human pca cell lines du145  (ATCC)

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: Characterization of PCa EVs. (A) EV size distribution measured by nanoparticle tracking analysis. (B) EV morphology visualized by transmission electron microscopy. Scale bars are 300 nm. (C) Western blot analysis was performed to investigate the expression levels of TSG101, Alix, Hsc70, CD9, calnexin, and cytochrome c in PC3 and DU145 cells and in their EVs. One representative of three experiments performed is shown.

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Expressing

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: PCa EVs promote lipolysis in adipocytes. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h. Lipid accumulation was then evaluated by cytofluorimetric analysis after staining with Bodipy 1 μM for 30 min. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h. FFA release was then evaluated by colorimetric assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (C) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h. Glycerol release was then evaluated by colorimetric assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (D) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 6 h. cAMP levels were then evaluated by ELISA assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (E) After PCa EV treatment (30 μg/mL for 12 h), Western blot analysis was performed to investigate the expression levels of PKA substrates, HSL, G0S2, and ATGL in 3T3‐L1 adipocytes. GAPDH expression was evaluated as a loading control. One representative of three experiments performed is shown. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control).

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Staining, Control, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: The secretome from EV‐treated adipocytes increases PCa cell growth. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (96 h). Cell proliferation was then evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control). (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (72 h). Clonogenic ability was then evaluated by colony formation assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control), ** p < 0.01 vs. Adipo CM (control).

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Trypan Blue Exclusion Assay, Control, Colony Assay

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: The secretome from EV‐exposed adipocytes enhances PCa cell invasive potential. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Cell migration was then evaluated by wound healing assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05. Scale bars are 200 μm. (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Cell migration was then evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control), *** p < 0.001 vs. Adipo CM (control). (C) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (48 h). Anoikis was then evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control). (D) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Cell invasion was then evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. Adipo CM (control).

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Migration, Wound Healing Assay, Transwell Assay, Control, Trypan Blue Exclusion Assay

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: FFAs in EV‐conditioned adipocyte secretome are taken up by PCa cells and mediate the above pro‐tumor effects. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). FFA uptake was then evaluated by colorimetric assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Dunnet's test. *** p < 0.001. (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Lipid accumulation was then evaluated by cytofluorimetric analysis after staining with Bodipy 1 μM for 30 min. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. *** p < 0.001 vs. C (control). (C) After delipidation, EV‐Adipo CM was given to PC3 and DU145 cells (72 h). Cell proliferation was then evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05, ** p < 0.01. (D) After delipidation, EV‐Adipo CM was given to PC3 and DU145 cells (24 h). Cell migration was then evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05.

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Colorimetric Assay, Staining, Control, Trypan Blue Exclusion Assay, Migration, Transwell Assay

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: FFAs in EV‐conditioned adipocyte secretome trigger the Akt signaling in PCa cells. (A) After EV‐Adipo CM treatment (24 h), Western blot analysis was performed to investigate the expression levels of p‐Akt in PC3 and DU145 cells. GAPDH expression was evaluated as a loading control. One representative of three experiments performed is shown. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. *** p < 0.001 vs. C (control). (B) After BEZ235 treatment (100 nM, 72 h) following EV‐Adipo CM treatment (3 h), PC3 and DU145 cell proliferation was evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05, *** p < 0.001. (C) After BEZ235 treatment (100 nM, 24 h) following EV‐Adipo CM treatment (3 h), PC3 and DU145 cell migration was evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05, *** p < 0.001.

