Journal: Cancer Biology & Therapy

Article Title: CPT1A mediates the succinylation of SP5 which activates transcription of PDPK1 to promote the viability and glycolysis of prostate cancer cells

doi: 10.1080/15384047.2024.2329372

Figure Lengend Snippet: CPT1A is highly expressed in PCa tissues and cell lines. (a) GEPIA was used to analyze the dysregulated expression of CPT1A in PCa. PRAD: prostate adenocarcinoma. T: tumor and N: normal. (b) The expression of CPT1A in PCa tissues and the normal tissues was detected by qPCR. (c) The expression of CPT1A in PCa cell lines and the prostate epithelial cell line (PNT2) was detected by qPCR. * p <.05 in (A). ** p < .01, *** p < .001 vs PNT2 group.

Article Snippet: HEK-293T cells, normal human epithelial prostate PNT2 cells and the PCa cell lines including DU145, LNCap, 22Rv1 and VCaP were purchased from ATCC (USA) and were maintained in Dulbecco’s modified eagle medium containing 10% fetal bovine serum and 1% penicillin/streptomycin (all from Thermo Fisher Scientific, USA).

Techniques: Expressing

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Cell surface HER2 expression on quiescent prostate cancer cells. (A) Cell surface HER2 expression on PCa cell lines (PC3, DU145, C4-2B, LNCaP) and benign prostate epithelial cells (PNT2) cultured in 10% FBS measured by flow cytometry and mean fluorescence intensity (MFI). Open histograms represent an isotype control antibody. Shaded histograms are a HER2 antibody. The vertical line and arrow represent positive expression as defined by the isotype control antibody. (B) Flow cytometry for surface HER2 and BrdU incorporated into DNA of PC3 cells (C) Histograms showing HER2 cell surface expression on the same cell lines cultured in the presence or absence of FBS. (D) Quantification of HER2 surface expression from panel (B) as defined by mean fluorescence intensity (MFI). (E) Immunofluorescence imaging of PC3 (left) or C4-2B (right) cells cultured in either 10% FBS or serum starved conditions showing endogenous p27 and HER2 expression. HER2 (red), p27 (green) and nuclei (DAPI, blue). Scale bar, 50 μm. (F) Cell surface HER2 as measured by flow cytometry of PCa cells cultured with either 1 µM abemaciclib or 0.01% ethanol vehicle. (G) Western blot for HER2 from indicated cell lines cultured for 2 days with or without FBS. β-actin was a loading control. Data represent mean ± S.D. of triplicate wells. A representative biological replicate experiment is shown.

Article Snippet: Normal human prostate epithelial PNT2 cells (Cat #: 95012613) was purchased from Sigma ( St. Louis, MO ).

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence, Immunofluorescence, Imaging, Western Blot

Journal: Cancers

Article Title: Therapeutic and Diagnostic Potential of Folic Acid Receptors and Glycosylphosphatidylinositol (GPI) Transamidase in Prostate Cancer

doi: 10.3390/cancers16112008

Figure Lengend Snippet: Folate receptor 1 expression in healthy PNT2 and prostate cancer cells (LNCaP). Folate receptor 1 (FR1/red) was quantified by qRT-PCR ( A ), Western blot ( B ) and immunofluorescence ( C , D ). The graphs show relative values calculated to healthy control cells. In ( B ), a representative Western blot membrane with the corresponding marker band (black) at 37 kDa is shown. The effective protein load was detected by BioRad-stain-free technology, enabling adjustment of protein loads by quantification of total protein levels. p -values of * 0.05, ** 0.01 and *** 0.001 were labeled with one to three asterisks indicating significances from at least 3 independent experiments. Scalebar 100 µm.

Article Snippet: For all experiments, the two cell lines, LNCaP (Merck, 89110211, prostate cancer human, Rahway, NJ, USA) and PNT2 (Merck, 95012613, healthy epithelial prostate cell), were used.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Membrane, Marker, Staining, Labeling

