qRT-PCR. n = 3. c The wild type and mutant of the FTO promoter were constructed, and the critical sequences are shown in the panel. Red rectangles indicate mutant sequences, and blue rectangles indicate the regions within the FTO promoter for PCR amplification. d ZFHX3 knockdown increased the activity of the wild-type promoter of FTO , but the effect on the mutant promoter of FTO was partly impaired compared to the wild-type promoter. n = 3. e Expression plasmids for control-vector (Flag) or Flag-ZFHX3 were transfected in 293T cells and the efficiency was detected by western blot. f Detection of ZFHX3-bound FTO promoter DNA in ZFHX3-overexpressed cells or control cells was completed using ChIP and regular PCR. g Binding of ZFHX3 to FTO promoter region 1 and region 2 was found using ChIP and qRT-PCR. n = 3. ** P < 0.01; *** P < 0.001; ns not significant.

Article Snippet: Human immortalized prostate epithelial cell line RWPE1 and other human PCa cell lines were purchased from KeyGEN Biotech (Jiangsu, China).

Techniques: Knockdown, Expressing, Western Blot, Quantitative RT-PCR, Mutagenesis, Construct, Amplification, Activity Assay, Control, Plasmid Preparation, Transfection, Binding Assay

Journal: Cell Death Discovery

Article Title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m 6 A demethylation

doi: 10.1038/s41420-024-02060-w

Figure Lengend Snippet: a , d The knockdown efficiency of FTO shRNA with lentivirus constructs in RWPE1 or siRNAs against FTO in LNCaP was confirmed by western blot. b Acini formation was decreased in the FTO knockdown cell line. The arrows indicated the normal acini structure. n = 3. c , f Western blot showed that MYC expression was repressed when FTO was knocked down by shRNA or siRNAs, separately in RWPE1 and LNCaP. e Sphere formation was decreased after knocking down FTO (siFTO-1) in LNCaP. The average number of spheres with a diameter >75 µm/well was counted. n = 3. g We determined the m 6 A abundance in mRNA in RWPE1 cells upon FTO inhibitor FB23-2 treatment under various concentrations as indicated for 72 h via dot blot assay. h FB23-2 suppressed cell proliferation of RWPE1 detected by SRB assay. The cells treated with different concentrations of FB23-2 were collected at indicated time. n = 3. i – k Knockdown of ZFHX3 by siRNA was accompanied by three concentrations of siRNAs against FTO as indicated. The efficiency of ZFXH3 and FTO knockdown was tested by western blot and FTO knockdown eliminated the promoting effect of ZFHX3’s inhibition on acini formation in RWPE1 cells. n = 3. * P < 0.05; ** P < 0.01; *** P < 0.001, ns not significant.

Article Snippet: Human immortalized prostate epithelial cell line RWPE1 and other human PCa cell lines were purchased from KeyGEN Biotech (Jiangsu, China).

Techniques: Knockdown, shRNA, Construct, Western Blot, Expressing, Dot Blot, Sulforhodamine B Assay, Inhibition

Journal: Cell Death Discovery

Article Title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m 6 A demethylation

doi: 10.1038/s41420-024-02060-w

Figure Lengend Snippet: a Immunoblotting of FTO in FTO knockdown RWPE1 and control RWPE1 cells is presented. b The distribution of peaks is shown with a significant change in m 6 A level in FTO knockdown RWPE1 cells compared to control RWPE1 cells. c A Venn diagram shows the shared genes between increased m 6 A peaks upon FTO deletion and FTO-relation genes analyzed by RNA-seq. A total of 236 genes were observed. d , e Pathway analysis and KEGG analysis of the above 236 shared genes showed that the cell cycle was altered in FTO knockdown cells. f The mapped reads represent enriched RNA fragments by MeRIP-seq. RNA methylation profiles were loaded in IGV software and m 6 A modification peak alterations in CDKN2C and E2F2 mRNA full length were visualized. g MeRIP-qRT-PCR was used to detect the m 6 A levels alterations of CDKN2C and E2F2 after knocking down FTO in RWPE1 cells. h Cell cycle distribution of FTO silencing cells and control cells was analyzed by flow cytometry. i E2F2 and CDKN2C were detected by western blot in ZFHX3 knockdown cells or control cells where FTO was silenced with siRNA targeting FTO or siCtrl. ** P < 0.01; *** P < 0.001, ns not significant.

