prostate carcinoma 3 cell line pc 3 castration resistant  (ATCC)

Journal: Scientific Reports

Article Title: Immune profiling identifies the contribution of extracellular vesicles to immune modulation and progression in prostate cancer

doi: 10.1038/s41598-025-31838-w

Figure Lengend Snippet: Characterisation of prostate cancer-derived extracellular vesicles. EVs post differential ultracentrifugation were characterised using nanoparticle tracking analysis (NTA), immunoblotting analysis, and scanning electron microscopy (SEM). a ). Particle concentration and size of which three replicates of each sample were analysed by NTA independently. Data analysed by one-way ANOVA test and presented as mean bars (n = 3) ± SEM, the significant p-value is reported; * indicates p < 0.05, ** indicates p < 0.01. n.s. = not significant. b ) Representative graphs of prostate cancer EVs distribution from NTA software. c )Immunoblotting analysis of EVs from PC-3, LNCaP and DU 145 cells and cell lysates. Detection of EVs associated positive markers, syntenin, CD63, and negative marker calnexin. SEM images of d ) PC-3 EVs, e ) LNCaP EVs and f) DU 145 EVs with a range of 90 nm to 130 nm (magnification 62000x).

Article Snippet: The androgen-dependent LNCaP metastatic prostate carcinoma cell line (CRL-1740), androgen-independent prostate carcinoma PC-3 cell line (CRL-1435) and androgen-independent prostate carcinoma DU 145 cell line (HTB-81) were purchased from the American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Western Blot, Electron Microscopy, Concentration Assay, Software, Marker

Journal: Scientific Reports

Article Title: Immune profiling identifies the contribution of extracellular vesicles to immune modulation and progression in prostate cancer

doi: 10.1038/s41598-025-31838-w

Figure Lengend Snippet: Effect of PC-3 and LNCaP EVs on monocyte populations. PBMCs were treated with PC-3 EVs, LNCaP EVs, DU 145 EVs and t-NEPC LNCaP EVs for 24 h. a ) Comparison of % CD14 + CD16 + rare monocytes from 10 healthy volunteers treated with EVs derived from PC-3, LNCaP and DU 145. b ) Comparison of % CD14 + CD16 + rare monocytes treated with EVs derived from PC-3, LNCaP and DU 145 in respect to the untreated samples (CTRL). c ) Comparison of % CD14 + CD16 + rare monocytes treated with EVs derived from PC-3 compared to t-NEPC LNCaP treated cells. Data analysed by one-way ANOVA and presented as mean bars (n = 10) ± SEM, the significant p -value is reported; **** p < 0.0001; *** indicates p < 0.001; n.s. = not significant.

Article Snippet: The androgen-dependent LNCaP metastatic prostate carcinoma cell line (CRL-1740), androgen-independent prostate carcinoma PC-3 cell line (CRL-1435) and androgen-independent prostate carcinoma DU 145 cell line (HTB-81) were purchased from the American Type Culture Collection (ATCC).

Techniques: Comparison, Derivative Assay

Journal: Scientific Reports

Article Title: Immune profiling identifies the contribution of extracellular vesicles to immune modulation and progression in prostate cancer

doi: 10.1038/s41598-025-31838-w

Figure Lengend Snippet: PC-3-EVs effects on innate lymphoid and NK cells compared to LNCaP-EVs. Effect of prostate cancer EVs on innate immune cells. a ) The percentage of CD4 + HLA-DR + after treatment with EVs derived from PC-3 and DU 145 compared to LNCaP treatment. b ) The percentage of CD8 + HLA-DR + treated with EVs derived from PC-3 and DU 145 in comparison to LNCaP treatment. c ) Comparison of the ratio of CD4 + HLA-DR + regarding the CD4 + . d ) Comparison of ratio of CD8 + HLA-DR + in regard to the CD8 + . Data analysed by one-way ANOVA and presented as mean bars (n = 10) ± SEM, the significant p -value is reported; * indicates p < 0.05; n.s. = not significant.