Journal: Cancers

Article Title: Therapeutic and Diagnostic Potential of Folic Acid Receptors and Glycosylphosphatidylinositol (GPI) Transamidase in Prostate Cancer

doi: 10.3390/cancers16112008

Figure Lengend Snippet: Glycosylphosphatidylinositol transamidase expression and folate receptor 1 localization in prostate cancer cells (LNCaP) and healthy prostate epithelial cells (PNT2). Glycosylphosphatidylinositol transamidase (GPI-T/blue) was quantified by qRT-PCR ( A ), Western blot ( B ) and immunofluorescence ( C , D ) in healthy PNT2 and prostate cancer cells (LNCaP). In ( D ), immunofluorescence against FR1 (red) is additionally shown; white arrows indicate membrane signals. Correlation between FR1-membrane fluorescence intensity and GPI-T fluorescence intensity in healthy PNT2 (gray squares) and LNCaP cancer cells (red circles) with corresponding correlation value Pearson R is shown in ( E ). The graphs ( A – C ) show the relative values calculated for the healthy control cell. ( B ) shows a representative Western blot membrane with the corresponding marker bands at 37 kDa and 50 kDa. The effective protein load was detected by BioRad-stain-free technology, enabling adjustment of protein loads by quantification of total protein levels. p -values of * 0.05, ** 0.01 and *** 0.001 were labeled with one to three asterisks indicating significances from at least 3 independent experiments. Scalebar 100 µm.

Article Snippet: For all experiments, the two cell lines, LNCaP (Merck, 89110211, prostate cancer human, Rahway, NJ, USA) and PNT2 (Merck, 95012613, healthy epithelial prostate cell), were used.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Membrane, Fluorescence, Marker, Staining, Labeling

Journal: Cancers

Article Title: Therapeutic and Diagnostic Potential of Folic Acid Receptors and Glycosylphosphatidylinositol (GPI) Transamidase in Prostate Cancer

doi: 10.3390/cancers16112008

Figure Lengend Snippet: Folate-functionalized lipoplexes for selective transfer of eGFP-plasmid DNA into cancer cells. As a control, eGFP plasmid DNA was transferred into healthy PNT2 and LNCaP cancer cells using unmodified standard lipoplexes. In addition, an increasing concentration of NHS-PEG-FA was used to characterize specific uptake via FR1 examined by eGFP-positive cells using microscopy ( A , B ). In addition, quantification was performed by flow cytometry, which provided information on the changes in transfection efficiency for each cell type ( C ) and the altered specificity between FR1-mediated uptake in PNT2 and LNCaP ( D ). Finally, in co-culture, this specific transfection of standard lipoplexes ( E ) and FA-functionalized lipoplexes ( F ) was further investigated. The healthy cells are shown demarcated in the white frames and could be identified by GPI-T immunofluorescence (weak blue) based on the lower fluorescence intensity compared to LNCaP cells (bright blue). Quantification of enhanced selectivity in co-culture is shown in ( G ). p -values of * 0.05, ** 0.01 and *** 0.001 were labeled with one to three asterisks indicating significances from at least 3 independent experiments. n.s., not significant. Scale bar 100 µm.

Article Snippet: For all experiments, the two cell lines, LNCaP (Merck, 89110211, prostate cancer human, Rahway, NJ, USA) and PNT2 (Merck, 95012613, healthy epithelial prostate cell), were used.

Techniques: Plasmid Preparation, Concentration Assay, Microscopy, Flow Cytometry, Transfection, Co-Culture Assay, Immunofluorescence, Fluorescence, Labeling

Journal: Scientific Reports

Article Title: 2D exfoliated black phosphorus influences healthy and cancer prostate cell behaviors

doi: 10.1038/s41598-021-85310-6

Figure Lengend Snippet: Effect of 2D BP on PNT-2 ( A , B ) and PC-3 ( C , D ) proliferation with and without infrared irradiation (NIR). The experiments performed on PNT-2 and PC-3 cell proliferation using Alamar Blue assay have shown that irradiated ( A – C ) and not irradiated ( B – D ) 2D BP (5 μg/mL) did not exert cytotoxic effects on PNT-2 and PC-3 after 1 and 3 days of exposure. The same result was obtained in presence of not irradiated 2D BP (25 μg/mL) at day 1 of cell culture ( A ). Not irradiated 2D BP (75 μg/mL) at day 3 and irradiated 2D BP (25–75 μg/mL) at all-time points significantly (° p ≤ 0.001; # p ≤ 0.0001) decreased cell viability of healthy prostatic cells (PNT-2) compared to control ( A , B ). Results are mean ± SEM of 3–4 experiments. The irradiated ( C ) and not irradiated ( D ) lowest concentration (5 μg/mL) of 2D BP induced a significant (° p ≤ 0.001; # p ≤ 0.0001) decrease in PC-3 (cancer cells) proliferation after 1 and 3 days of exposure compared to control. This effect results more significant in presence of irradiation ( A ). The best inhibition in PC-3 proliferation was obtained after 3 days of irradiated 2D BP exposure at concentration of 75 μg/mL # p ≤ 0.0001 ( A ). Results are mean ± SEM of 3–4 experiments.

