androgen independent prostate cancer cell line pc3  (ATCC)

Journal: bioRxiv

Article Title: LSH-mediated resolution of R-loops mitigates transcription-replication conflicts to preserve genomic stability in prostate cancer cells

doi: 10.1101/2025.06.04.657812

Figure Lengend Snippet: A Schematic of the Tet-on LSH-shRNA system for regulating LSH expression in PC3 cells. Treatment with doxycycline for five days (DOX_5d) resulted in a significant reduction in LSH expression compared to cells that did not receive doxycycline treatment (DOX_0d). Following a five-day washout period, LSH expression was fully rescued to normal level (RESC). B Doxycycline- inducible knockdown of LSH was assessed using Western blotting to track alterations in protein levels, with endogenous β-ACTIN serving as the loading control. C-D Representative images and the percentage of cells with micronuclei ( C ) or DNA bridge ( D ) in Tet-on LSH-shRNA PC3 cells treated as described in panel A. The scale bar represents 5 μm. Data are presented as mean ± s.d. (n = 4 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. E Representative images of γ-H2AX immunostaining and bar graph show the percentage of PC3 cells containing more than five γ-H2AX foci, treated as described in panel A. The scale bar represents 5 μm. Data are presented as mean ± s.d. (n = 4 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann– Whitney test. F Representative images of the alkaline comet assay and quantification of tail moment in PC3 cells, treated as described in panel A. The scale bar represents 5 μm. Data are presented as mean ± s.d. (n = 4 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. G Representative images of Edu staining and quantification of nuclear Edu signal intensity in PC3 cells, treated as described in panel A. The scale bar represents 10 μm. Signal intensity data are depicted as a scatter plot with mean ± s.d. (n = 4 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. AU denotes arbitrary units. H-I Quantification of replication fork velocity ( H ) and fork asymmetry ( I ) is presented using Tukey-style box plots. Schematic diagram of the DNA fiber assay and representative images of stretched DNA fibers in PC3 cells treated as described in panel A. Cells were sequentially labeled at the indicated time points with two different thymidine analogues— CldU (red) and IdU (green). The scale bar represents 10 μm. The central line of a Tukey-style box plot indicates the median value, while the boxes and whiskers represent the 25th to 75th percentiles and the 5th to 95th percentiles, respectively. Outliers are displayed as individual points. Data are representative of n = 4 biologically independent experiments. ***p < 0.001; n.s. indicates not significant, as determined by the One-way ANOVA with Tukey’s multiple comparison test. J Scatter plots depicting the distances covered by right-moving and left-moving bi-directional replication forks following the CldU pulse in PC3 cells treated as described in panel H. The central areas, delineated by red lines, represent bi-directional forks with a length difference of less than 25%. The percentage of symmetric forks is also indicated. Data are representative of n = 4 biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: The cell lines utilized in this study included the androgen-independent prostate cancer cell line PC3 (ATCC), the androgen-dependent prostate cancer cell line LNCaP (ATCC), and the human embryonic kidney epithelial cell line HEK293T (ATCC).

Techniques: shRNA, Expressing, Knockdown, Western Blot, Control, Two Tailed Test, MANN-WHITNEY, Immunostaining, Alkaline Single Cell Gel Electrophoresis, Staining, Labeling, Analogues, Comparison

Journal: bioRxiv

Article Title: LSH-mediated resolution of R-loops mitigates transcription-replication conflicts to preserve genomic stability in prostate cancer cells