Article Snippet: The androgen-dependent LNCaP metastatic prostate carcinoma cell line (CRL-1740), androgen-independent prostate carcinoma PC-3 cell line (CRL-1435) and androgen-independent prostate carcinoma DU 145 cell line (HTB-81) were purchased from the American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Comparison

Journal: Scientific Reports

Article Title: Immune profiling identifies the contribution of extracellular vesicles to immune modulation and progression in prostate cancer

doi: 10.1038/s41598-025-31838-w

Figure Lengend Snippet: T-NEPC LNCaP EVs effects on innate lymphoid and NK cells. Effect of t-NEPC LNCaP EVs on innate immune cells. a ) The percentage of CD4 + HLA-DR + after treatment with EVs derived from PC-3 compared to t-NEPC LNCaP treatment. b ) The percentage of CD8 + HLA-DR + treated with EVs derived from PC-3 compared to t-NEPC LNCaP treatment. c ) Comparison of the ratio of CD4 + HLA-DR + regarding the CD4 + . d ) Comparison of ratio of CD8 + HLA-DR + in regard to the CD8 + . Data analysed by parametric t-test and presented as mean bars (n = 10) ± SEM, the significant p -value is reported; * indicates p < 0.05; n.s. = not significant.

Article Snippet: The androgen-dependent LNCaP metastatic prostate carcinoma cell line (CRL-1740), androgen-independent prostate carcinoma PC-3 cell line (CRL-1435) and androgen-independent prostate carcinoma DU 145 cell line (HTB-81) were purchased from the American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Comparison

Journal: Scientific Reports

Article Title: Immune profiling identifies the contribution of extracellular vesicles to immune modulation and progression in prostate cancer

doi: 10.1038/s41598-025-31838-w

Figure Lengend Snippet: Release of cytokines by immune cells after treatment with prostate cancer EVs. Seven pro- and anti-inflammatory cytokines were evaluated from the conditioned medium of PBMCs from 10 healthy donors and samples treated with PC-3-EVs, LNCaP-EVs and DU 145 EVs for 24h. a ) Level of IL-6 between PBMCs treated with PC-3 EVs and DU 145 EVs compared to LNCaP EVs. b ) Level of MIP-1α between PBMCs treated with PC-3 EVs and DU 145 EVs compared to LNCaP EVs. c ) Level of IL-7 between PBMCs treated with PC-3 EVs and DU 145 EVs compared to LNCaP EVs. d ) Level of IFN-α2 between PBMCs treated with PC-3 EVs and DU 145 EVs compared to LNCaP EVs. e ) Level of IL-10 between PBMCs treated with PC-3 EVs and DU 145 EVs compared to LNCaP EVs. f ) Level of MCP-1 between PBMCs treated with PC-3 EVs and DU 145 EVs compared to LNCaP EVs. g ) Level of G-CSF between PBMCs treated with PC-3 EVs and DU 145 EVs compared to LNCaP EVs. Data analysed by one-way ANOVA and presented as mean bars (n = 10) ± SEM, the significant p -value is reported; **** indicates p < 0.0001; ***indicates p < 0.001; ** indicates p < 0.01; *indicates p < 0.05.

Article Snippet: The androgen-dependent LNCaP metastatic prostate carcinoma cell line (CRL-1740), androgen-independent prostate carcinoma PC-3 cell line (CRL-1435) and androgen-independent prostate carcinoma DU 145 cell line (HTB-81) were purchased from the American Type Culture Collection (ATCC).

Techniques:

Journal: bioRxiv

Article Title: Reverse transcriptase inhibitors induce autophagy in a LINE-1 ORF1p-dependent manner