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Western Blot, Expressing, Control, Trypan Blue Exclusion Assay, Migration, Transwell Assay

Journal: bioRxiv

Article Title: IGFs regulate cancer cell immune evasion in prostate cancer

doi: 10.1101/2024.12.03.626600

Figure Lengend Snippet: Subconfluent DU145, 22Rv1 and Myc-CaP cultures were serum-starved for 24 hr, stimulated with 30 nM IGF-1 or solvent (control) for 24 hr and RNA was extracted for RNA-seq. A. IGF-induced DEGs unique to and shared between the 3 PCa cell lines showing number (%). B. Expression heatmap of 57 DEGs in common between the 3 cell lines including 31 significantly upregulated and 26 downregulated DEGs and annotation into three curated immune relevant categories: cytokine signaling, immune response associated and Antigen Processing Machinery (APM). C. GSEA was performed using the three curated immune-associated pathway datasets (Supplementary Table S11). Pie charts represent the distribution of enriched pathways in each cell line. D. Heatmap of selected enriched pathways and enrichment scores at padj<0.05 and <0.01 for each cell line. E. GSEA enrichment plots showing response to interferon gamma (IFNγ), systemic lupus erythematosus (SLE), regulation of JNK cascade and antigen processing and cross presentation.

Article Snippet: Human PCa cell lines DU145 and 22Rv1 were from Cancer Research UK Laboratories (Clare Hall Hertfordshire UK) and Professor Sir Walter Bodmer (University of Oxford, UK) respectively.

Techniques: Solvent, Control, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: IGFs regulate cancer cell immune evasion in prostate cancer

doi: 10.1101/2024.12.03.626600

Figure Lengend Snippet: RNA was extracted from DU145, 22Rv1, and Myc-CaP cells 24 hr after serum-starving and IGF-treatment as in , and gene expression was assessed by RT-qPCR. Expression of: A . TAP1 , TAP2 mRNA in all cell lines and Tapbp in MycCaP; B. ERAP mRNA, C. β2M mRNA. D, E . Class I complexes are stabilized at the cell surface by high affinity peptide binding, while peptide-free (empty) heterodimers are generally unstable at the cell surface at physiological temperature. We used Class I antibody to H-2Kq, appropriate to the genotype of FVB mice from which Myc-CaP cells were derived . Analysis by flow cytometry of H2Kq surface expression in Myc-CaP cells treated with 30 nM IGF-1 or solvent for 2 or 5 days, showing representative flow cytometry plots at 5 days and n=3 independent experiments. D: percentage H2Kq positivity, E: H2Kq MFI. All graphs represent mean ± SEM of three independent analyses (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, nonsignificant by one-way ANOVA).

Article Snippet: Human PCa cell lines DU145 and 22Rv1 were from Cancer Research UK Laboratories (Clare Hall Hertfordshire UK) and Professor Sir Walter Bodmer (University of Oxford, UK) respectively.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Binding Assay, Derivative Assay, Flow Cytometry, Solvent

Journal: bioRxiv

Article Title: IGFs regulate cancer cell immune evasion in prostate cancer

doi: 10.1101/2024.12.03.626600

Figure Lengend Snippet: A . PCa cells were serum starved for 24 hrs, treated with 30 nM IGF-1 or solvent and expression of CD274 assessed by qPCR (n=3 independent assays, mean ± SEM). B. DU145 cells were treated as in A and PD-L1 surface expression measured by flow cytometry. Left, representative flow histogram; right, quantification of MFI (n = 3 independent assays, mean ± SEM). C. DU145 cells were pre-treated with 1 μg/mL Actinomycin D for 2 hrs and then 30 nM IGF-1 was added. After 24 hr PD-L1 surface expression was measured using flow cytometry. Graphs represent mean ± SEM. D. DU145 cells were pre-treated with 300 nM IGF-1R inhibitor BMS-754807, 5 μM AKT inhibitor AZD5363 and 50 nM MEK inhibitor trametinib for 1 hr and 30 nM IGF-1 was added for 24 hrs. Parallel cultures were used for RNA extraction and RT-qPCR for CD274 (PD-L1) mRNA quantification (upper graph) and surface protein expression measured by flow cytometry (lower, n=3 independent assays, mean ± SEM). (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, nonsignificant by one-way ANOVA).

Article Snippet: Human PCa cell lines DU145 and 22Rv1 were from Cancer Research UK Laboratories (Clare Hall Hertfordshire UK) and Professor Sir Walter Bodmer (University of Oxford, UK) respectively.

Techniques: Solvent, Expressing, Flow Cytometry, RNA Extraction, Quantitative RT-PCR