doi: 10.1101/2025.06.04.657812

Figure Lengend Snippet: A Representative S9.6 immunostaining images showing R-loop levels in PC3 Tet-on LSH-shRNA cells under different conditions: untreated (Dox_0d), doxycycline-treated for five days (Dox_5d), and after a five-day doxycycline washout period (RESC). The scale bar represents 5 μm. Signal intensity data are depicted as a scatter plot with mean ± s.d. (n = 4 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann– Whitney test. AU denotes arbitrary units. B Western blot analysis of PC3 Tet- on LSH-shRNA cells demonstrated the successful overexpression of both wild-type (WT) RNASEH1 and its WKKD/D210N mutant variants, all of which were tagged with V5. Detection was performed using an anti-V5 antibody, with β-Actin serving as the loading control. C Representative images of S9.6 immunostaining were utilized to assess R-loop levels in DOX_0d and DOX_5d cells overexpressing either WT or WKKD RNASEH1. The scale bar represents 5 μm. Signal intensity data are depicted as a scatter plot with mean ± s.d. (n = 4 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. AU denotes arbitrary units. D Representative images of γ-H2AX immunostaining were obtained, alongside the percentage of cells exhibiting more than five γ-H2AX foci in DOX_0d and DOX_5d cells overexpressing either WT or WKKD RNASEH1. The scale bar represents 5 μm. Data are presented as mean ± s.d. (n = 4 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann– Whitney test. E Representative images of the alkaline comet assay, along with quantification of the tail moment, were obtained from DOX_0d and DOX_5d cells overexpressing either WT or WKKD RNASEH1. Data are presented as mean ± s.d. (n = 4 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann– Whitney test. F Representative images of EdU staining and the quantification of nuclear EdU signal intensity were obtained from DOX_0d and DOX_5d cells overexpressing either WT or WKKD RNASEH1. The scale bar represents 10 μm. Signal intensity data are depicted as a scatter plot with mean ± s.d. (n = 4 biologically independent experiments). *p < 0.05; ***p < 0.001, as determined by the two-tailed Mann–Whitney test. AU denotes arbitrary units. G-H Quantification of replication fork velocity ( G ) and fork asymmetry ( H ) is presented using Tukey-style box plots. Representative images depict stretched DNA fibers from DOX_0d and DOX_5d cells overexpressing either WT or WKKD RNASEH1. The cells were sequentially labeled with CldU (red) and IdU (green). The scale bar represents 10 μm. The central line of a Tukey-style box plot indicates the median value, while the boxes and whiskers represent the 25th to 75th percentiles and the 5th to 95th percentiles, respectively. Outliers are displayed as individual points. Data are representative of n = 4 biologically independent experiments. *p < 0.05; ***p < 0.001; n.s. indicates not significant, as determined by the One- way ANOVA with Tukey’s multiple comparison test. I Scatter plots depicting the distances covered by right-moving and left-moving bi-directional replication forks following the CldU pulse in DOX_0d and DOX_5d cells overexpressing either WT or WKKD RNASEH1. The central areas, delineated by red lines, represent bi-directional forks with a length difference of less than 25%. The percentage of symmetric forks is also indicated. Data are representative of n = 4 biologically independent experiments. J-K Representative images of FANCD2 ( J ) and BLM ( K ) immunostaining along with the percentage of cells exhibiting more than ten foci are presented for DOX_0d and DOX_5d cells overexpressing either WT or WKKD RNASEH1. The scale bar represents 5 μm. Data are presented as a bar graph with mean ± s.d. (n = 4 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. Source data are provided as a Source Data file.

Article Snippet: The cell lines utilized in this study included the androgen-independent prostate cancer cell line PC3 (ATCC), the androgen-dependent prostate cancer cell line LNCaP (ATCC), and the human embryonic kidney epithelial cell line HEK293T (ATCC).

Techniques: Immunostaining, shRNA, Two Tailed Test, MANN-WHITNEY, Western Blot, Over Expression, Mutagenesis, Control, Alkaline Single Cell Gel Electrophoresis, Staining, Labeling, Comparison

Journal: bioRxiv

Article Title: LSH-mediated resolution of R-loops mitigates transcription-replication conflicts to preserve genomic stability in prostate cancer cells