Article Snippet: Human immortalized prostate epithelial cell line RWPE1 and other human PCa cell lines were purchased from KeyGEN Biotech (Jiangsu, China).

Techniques: Western Blot, Knockdown, Control, RNA Sequencing, Methylation, Software, Modification, Quantitative RT-PCR, Flow Cytometry

Journal: Cell Death Discovery

Article Title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m 6 A demethylation

doi: 10.1038/s41420-024-02060-w

Figure Lengend Snippet: a , b The potential m 6 A sites of ZFHX3 and the consensus motifs modified by m 6 A predicted by SRAMP are shown. c The m 6 A peaks in the black rectangles (R1, R2, and R3) visualized by IGV are those that co-localized with predicted sites of ZFHX3 . d The m 6 A levels of fragments in ZFHX3 (R1, R2, and R3) were elevated by MeRIP-qRT-PCR after knocking down FTO in RWPE1 cells. n = 3. e Western blot and RT-qPCR assays showed that ZFHX3 expression at the protein level or mRNA level was elevated after depletion of FTO (shFTO) in RWPE1 cells. n = 3. f The mRNA half-life ( t 1/2 ) was increased in RWPE1 cells with depleted expression of FTO (shFTO). g ZFHX3 protein expression or mRNA expression was upregulated after knockdown of FTO (siFTO-1 and siFTO-2) in LNCaP cells. n = 3. h The mRNA half-life ( t 1/2 ) was increased in LNCaP cells with depleted expression of FTO (siFTO-1). *** P < 0.001.

Article Snippet: Human immortalized prostate epithelial cell line RWPE1 and other human PCa cell lines were purchased from KeyGEN Biotech (Jiangsu, China).

Techniques: Modification, Quantitative RT-PCR, Western Blot, Expressing, Knockdown

Journal: Oncotarget

Article Title: Prostate cancer diagnosis using epigenetic biomarkers, 3D high-content imaging and probabilistic cell-by-cell classifiers

doi: 10.18632/oncotarget.18985

Figure Lengend Snippet: Imaging was performed to acquire four categories of tissue frames corresponding to a sampling spectrum of epithelial and stromal compartments: epithelia only (E), epithelia with minor bordering stroma (E+s), mixed epithelia and stroma at various ratios (ES), and stroma only (S).

Article Snippet: Cultured cells included primary human prostate epithelial cells (HPrEpiC, ScienCell, Carlsbad, CA) as normal primary cells, and LNCaP (American Type Culture Collection, Manassas, VA), an androgen-sensitive prostate cancer cell line.

Techniques: Imaging, Sampling

Journal: Oncotarget

Article Title: Prostate cancer diagnosis using epigenetic biomarkers, 3D high-content imaging and probabilistic cell-by-cell classifiers

doi: 10.18632/oncotarget.18985

Figure Lengend Snippet: Differential levels and cellular heterogeneity of Biomarkers I and II between biopsied benign and cancerous prostatic tissues (represented by AC with GS6), as visualized by confocal scanning microscopy (A). Each marker (false-colored) was recorded in a separate channel. For each tissue sample all channels —including the multi-color overlay image— are presented as maximum intensity projections. (B) Quantitative presentation of biomarker levels as bar plots indicate for Biomarkers I: significant increase in DNA content (DAPI) and AMACR levels and simultaneous loss of the two epigenetic DNA modifications 5mC and 5hmC in both basal and luminal epithelial cells in AC versus benign tissue; luminal cells seemingly exhibit a stronger loss of 5hmC compared to basal cells. Biomarkers II: concurrent loss of suppressive chromatin-state marker H3K27me3 and decrease of chromatin-associated SAFB levels, while H3K9me3 and nuclear AR levels are highly elevated in AC versus benign tissue. Scale bar is 10 μm.

Article Snippet: Cultured cells included primary human prostate epithelial cells (HPrEpiC, ScienCell, Carlsbad, CA) as normal primary cells, and LNCaP (American Type Culture Collection, Manassas, VA), an androgen-sensitive prostate cancer cell line.