Article Snippet: The experiments were performed using PNT-2 (human Normal prostate epithelium immortalized with SV40) and PC-3 (human Caucasian prostate adenocarcinoma) cell lines purchased from Sigma-Aldrich (Milano, Italy).

Techniques: Irradiation, Alamar Blue Assay, Cell Culture, Concentration Assay, Inhibition

Journal: Scientific Reports

Article Title: 2D exfoliated black phosphorus influences healthy and cancer prostate cell behaviors

doi: 10.1038/s41598-021-85310-6

Figure Lengend Snippet: Effect of not irradiated (-NIR) 2D BP on PNT-2 and PC-3 cell density (qualitative analysis). PNT-2 and PC-3 cells grown in absence ( A , C ) and in presence ( B , D ) of 2D BP. The exposure of PNT-2 cells to 2D BP does not change cell density ( B ) compared to control ( A ). By contrasts, 24 h of 2D BP treatment reduces PC-3 density ( C ) compared to cells in the control ( D ). The images are representative of three experiments. Cell morphological features at 72 h 2D BP treatment are reported in Fig. of Supporting information section.

Article Snippet: The experiments were performed using PNT-2 (human Normal prostate epithelium immortalized with SV40) and PC-3 (human Caucasian prostate adenocarcinoma) cell lines purchased from Sigma-Aldrich (Milano, Italy).

Techniques: Irradiation

Journal: Scientific Reports

Article Title: 2D exfoliated black phosphorus influences healthy and cancer prostate cell behaviors

doi: 10.1038/s41598-021-85310-6

Figure Lengend Snippet: Reactive Oxygen Species (ROS) production from PNT-2 ( A , C ) and PC-3 ( B , D ) cells with (+ NIR) and without infrared (-NIR) irradiation. The exposure of PNT-2 and PC-3 cells (healthy and cancer cells) to H 2 O 2 /Fe 2+ (2 mM) significantly (* p ≤ 0.05, # p ≤ 0.0001) increases ROS production compared to control ( A – D ). As irradiated 2D BP with H 2 O 2 /Fe 2+ (2 mM) irradiated 2D BP without H 2 O 2 /Fe 2+ (2 mM) stimulation was able to induce a significant ( #,§ p ≤ 0.0001) increase in ROS generation in PC-3 cells ( B , D ). Not irradiated 2D BP significantly (# p ≤ 0.0001) reduced basal ROS levels compared to control on PNT-2 cell line ( C ). Conversely, not irradiated 2D BP did not inhibit ROS generation induced by H 2 O 2 /Fe 2+ (2 mM) in PNT-2 cells compared to H 2 O 2 /Fe 2+ (2 mM) alone ( C ). Results are mean ± SEM of 3–4 experiments.

Article Snippet: The experiments were performed using PNT-2 (human Normal prostate epithelium immortalized with SV40) and PC-3 (human Caucasian prostate adenocarcinoma) cell lines purchased from Sigma-Aldrich (Milano, Italy).

Techniques: Irradiation

Journal: Scientific Reports

Article Title: 2D exfoliated black phosphorus influences healthy and cancer prostate cell behaviors

doi: 10.1038/s41598-021-85310-6

Figure Lengend Snippet: Nitrites production from PNT-2 and PC-3 cells without ( A ) and with ( B ) infrared irradiation (NIR). The results suggest that 48 h of exposure to 2D BP without and with irradiation significantly ( § p ≤ 0.0001) increase basal nitrites values in PC-3 cells compared to PNT-2. This effect is more evident in presence of pathological conditions obtained through LPS (1 μg/ml) stimulation ( # p ≤ 0.0001) and irradiation ( # ,° p ≤ 0.0001), ( B ). Results are mean ± SEM of 3–4 experiments.

Article Snippet: The experiments were performed using PNT-2 (human Normal prostate epithelium immortalized with SV40) and PC-3 (human Caucasian prostate adenocarcinoma) cell lines purchased from Sigma-Aldrich (Milano, Italy).

Techniques: Irradiation

Journal: Scientific Reports

Article Title: 2D exfoliated black phosphorus influences healthy and cancer prostate cell behaviors

doi: 10.1038/s41598-021-85310-6

Figure Lengend Snippet: GPX-3 expression in PNT-2 ( A , B ) and PC-3 ( C , D ) cells in presence of 2D BP without infrared (-NIR) irradiation. PNT-2 and PC-3 cell GPX-3 expression in absence (A,C) and in presence 2D BP exposure at concentrations of 5 (B,D) μg/mL after of 24 h of cell culture. The images are representative of three experiments.

Article Snippet: The experiments were performed using PNT-2 (human Normal prostate epithelium immortalized with SV40) and PC-3 (human Caucasian prostate adenocarcinoma) cell lines purchased from Sigma-Aldrich (Milano, Italy).

