pnt2 human cell line  (Millipore)

Journal: British Journal of Cancer

Article Title: Peroxisomal β-oxidation enzyme, DECR2, regulates lipid metabolism and promotes treatment resistance in advanced prostate cancer

doi: 10.1038/s41416-023-02557-8

Figure Lengend Snippet: a Illustration of fatty acid oxidation in the peroxisome and mitochondria. Thioridazine (TDZ) is an inhibitor of perFAO. b Heatmap of peroxisomal β-oxidation (perFAO) gene expression in Taylor and Grasso cohorts. We manually curated a list of perFAO genes based on Gene Ontology pathway. Cell viability of ( c ) castrate-resistant C42B, 22Rv1 and V16D, and enzalutamide-resistant MR49F prostate cancer cell lines across a range of TDZ doses. d C42B and 22Rv1 prostate cancer cell lines treated with 2.5 µM TDZ were assessed for cell migration using transwell migration assay. Scale bar: left, 100 µm; right, 200 µm. e Immunostaining for proliferative marker Ki67 in vehicle (VEH) or TDZ-treated (20 μM) patient-derived explants (PDEs). Immunohistochemical staining and quantification of the proliferative marker Ki67 is shown ( n = 11). Scale bar, 50 µm. f DECR2 expression with respect to tumour progression in four independent datasets. DECR2 levels were analysed in normal, primary, and metastatic castrate-resistant or neuroendocrine tissue samples. g The association of DECR2 expression and disease-free survival in the MSKCC (Taylor) cohort. h DECR2 protein expression in non-malignant prostate cell lines (PNT1 and PNT2) and prostate cancer cell lines (LNCaP, VCaP, C42B, 22Rv1, V16D, PC3), including enzalutamide-resistant prostate cancer cell line (MR49F). i Left : Representative DECR2 IHC staining of benign prostate tissues and prostate cancer tissues. Scale bar, 50 µm. Middle : DECR2 protein expression in a validation cohort consisting of benign prostate tissues ( n = 3) and prostate cancer tissues ( n = 10). Right : Intra-tissue IHC analysis of DECR2 expression in prostate cancer tissues ( n = 10). All cell line data are representative of at least two independent experiments and presented as mean ± s.e.m of triplicate wells. Statistical analysis was performed using ordinary one-way ANOVA or two-tailed student’s t test. Data in ( e ) were statistically analysed using paired t-test. Data in ( g ) were statistically analysed using a two-sided log-rank test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Human immortalised normal prostate epithelial cell lines PNT1 and PNT2 were obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Gene Expression, Migration, Transwell Migration Assay, Immunostaining, Marker, Derivative Assay, Immunohistochemical staining, Staining, Expressing, Immunohistochemistry, Biomarker Discovery, Two Tailed Test

Journal: bioRxiv

Article Title: TGF-β induces matrisome pathological alterations and EMT in patient-derived prostate cancer tumoroids

doi: 10.1101/2023.04.03.534859

Figure Lengend Snippet: (A) Representative two-photon images of PNT2 cells taken after 10 days of colonization of two tumour dECMs obtained from two different patients. The images represent maximum projections of 3D z-stack images. Cells were stained with DAPI. The colour code refers to the depth to which nuclei are found inside the dECM. Scale bars: 100 µm. (B) Representative confocal image of PNT2 cells cultured for 10 days on tumour dECM. The cells are stained for vimentin (VIM, red), F-actin (green) and nuclei are counterstained DAPI (blue). Scale bar: 25 µm. (C) Flow cytometry dot plots of vimentin and E-cadherin expression for PNT2 cells, after a week of incubation with 2D and 3D dECMs, in the presence or absence of TGF-β. CTRL cells grown directly on the surface of culture flasks are also shown. (D) Vimentin expression quantification based on flow cytometry analysis. Statistical analysis was performed by two-way ANOVA. (E) Representative dot plot of PCTs containing E-cadherin (E-CAD), vimentin and dual positive cells. Histogram representation of the epithelial cells from the PCTs expressing vimentin, for control condition and cultured in 3D dECMs in the presence or absence of TGF-β inhibitor (F) Vimentin expression quantification based on flow cytometry analysis, for E-CAD-positive cells. Statistical analysis was performed by one-way ANOVA. (G) Graphical representation of the experimental design and the results obtained.

Article Snippet: PNT2 human cell line was purchased from Sigma Aldrich (95012613-1VL) and cultured in RPMI 1640 containing 2 mM Glutamine and 10 % Foetal Bovine Serum (FBS), as suggested by the manufacturer.

Techniques: Staining, Cell Culture, Flow Cytometry, Expressing, Incubation

Journal: Cells

Article Title: N -Glycomic Analysis of the Cell Shows Specific Effects of Glycosyl Transferase Inhibitors

doi: 10.3390/cells10092318

Figure Lengend Snippet: Confocal microscopy of PNT2 cells were the top cells without 6AF treatment (control) and the bottom cells with 6AF treatment. Cells were stained with CellMask™ (green) and 7-Azido-4-Methylcoumarin (red) (ThermoFisher, Waltham, MA, USA). The merge section is the overlap between CellMask™ and 7-Azido-4-Methylcoumarin.

Article Snippet: Dithiothreitol (DTT), iodoacetamide (IAA), and a human immortalized prostate PNT2 cell line were purchased from Sigma-Aldrich, St. Lousi, MO, USA.