doi: 10.1101/2025.08.07.669170

Figure Lengend Snippet: SPV122.2 and EFV (NNRTI class), and Abacavir (NRTI class) up-regulate LINE-1 mRNA and ORF1p levels in PC3 cells. A. Structure of a functional human L1 Hs retrotransposon. The schematic shows the binding sites for qPCR primers (L1ORF1 For, L1 ORF1 Rev, L1ORF2 For and L1ORF2 Rev), represented as small convergent black arrows on L1 cDNA, and for siRNAs s552967, s552968 and s552969 (red lines; coordinates in the Methods section) on full-length L1 mRNA. B. RT-PCR quantification of endogenous full-length L1 mRNA transcripts using ORF1 - and ORF2 -specific primers shown in A and RNA extracted from PC3 cells cultured with EFV, SPV122.2 or ABC (2 cycles, 96 h each) relative to their respective controls (controls are set as 1). Throughout this study, DMSO-soluble NNRTIs were compared to cultures treated with 0.2% DMSO alone (indicated as DMSO), while the hydrosoluble ABC was compared to untreated control cultures (indicated as CTR). Data are presented as the mean ± SD from 8 assays. Statistical significance was assessed using the two-way ANOVA and Multiple Comparison Test. **, p<0.01; ***, p<0.001; ****, p<0.0001. C. Representative immunoblot assay of ORF1p in PC3 whole cell extracts cultured with and without SPV122.2, EFV or ABC; tubulin was used as a loading control. In three independent immunoblotting assays, ORF1p signals were measured by densitometry and normalized to those of the loading control (either GAPDH, or tubulin, or both were used in different experiments). Histograms represent the mean ± SD ratios, setting the values obtained in untreated and in DMSO-treated cells as 1. Statistical significance was calculated using the unpaired Student’s t- test, with **, p<0.01 (ABC vs. CTR) and ****, p<0.0001 (SPV122.2 vs DMSO and EFV vs. DMSO). D-E. Immunofluorescence (IF) fields immunostained for ORF1p in SPV122.2 vs. DMSO-treated (D), and ABC-treated vs. untreated (CTR) (E) PC3 cultures. Bars: 10 μm. The scatter plots on the right, display the distribution of ORF1p signal intensity per cell measured in the experiments in D and E (30-50 counted cells per sample). The ORF1p fluorescence intensity was measured both in whole cells (in D, panel a, the white dashed line delimits one exemplifying cell profile), and after selecting DAPI-stained nuclei (the yellow dashed line in D, panel b, delimits one exemplifying nucleus) as the Region of Interest (RoI). The distributions of signals were statistically analyzed using the Mann-Whitney test: **, p<0.01; ***, p<0.001; ****, p<0.0001. The same trend was replicated in three independent experiments for SPV122.2 (140 DMSO and 120 SPV122.2 analyzed cells), and in four independent experiments for ABC (180 CTR and 250 ABC analyzed cells).

Article Snippet: The prostate carcinoma cell line PC3 (ATCC CRL-1435) was cultured in RPMI 1640 supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin in a humidified atmosphere at 37°C in 5% CO 2 .

Techniques: Functional Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Control, Comparison, Western Blot, Immunofluorescence, Fluorescence, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Reverse transcriptase inhibitors induce autophagy in a LINE-1 ORF1p-dependent manner

doi: 10.1101/2025.08.07.669170

Figure Lengend Snippet: SPV122.2 and ABC inhibitors up-regulate γ−H2AX accumulation in nuclei in an autophagy-dependent manner. A. Schedule of treatments used to analyze the implication of autophagy in the induction of DNA damage by RTIs. (3-MA, 3-Methyladenine). B. IF analysis of γ−H2AX in SPV122.2-treated PC3 cells: γ−H2AX nuclear signals increased significantly compared to DMSO-treated cultures. Addition of the autophagy inhibitor 3-MA (SPV122.2+3-MA panels) prevents that increase, bringing γ−H2AX signals back to the level measured in DMSO controls. Bar, 10 μm. The graphs below show the distribution of γ−H2AX intensity signals per cell under all tested conditions (>70 counted cells per condition in 2 independent assays; ****, p<0.0001; ns, non significant). C . IF analysis of γ−H2AX in ABC-treated PC3 cultures: nuclear γ−H2AX signals significantly increase compared to untreated CTR, and 3-MA addition counteracts that increase. Bar, 10 μm. At least 90 cells per condition were analyzed in 3 independent assays. **, p<0.01; ****, p<0.0001; ns, non significant. The Kruskal-Wallis test (Dunn’s multiple comparison test) was used to statistically analyze the data presented in B and C .

Article Snippet: The prostate carcinoma cell line PC3 (ATCC CRL-1435) was cultured in RPMI 1640 supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin in a humidified atmosphere at 37°C in 5% CO 2 .