Techniques: Expressing, Irradiation, Cell Culture

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicles From Prostate Cancer‐Corrupted Osteoclasts Drive a Chain Reaction of Inflammatory Osteolysis and Tumour Progression at the Bone Metastatic Site

doi: 10.1002/jev2.70091

Figure Lengend Snippet: Metastatic PCa cells promote OC activity and induce pathological phenotype. (a) Representative IVIS image showing PCa‐transplanted xenograft mice ( N = 3) on Day 35. Tumours in the hind limbs were evaluated based on the photon radiance of cancer cell bioluminescence. (b) Representative images of HE‐, osteoclastic and osteoblastic marker TRAP/ALP‐ and cancer‐specific marker LAT1 stained sections of bone metastatic sites in the xenografted mouse which was shown in (a). All images were obtained with an all‐in‐one fluorescence microscope using a 4x objective for low‐, 10x objective for middle‐, and 40x objective for high‐magnification. Black arrowheads indicate the tumour metastatic site, and white arrowheads indicate OCs. The metastatic tumour developed in the femur. Scale bar indicates 300 µm. (c) Schematic images of the transwell co‐culture system for obtaining cancer‐associated OCs (CAOCs) and normal osteoclasts (NOCs). OC precursor RAW 264.7 cells were grown into mature OCs in the lower compartment. Mature OCs with osteolytic PC3M cells (OCP cells) and mature OCs with osteoblastic C4‐2B cells (OCC cells) were used as CAOCs. Mature OCs with blank transwells (OC cells) and mature OCs with PNT2 (OCN cells) were prepared as control NOCs. (d) Schematic images of transwell co‐culture for observing CAOCs and NOCs. (e) Representative TRAP staining images of each OC. Bars represent 100 µm. ( f) Representative TRAP staining images of each OCs treated with denosumab at the concentration of 10 µg/mL. Bars represent 100 µm.

Article Snippet: PC3M, C4‐2B, and the immortalized normal human prostatic epithelial cell line PNT2 (DS Pharma Biomedical Co., Ltd, Osaka, Japan) were cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10%‐inactivated foetal bovine serum (FBS, Gibco, Thermo Fisher Scientific) and 1% antibiotic‐antimycotic (AA) solution (Gibco, Thermo Fisher Scientific) at 37 °C in atmosphere of 95% air and 5% CO 2 .

Techniques: Activity Assay, Marker, Staining, Fluorescence, Microscopy, Co-Culture Assay, Control, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicles From Prostate Cancer‐Corrupted Osteoclasts Drive a Chain Reaction of Inflammatory Osteolysis and Tumour Progression at the Bone Metastatic Site

doi: 10.1002/jev2.70091

Figure Lengend Snippet: Inflammatory‐related signalling pathways are enhanced in CAOCs. (a) Schematic protocol of co‐culture and gene expression analysis of CAOCs. (b) Heat map showing differentially expressed genes (CAOC vs. OCN, change >2.0‐fold and p < 0.05) in RAW 264.7, co‐cultured with PCa cells ( n = 2) in the presence of RANKL. (c) The number of differentially expressed genes in mature OCs co‐cultured with PC3M cells (OCP cells) or C4‐2B cells (OCC cells) compared to that in OCN cells co‐cultured with PNT2 cells (OCN cells). (d) PCA of gene expression of each type of OCs. (e) Pathway analysis of selected genes that were significantly upregulated in CAOCs compared to OCN cells. (f) GSEA of CAOC versus NOC, highlighting pro‐inflammatory phenotypes. NES: normalized enrichment score. p value was calculated using GSEA. (g) Expression levels of Il‐1b , Casp1 , Il‐6 and Tnf in gene sets of inflammation‐related genes. OC co‐cultured with a blank insert (OC cells) or PNT2 (OCN cells), and OC co‐cultured with PC3M (OCP cells) or C4‐2B (OCC cells) are presented. The blue, green, yellow and red dots represent the OC, OCN, OCP and OCC cell data, respectively.

Article Snippet: PC3M, C4‐2B, and the immortalized normal human prostatic epithelial cell line PNT2 (DS Pharma Biomedical Co., Ltd, Osaka, Japan) were cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10%‐inactivated foetal bovine serum (FBS, Gibco, Thermo Fisher Scientific) and 1% antibiotic‐antimycotic (AA) solution (Gibco, Thermo Fisher Scientific) at 37 °C in atmosphere of 95% air and 5% CO 2 .

Techniques: Co-Culture Assay, Gene Expression, Cell Culture, Expressing