Techniques: Confocal Microscopy, Staining

Journal: Cells

Article Title: N -Glycomic Analysis of the Cell Shows Specific Effects of Glycosyl Transferase Inhibitors

doi: 10.3390/cells10092318

Figure Lengend Snippet: The relative abundance profile of each glycosylation inhibitor with its control counterpart plotted on stack columns with ( A ) Caco-2, ( B ) A549, and ( C ) PNT2 cell lines. Stack column plots are color coded by glycan subtypes.

Article Snippet: Dithiothreitol (DTT), iodoacetamide (IAA), and a human immortalized prostate PNT2 cell line were purchased from Sigma-Aldrich, St. Lousi, MO, USA.

Techniques:

Journal: eLife

Article Title: Human DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.7554/eLife.54166

Figure Lengend Snippet: ( A ) Violin plots of DECR1 protein overexpression in primary PCa tissues compared to benign prostate tissues in two independent datasets. ( B ) Representative DECR1 IHC staining of benign prostate tissues and PCa tissues (negative control stain included in bottom right box). Scale bar, 50 µm. ( C ) Violin plot of DECR1 protein expression in a validation cohort consisting of benign prostate tissues (n = 3) and PCa tissues (n = 8) ( top panel ). Intra-tissue IHC analysis of DECR1 expression in PCa tissues (n = 8) ( bottom panel ). ( D ) DECR1 protein expression in non-malignant prostate cell lines (PNT1 and PNT2) and PCa cell lines (LNCaP, VCaP, 22RV1, C42B and PC3). ( E ) Immunocytochemistry staining of LNCaP cells to determine the subcellular localization of DECR1: nuclei were labelled using DAPI; mitochondria were labelled using MitoTracker Red; and DECR1 proteins were labelled using Alexa Fluor 488 secondary antibody, (Scale bar = 10 μm). Immunoblot of PNT1 and LNCaP cells separated into cytosolic, mitochondrial and nuclear fractions and incubated with poly (ADP-ribose) polymerase (PARP) and cytochrome-C antibodies to mark nuclear and mitochondrial fractions. Data are represented as violin plots in GraphPad prism: the horizontal line within the violin represents the median. Statistical analysis was performed using a Mann-Whitney two-tailed t-test (A and C top panel ), or two-tailed paired t-test (C bottom panel ): *p<0.05, **p<0.01 and ***p<0.001.

Article Snippet: The human normal prostate epithelial cell lines PNT1 and PNT2 were obtained from the European Collection of Authenticated Cell Cultures (ECACC), and prostate carcinoma cells LNCaP, VCaP, and 22RV1 were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA).

Techniques: Over Expression, Immunohistochemistry, Negative Control, Staining, Expressing, Biomarker Discovery, Immunocytochemistry, Western Blot, Incubation, MANN-WHITNEY, Two Tailed Test

Journal: eLife

Article Title: Human DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.7554/eLife.54166

Figure Lengend Snippet: ( A ) Cell viability after DECR1 knockdown in non-malignant PNT1 prostate cells; hormone-responsive PCa cell lines (LNCaP and VCaP); castrate-resistant V16D and 22RV1 cell lines and enzalutamide-resistant MR94F cells cultured in full serum media. ( B ) Cell viability and cell death of stable DECR1 knockdown LNCaP cells cultured in full serum media. ( C ) Cell viability of stable DECR1-overexpressed LNCaP cells cultured in full serum media. Cell viability and cell death were measured using trypan blue exclusion following 96 hr DECR1 knockdown. Percentages are represented relative to the control siRNA; n = 3 independent experiments per cell line. ( D ) Clonogenic cell survival of LNCaP cells was assessed using colony formation assay. Stable DECR1-overexpressed cells or ( E ) stable DECR1 knockdown was achieved using two different short hairpin (sh) vectors and DECR1 expression was confirmed using western blot. Cells were cultured for 2 weeks, washed with PBS, fixed with paraformaldehyde and stained with 1% crystal violet for 30 min. Colonies with more than 50 cells were counted manually; data shown is representative of n = 2 independent experiments. ( F ) LNCaP and 22RV1 cell growth in 3D spheres. Spheroids were prepared using the hang drop assay following 48 hr DECR1 knockdown. Spheroid volumes were determined after five days of culturing the cells in 20 µl drops; at least 25 spheres per cell line were assessed using the ReViSP software, n = 3 independent experiments per cell line. ( G ) LNCaP, 22RV1 and MR49F cell migration and ( H ) 22RV1 cell invasion were assessed using a transwell migration/invasion assay. Cells were transfected with DECR1 siRNA or control siRNA for 48 hr prior to the assay; data shown is representative of n = 3 independent experiments. ( I ) Violin plots of mKi67 and DECR1 mRNA expression in subcutaneous LNCaP tumors (n = 5 mice, shControl; n = 4 mice, shDECR1). ( J ) Representative Ki67 IHC staining of subcutaneous LNCaP tumors. Scale bar, 100 μm. Data in bar graphs are represented as the mean ± s.e.m. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. ( K ) Tumor growth of intraprostatically injected LNCaP cells (shControl and shDECR1). (J) Lung luminescence readings following DECR1 knockdown in mice. Data are presented as mean ± s.e.m. Statistical analysis was performed using two-way ANOVA or two-tailed student’s t-test: *p<0.05 and ***p<0.001.

Article Snippet: The human normal prostate epithelial cell lines PNT1 and PNT2 were obtained from the European Collection of Authenticated Cell Cultures (ECACC), and prostate carcinoma cells LNCaP, VCaP, and 22RV1 were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA).

Techniques: Knockdown, Cell Culture, Control, Colony Assay, Expressing, Western Blot, Staining, Software, Migration, Invasion Assay, Transfection, Immunohistochemistry, Injection, Two Tailed Test