doi: 10.1101/2025.06.04.657812

Figure Lengend Snippet: A Western blot analysis confirmed the knockout (KO) of LSH in PC3 cells and the overexpression of wild-type RNH1_V5 in LSH KO cells (KO+RNH1). LSH was detected with an anti-LSH antibody, RNH1_V5 was identified using an anti-V5 antibody, and β-Actin was used as the loading control. B The genomic distribution of R-loop CUT&Tag peaks is presented for LSH WT and KO cells. UTR refers to the untranslated region. The data are representative of n = 3 biologically independent experiments. C The differences in the genomic distribution of R-loop CUT&Tag peaks between LSH WT and KO cells are illustrated. UTR refers to the untranslated region. The data are representative of n = 3 biologically independent experiments. D Genome browser view of R-loop CUT&Tag data representing three independent biological replicates of LSH WT and KO cells. Samples treated with the RNH1 enzyme (RNH1) and KO+RNH1 cells were utilized as negative control groups. E The average R-loop CUT&Tag signals are shown over 6 kb regions centered on the transcription start site (TSS) in LSH WT and KO cells. Samples treated with the RNH1 enzyme (RNH1) and KO+RNH1 cells served as negative control groups to confirm the specificity of the detected R-loop signals. F The average R-loop CUT&Tag read density and heatmap for LSH WT and KO cells are presented within a 1 kb window around the center of peak regions. Samples treated with the RNH1 enzyme (RNH1) and KO+RNH1 cells were used as negative control groups to confirm the specificity of the detected R-loop signals. G The counts and fold changes of R-loop CUT&Tag peak gains (green) and losses (red) are presented for LSH KO cells compared to LSH WT cells. Additionally, the comparison between the WT group and the RNH1-treated group was carried out to confirm the specificity of the detected differential peaks. H Pathway analysis of genes exhibiting R-loop gains at the promoter region was conducted, comparing LSH KO and WT cells. The data are representative of n = 3 biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: The cell lines utilized in this study included the androgen-independent prostate cancer cell line PC3 (ATCC), the androgen-dependent prostate cancer cell line LNCaP (ATCC), and the human embryonic kidney epithelial cell line HEK293T (ATCC).

Techniques: Western Blot, Knock-Out, Over Expression, Control, Negative Control, Comparison

Journal: bioRxiv

Article Title: LSH-mediated resolution of R-loops mitigates transcription-replication conflicts to preserve genomic stability in prostate cancer cells

doi: 10.1101/2025.06.04.657812

Figure Lengend Snippet: A Western blot analysis was conducted on PC3 Tet-on LSH-shRNA cells overexpressing either WT or D210N RNASEH1, both of which were tagged with V5. The cells were left untreated (DOX_0d) or treated with doxycycline for five days (DOX_5d). β-Actin was used as the loading control. B Representative images of S9.6/LSH proximity ligation assay (PLA) in DOX_0d and DOX_5d cells overexpressing either WT or D210N RNASEH1. PLA foci (red) indicate the association of the LSH antibody within a 40 nm distance of the S9.6 antibody. The scale bar represents 10 μm. The quantification of PLA foci per nucleus for each experimental condition is presented as a scatter plot with mean ± s.d. (n = 3 biologically independent experiments). ****p < 0.0001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. C Western blot analysis was performed to evaluate RNH1 or SETX knockdown in PC3 cells following siRNA transfection, with cells transfected with negative control (NC) siRNA serving as the control group. β-Actin was used as the loading control. D Representative images of S9.6/LSH PLA analysis in PC3 cells transfected with siRNA targeting RNH1 or SETX. The scale bar represents 10 μm. The quantification of PLA foci per nucleus for each experimental condition is presented as a scatter plot with mean ± s.d. (n = 3 biologically independent experiments). ****p < 0.0001, as determined by the two-tailed Mann–Whitney test. E Western blot analysis of an RNA/DNA hybrid immunoprecipitation (IP) experiment was performed using the S9.6 antibody in PC3 cells overexpressing either WT or D210N RNASEH1. Both input and IP fractions were probed with LSH and lamin B1 antibodies, with LAMIN B1 serving as a negative control. Inputs are displayed on the left, and the S9.6-immunoprecipitated samples are shown on the right. The IgG Lane corresponds to a control IP using an IgG antibody. F Coomassie-stained SDS-PAGE gel of purified recombinant LSH_WT protein. G Electrophoretic mobility shift assay (EMSA) was conducted to analyze the binding of 0, 5, 10, and 20 μM of purified recombinant LSH_WT protein to 200 nM R-loops or RNA: DNA hybrids, both with and without 5’- RNA overhangs. The RNA 5’-terminus was labeled with 6-FAM fluorescence; DNA is depicted in black, while RNA is shown in red. The samples were loaded onto a 15% polyacrylamide gel and visualized using the Cy2 channel. H-J Representative immunofluorescence images depicting RAD51 ( H ), BRCA1 ( I ), and 53BP1 ( J ) foci in PC3 Tet-on LSH-shRNA cells treated with DMSO or with CPT (10 μM for 2 hours). The cells were left untreated (Dox_0d), treated with doxycycline for five days (Dox_5d), or subjected to a five-day doxycycline washout period (RESC). The scale bar represents 10 μm. The quantification of the number of RAD51, BRCA1, and 53BP1 foci per nucleus for each experimental condition is presented as a scatter plot with mean ± s.d. (n = 3 biologically independent experiments). ****p < 0.0001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. Source data are provided as a Source Data file.