Techniques: Comparison

Journal: bioRxiv

Article Title: Reverse transcriptase inhibitors induce autophagy in a LINE-1 ORF1p-dependent manner

doi: 10.1101/2025.08.07.669170

Figure Lengend Snippet: PC3 cell treatment with SPV122.2 and ABC results in an interaction between ORF1p and γ−H2AX. A. PLA assays probing ORF1p and γ−H2AX in DMSO- (a) and SPV122.2- (b) treated cultures. B. PLA assays between ORF1p and γ−H2AX in untreated CTR (a) and ABC-treated (b) cultures. The PLA probe (red) selectively hybridizes where ligation products are formed. Bars, 10 μm. Treatment with both RTIs up-regulate the formation of ORF1p/γ−H2AX ligation products compared to control cultures. Graphs display the distribution of PLA signals (intensity / cell) measured in 150 to 260 cells per condition in 2 independent assays and statistically analyzed using the Mann-Whitney test. ****, p<0.0001.

Article Snippet: The prostate carcinoma cell line PC3 (ATCC CRL-1435) was cultured in RPMI 1640 supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin in a humidified atmosphere at 37°C in 5% CO 2 .

Techniques: Ligation, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Reverse transcriptase inhibitors induce autophagy in a LINE-1 ORF1p-dependent manner

doi: 10.1101/2025.08.07.669170

Figure Lengend Snippet: SPV122.2 and ABC induce the formation of ligation products between ORF1p and BCLAF1 proteins in PC3 cells . A. PLA assays probing ORF1p and BCLAF1 in DMSO-treated (a) and SPV122.2-treated (b) cultures. B. PLA assays probing ORF1p and BCLAF1 in untreated (CTR, a) and ABC-treated (b) PC3 cultures. The PLA probe is depicted in red. Bars, 10 μm. Graphs quantify the PLA signal intensity per cell under each condition, demonstrating an increase in ligation products upon treatment with both RTIs. Cells (>90 cells per sample) were statistically analyzed using the Mann-Whitney test. ****, p<0.0001.

Article Snippet: The prostate carcinoma cell line PC3 (ATCC CRL-1435) was cultured in RPMI 1640 supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin in a humidified atmosphere at 37°C in 5% CO 2 .

Techniques: Ligation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Reverse transcriptase inhibitors induce autophagy in a LINE-1 ORF1p-dependent manner

doi: 10.1101/2025.08.07.669170

Figure Lengend Snippet: Induction of micronuclei containing ORF1p and p62 in RTI-treated PC3 cultures. A. Histograms represent the fraction of cells with micronuclei (mean ± SD) in PC3 cultures treated with RTIs (SPV122.2, ABC), compared to controls (DMSO-treated or untreated, CTR). >450 cells per condition were counted in 3 experiments. SPV122.2-treated were compared to DMSO, and ABC-treated to untreated cultures, using the unpaired t -test. **, p <0.01; ***, p <0.001. B. Examples of cells stained for lamin B1, γ-H2AX and p62 after treatment with SPV122.2 (panel a) and ABC (panel b): both γ-H2AX and p62 are contained in micronuclei. C. Examples of parallel cultures treated with SPV122.2 (panels a-b: two examples are shown) or with ABC (panel c), immunostained for γ-H2AX and ORF1p: both proteins are contained in micronuclei. Micronuclei in B and C are membrane-enclosed, as depicted by lamin B1 staining.

Article Snippet: The prostate carcinoma cell line PC3 (ATCC CRL-1435) was cultured in RPMI 1640 supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin in a humidified atmosphere at 37°C in 5% CO 2 .

Techniques: Staining, Membrane

Journal: bioRxiv

Article Title: Reverse transcriptase inhibitors induce autophagy in a LINE-1 ORF1p-dependent manner

doi: 10.1101/2025.08.07.669170

Figure Lengend Snippet: p62 and ORF1p accumulate at sites of lamin B1 ruptures induced by SPV122.2 and ABC . A. Exemplifying IF fields from PC3 cultures untreated (a), or treated with SPV122.2 (b) or with ABC (c), immunostained for p62 and lamin B1: p62 accumulates at sites where lamin B1 is fragmented. B. Parallel cultures were immunostained for ORF1p and lamin B1: in panel b (SPV122.2), ORF1p signals interpose between discontinuous signals caused by fragmented lamin B1, and result in a punctuated pattern; panels c (SPV122.2) and d (ABC) show examples of more extensive lamin B1 fragmentation, with intense ORF1p accumulation.