Techniques: Microscopy, Marker, Biomarker Discovery

Journal: Oncotarget

Article Title: Prostate cancer diagnosis using epigenetic biomarkers, 3D high-content imaging and probabilistic cell-by-cell classifiers

doi: 10.18632/oncotarget.18985

Figure Lengend Snippet: The data was separated into subsets representing the first biopsy (blue), the second biopsy (red), and finally prostatectomy (green). Each dot represents one cell. The results show a high overlap when epithelial and stromal compartments are analyzed together (ES). The overlap is reduced in the case of only a minor involvement of stroma (E+s). The best segregation is seen when the epithelial compartment is analyzed by itself, indicating the highest change (variance) for the analyzed markers. The latter subdata is missing data from two biopsies, as most patients for which epithelial (E) compartments could be analyzed were initially diagnosed with lots of AC and thus only underwent one biopsy prior to prostatectomy.

Article Snippet: Cultured cells included primary human prostate epithelial cells (HPrEpiC, ScienCell, Carlsbad, CA) as normal primary cells, and LNCaP (American Type Culture Collection, Manassas, VA), an androgen-sensitive prostate cancer cell line.

Techniques:

Journal: Oncotarget

Article Title: Prostate cancer diagnosis using epigenetic biomarkers, 3D high-content imaging and probabilistic cell-by-cell classifiers

doi: 10.18632/oncotarget.18985

Figure Lengend Snippet: Performance of Logistic regression model with the development data set and Biomarkers I, utilizing epithelial cell only (A) and all subsets of all imaged cells (B). 5mC shows best performance as single marker in both cases, and is only exceeded by the combined marker panel.

Article Snippet: Cultured cells included primary human prostate epithelial cells (HPrEpiC, ScienCell, Carlsbad, CA) as normal primary cells, and LNCaP (American Type Culture Collection, Manassas, VA), an androgen-sensitive prostate cancer cell line.

Techniques: Marker

Journal: Oncotarget

Article Title: Prostate cancer diagnosis using epigenetic biomarkers, 3D high-content imaging and probabilistic cell-by-cell classifiers

doi: 10.18632/oncotarget.18985

Figure Lengend Snippet: Logistic regression model coefficients for epithelial cells only

Article Snippet: Cultured cells included primary human prostate epithelial cells (HPrEpiC, ScienCell, Carlsbad, CA) as normal primary cells, and LNCaP (American Type Culture Collection, Manassas, VA), an androgen-sensitive prostate cancer cell line.

Techniques:

Journal: Oncotarget

Article Title: Prostate cancer diagnosis using epigenetic biomarkers, 3D high-content imaging and probabilistic cell-by-cell classifiers

doi: 10.18632/oncotarget.18985

Figure Lengend Snippet: Predictions of the logistic model based on epithelial cells only

Article Snippet: Cultured cells included primary human prostate epithelial cells (HPrEpiC, ScienCell, Carlsbad, CA) as normal primary cells, and LNCaP (American Type Culture Collection, Manassas, VA), an androgen-sensitive prostate cancer cell line.

Techniques:

Journal: Oncotarget

Article Title: Prostate cancer diagnosis using epigenetic biomarkers, 3D high-content imaging and probabilistic cell-by-cell classifiers

doi: 10.18632/oncotarget.18985

Figure Lengend Snippet: Validation of KNN classification for predicting tissue pathological categories (including cancer stages) using epithelial cells only and Biomarkers I

Article Snippet: Cultured cells included primary human prostate epithelial cells (HPrEpiC, ScienCell, Carlsbad, CA) as normal primary cells, and LNCaP (American Type Culture Collection, Manassas, VA), an androgen-sensitive prostate cancer cell line.

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Prostate cancer diagnosis using epigenetic biomarkers, 3D high-content imaging and probabilistic cell-by-cell classifiers

doi: 10.18632/oncotarget.18985

Figure Lengend Snippet: Validation of KNN classification for predicting GS based on epithelial cells only with Biomarkers I

Article Snippet: Cultured cells included primary human prostate epithelial cells (HPrEpiC, ScienCell, Carlsbad, CA) as normal primary cells, and LNCaP (American Type Culture Collection, Manassas, VA), an androgen-sensitive prostate cancer cell line.

Techniques: Biomarker Discovery