Article Snippet: The cell lines utilized in this study included the androgen-independent prostate cancer cell line PC3 (ATCC), the androgen-dependent prostate cancer cell line LNCaP (ATCC), and the human embryonic kidney epithelial cell line HEK293T (ATCC).

Techniques: Western Blot, shRNA, Control, Proximity Ligation Assay, Two Tailed Test, MANN-WHITNEY, Knockdown, Transfection, Negative Control, Immunoprecipitation, Staining, SDS Page, Purification, Recombinant, Electrophoretic Mobility Shift Assay, Binding Assay, Labeling, Fluorescence, Immunofluorescence

Journal: bioRxiv

Article Title: LSH-mediated resolution of R-loops mitigates transcription-replication conflicts to preserve genomic stability in prostate cancer cells

doi: 10.1101/2025.06.04.657812

Figure Lengend Snippet: A Schematic representation of a human LSH mutation at the ATP binding site, illustrating a point mutation that replaces lysine (K) at amino acid 254 with alanine (A). B LSH 3’ UTR siRNA was designed to knock down endogenous LSH expression in PC3 cells (left panel), while these cells overexpressed either exogenous LSH(WT)-Flag or LSH(K254A)-Flag (middle panel). Following transient transfection with LSH 3’ UTR siRNA, western blot analysis (right panel) of PC3 cells indicated that the expression of exogenous LSH(WT)-Flag or LSH(K254A)-Flag protein was comparable to endogenous LSH expression, with β-Actin serving as the loading control. C Representative images of S9.6 immunostaining were used to evaluate the R-loop levels in PC3 cells treated as described in panel B. The scale bar represents 5 μm. Data are presented as a scatter plot with mean ± s.d. ( n = 3 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. D Coomassie-stained SDS-PAGE gel of purified recombinant LSH_WT and LSH_K254A proteins. E Electrophoretic mobility shift assays (EMSA) were conducted to analyze the binding of purified mutant LSH_K254A recombinant protein at concentrations of 0, 5, 10, and 20 μM to 200 nM R- loops or RNA: DNA hybrids, with or without 5’-RNA overhangs. The RNA 5’- terminus was labeled with 6-FAM fluorescence, with DNA represented in black and RNA in red. The samples were loaded onto a 15% polyacrylamide gel and visualized using the Cy2 channel. F Representative images of the alkaline comet assay along with quantification of tail moment in PC3 cells treated as outlined in panel B. Data are presented as a bar graph with mean ± s.d. ( n = 3 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. G Representative images of γ-H2AX immunostaining, complemented by a bar graph, illustrate the percentage of PC3 cells that contain more than five γ-H2AX foci, as treated in panel B. The scale bar represents 5 μm. Data are presented as a bar graph with mean ± s.d. ( n = 3 biologically independent experiments). ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. H Quantification of replication fork velocity and asymmetry in PC3 cells treated as described in panel B. The data are presented as a Tukey-style box plot, where the center line represents the median value, and the boxes and whiskers indicate the 25th to 75th percentiles and the 5th to 95th percentiles, respectively. Outliers are represented as individual points. The data are representative of n = 3 biologically independent experiments. ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. I DNA-RNA hybrids CUT&Tag-qPCR analysis at R-loop loci and repetitive sequences in PC3 cells treated as described in panel B. Signal values were normalized to the controls and plotted as mean ± s.d. (n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001; n.s. indicates not significant, as determined by the One-way ANOVA with Tukey’s multiple comparison test. J ChIP–qPCR analysis of γ-H2AX at R-loops loci and repetitive sequences in PC3 cells treated as described in panel B. Signal values were normalized to the controls and plotted as mean ± s.d. (n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. Source data are provided as a Source Data file.

Article Snippet: The cell lines utilized in this study included the androgen-independent prostate cancer cell line PC3 (ATCC), the androgen-dependent prostate cancer cell line LNCaP (ATCC), and the human embryonic kidney epithelial cell line HEK293T (ATCC).