Article Snippet: The prostate carcinoma cell line PC3 (ATCC CRL-1435) was cultured in RPMI 1640 supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin in a humidified atmosphere at 37°C in 5% CO 2 .

Techniques:

Journal: bioRxiv

Article Title: Reverse transcriptase inhibitors induce autophagy in a LINE-1 ORF1p-dependent manner

doi: 10.1101/2025.08.07.669170

Figure Lengend Snippet: SPV122.2 and ABC induce the formation of ligation products between ORF1p and lamin B1. A. PLA assays show that SPV122.2 treatment (panel b) up-regulates ORF1p/lamin B1 ligation products compared to PC3 cultures lacking SPV122 (DMSO-treated, panel a). B. PLA assays as in A depict a similar increase in ABC-treated (b) compared to untreated (CTR, a) PC3 cultures. In A and B , red signals locate ligation products to the vicinity of the nuclear envelope, including at sites where the nuclear envelope shows ruptures (framed, 2x zoom-in). Bars, 10 μm. Adjacent graphs show the distribution of PLA signal intensities per cell in presence and absence of SPV122.2 (A) or ABC (B), respectively (>100 analyzed cells per condition). Statistical analysis was performed using the Mann-Whitney test. ****, p<0.0001

Article Snippet: The prostate carcinoma cell line PC3 (ATCC CRL-1435) was cultured in RPMI 1640 supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin in a humidified atmosphere at 37°C in 5% CO 2 .

Techniques: Ligation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Reverse transcriptase inhibitors induce autophagy in a LINE-1 ORF1p-dependent manner

doi: 10.1101/2025.08.07.669170

Figure Lengend Snippet: Both autophagy and LINE-1 expression are required for the induction of lamin B1 fragmentation by SPV122.2 and ABC. A. Confocal microscopy images of lamin B1 in PC3 cultures: intact nuclear envelopes are depicted by the continuity of lamin B1 staining in DMSO-treated (a) and 3-MA-treated (b) cells; SPV122.2 (c) induces lamin B1 fragmentation and micronuclei formation; 3- MA addition (d) prevents SPV122.2-induced fragmentation. B. RT-PCR (left) and IF (right) analyses of LINE-1 mRNA and ORF1p protein, respectively, after transfection with LINE-1-specific (indicated as L1, map in A) and luciferase-specific (GL2, control) siRNAs in PC3 cells. Histograms represent the relative expression of LINE-1 mRNA in control (GL2, taken as 1) and L1-interfered cells after normalization to GAPDH. Ratios were calculated using the 2-ΔΔCT method and p-values by the unpaired t -test. ***, p<0.001 (n, 8). The ORF1p signal intensity was measured by quantitative IF at the single cell level, selecting whole cells as the Region of Interest (RoI) (details in Methods). Graphs represent the distribution of ORF1p signal intensities (in arbitrary units) in individual cells (120 to 180 cells per condition were counted in 2 independent experiments), and statistically analyzed using the Mann-Whitney test: ****, p<0.0001. C. siRNA-mediated LINE-1 down-regulation prevents SPV122.2-dependent lamin B1 fragmentation. The upper panel shows representative confocal images of lamin B1 in DMSO-treated controls, either non-interfered (indicated as -, panel a), or interfered with neutral siRNA (+GL2, panel b), or with LINE-1-specific siRNA (+L1, panel c). In the lower panel lamin B1 is immunostained in SPV122.2-treated cultures either non-interfered (d), or interfered with GL2 (e), or interfered with LINE-1-specific siRNAs (L1, f). Sites of lamin B1 discontinuity are indicated by white arrows. The graph quantifies the fraction of cells exhibiting discontinuous lamin B1 (mean % ± SD values) from >100 cells/condition in 3 assays. Samples were statistically analyzed using the unpaired t -test. *, p <0.05. Bars, 20 μm.

Article Snippet: The prostate carcinoma cell line PC3 (ATCC CRL-1435) was cultured in RPMI 1640 supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin in a humidified atmosphere at 37°C in 5% CO 2 .

Techniques: Expressing, Confocal Microscopy, Staining, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Control, MANN-WHITNEY