Techniques: Mutagenesis, Binding Assay, Knockdown, Expressing, Transfection, Western Blot, Control, Immunostaining, Two Tailed Test, MANN-WHITNEY, Staining, SDS Page, Purification, Recombinant, Electrophoretic Mobility Shift Assay, Labeling, Fluorescence, Alkaline Single Cell Gel Electrophoresis, Comparison, ChIP-qPCR

Journal: bioRxiv

Article Title: LSH-mediated resolution of R-loops mitigates transcription-replication conflicts to preserve genomic stability in prostate cancer cells

doi: 10.1101/2025.06.04.657812

Figure Lengend Snippet: LSH interacts with FANCD2 to resolve transcription- replication conflicts mediated by R-loops at stalled replication forks. A Representative images of S9.6 immunostaining to evaluate R-loop levels in PC3 Tet-on LSH-shRNA cells following transient transfection with siRNAs targeting UAP56, THOC1, FANCD2, or SETX. The cells were left untreated (DOX_0d) or treated with doxycycline for five days (DOX_5d). The scale bar represents 5 μm. Signal intensity data are depicted as a scatter plot with mean ± s.d. (n = 3 biologically independent experiments). ** p < 0.01; ***p < 0.001; n.s. indicates not significant, as determined by the two-tailed Mann– Whitney test. AU denotes arbitrary units. B Representative images of PCNA/RNAPIIS2P PLA in cells treated as described in panel A. The scale bar represents 5 μm. Quantification of the number of PLA foci per nucleus for each experimental condition is presented as a scatter plot with mean ± s.d. (n = 3 biologically independent experiments). ** p < 0.01; ****p < 0.0001; n.s. indicates not significant, as determined by the two-tailed Mann–Whitney test. C Western blot analysis was performed in PC3 Tet-on LSH-shRNA cells overexpressing either wild-type (WT) or WKKD RNASEH1, following transient transfection with siRNAs targeting UAP56 or SETX. GAPDH was used as the loading control. D Representative images of LSH/FANCD2 PLA in cells treated as described in panel C. The scale bar represents 5 μm. Quantification of the number of PLA foci per nucleus for each experimental condition is presented as a scatter plot with mean ± s.d. (n = 3 biologically independent experiments). ***p < 0.001; ****p < 0.0001, as determined by the two-tailed Mann–Whitney test. E Representative images of LSH/PCNA PLA in PC3 Tet-on LSH-shRNA cells, which were left untreated (DOX_0d) or treated with doxycycline for five days (DOX_5d). Negative controls, utilizing only LSH or PCNA antibodies, were performed to verify the specificity of the detected PLA signals. The scale bar represents 5 μm. Quantification of the number of PLA foci per nucleus for each experimental condition is presented as a scatter plot with mean ± s.d. (n = 3 biologically independent experiments). ****p < 0.0001, as determined by the two-tailed Mann–Whitney test. F Representative images of LSH/γH2AX PLA in cells treated as described in panel C. The scale bar represents 5 μm. Quantification of the number of PLA foci per nucleus for each experimental condition is presented as a scatter plot with mean ± s.d. (n = 3 biologically independent experiments). *p < 0.05; ****p < 0.0001, as determined by the two-tailed Mann–Whitney test. G Representative images of LSH/γ-RPA32 S4/S8P PLA in cells treated as described in panel C. The scale bar represents 5 μm. Quantification of the number of PLA foci per nucleus for each experimental condition is presented as a scatter plot with mean ± s.d. (n = 3 biologically independent experiments). ***p < 0.001; ****p < 0.0001, as determined by the two-tailed Mann–Whitney test. Source data are provided as a Source Data file.

Article Snippet: The cell lines utilized in this study included the androgen-independent prostate cancer cell line PC3 (ATCC), the androgen-dependent prostate cancer cell line LNCaP (ATCC), and the human embryonic kidney epithelial cell line HEK293T (ATCC).

Techniques: Immunostaining, shRNA, Transfection, Two Tailed Test, MANN-WHITNEY, Western Blot, Control

Journal: bioRxiv

Article Title: LSH-mediated resolution of R-loops mitigates transcription-replication conflicts to preserve genomic stability in prostate cancer cells

doi: 10.1101/2025.06.04.657812

Figure Lengend Snippet: A The Venn diagram illustrates the number of overlapping genes (n = 471) that are downregulated in LSH KO cells (KO vs. WT, red) and restored in KO+RNH1 cells (blue). Data are representative of n = 3 biologically independent experiments. B Pathway analysis of the overlapping genes was conducted, with a primary focus on cancer signaling pathways, transcriptional activity, and cellular biological processes. Data are representative of n = 3 biologically independent experiments. C Heat map displaying the relative expression levels of overlapping genes in WT, KO, and KO+RNH1 PC3 cells, as outlined in the pathway analysis from panel B. Data are representative of n = 3 biologically independent experiments. D The average R-loop CUT&Tag signals over 6 kb regions centered on the TSS of overlapping genes were analyzed in WT, KO, and KO+RNH1 PC3 cells. Samples treated with the RNH1 enzyme served as a negative control to confirm the specificity of the detected R-loop signals. Data are representative of n = 3 biologically independent experiments. TSS denotes transcription start site. E Co-IP experiments were performed in PC3 cells to evaluate the interaction between LSH and MYC as well as E2F3 proteins. Whole-cell lysates were collected and subjected to immunoprecipitation using either an anti-LSH antibody or an IgG control. Immunoblotting was subsequently carried out with anti-MYC and anti-E2F3 antibodies. Input samples are indicated, and the data represent n = 3 biologically independent experiments. F Enrichment plots for MYC and E2F targets were generated to compare WT and KO cells and further validated by the comparison of KO cells with KO+RNH1 cells. Data are representative of n = 3 biologically independent experiments. G The heat map illustrates the relative expression levels of five selected MYC and E2F target genes among the overlapping genes in WT, KO, and KO+RNH1 PC3 cells. Data are representative of n = 3 biologically independent experiments. H Genome browser snapshots of CUT&Tag-seq data depict R-loop accumulation at the selected MYC target genes, as illustrated in panel G, for WT, KO, and KO+RNH1 PC3 cells. I ChIP–qPCR analysis of γ-H2AX at the selected MYC target genes, as illustrated in panel G, for WT, KO, and KO+RNH1 PC3 cells. Values were normalized to the WT group and presented as a bar graph with mean ± s.d. (n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001; ****p < 0.0001, as determined by the One-way ANOVA with Tukey’s multiple comparison test. J ChIP–qPCR analysis of FANCD2 at the selected MYC target genes, as shown in panel G, for WT and KO PC3 cells. Data are presented as a bar graph with mean ± s.d. (n = 3 biologically independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001, as determined by the two- tailed Mann–Whitney test. K ChIP–qPCR analysis of RNAPIIS2P at the promoter and gene body regions of the selected MYC target genes, as shown in panel G, for WT, KO, and KO+RNH1 PC3 cells. Values were normalized to the WT group and presented as a bar graph with mean ± s.d. (n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001; ****p < 0.0001, as determined by the One-way ANOVA with Tukey’s multiple comparison test. L Genome browser snapshots of CUT&Tag-seq data depict R-loop accumulation at the selected E2F target genes, as illustrated in panel G, for WT, KO, and KO+RNH1 PC3 cells. M ChIP–qPCR analysis of γ-H2AX at the selected E2F target genes, as illustrated in panel G, for WT, KO, and KO+RNH1 PC3 cells. Values were normalized to the WT group and presented as a bar graph with mean ± s.d. (n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001; ****p < 0.0001, as determined by the One-way ANOVA with Tukey’s multiple comparison test. N ChIP–qPCR analysis of FANCD2 at the selected E2F target genes, as shown in panel G, for WT and KO PC3 cells. Data are presented as a bar graph with mean ±s.d. (n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001, as determined by the two-tailed Mann–Whitney test. O ChIP–qPCR analysis of RNAPIIS2P at the promoter and gene body regions of the selected E2F target genes, as shown in panel G, for WT, KO, and KO+RNH1 PC3 cells. Values were normalized to the WT group and presented as a bar graph with mean ± s.d. (n = 3 biologically independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, as determined by the One-way ANOVA with Tukey’s multiple comparison test. Source data are provided as a Source Data file.

Article Snippet: The cell lines utilized in this study included the androgen-independent prostate cancer cell line PC3 (ATCC), the androgen-dependent prostate cancer cell line LNCaP (ATCC), and the human embryonic kidney epithelial cell line HEK293T (ATCC).

Techniques: Protein-Protein interactions, Activity Assay, Expressing, Negative Control, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Western Blot, Generated, Comparison, ChIP-qPCR, Two Tailed Test, MANN-WHITNEY

Journal: Cancer Cell International

Article Title: Inhibition of cancer cell-derived exosomal microRNA-183 suppresses cell growth and metastasis in prostate cancer by upregulating TPM1

doi: 10.1186/s12935-020-01686-x

Figure Lengend Snippet: Overexpression of miR-183 promoted cell proliferation and invasion in prostate cancer. a Determination of miR-183 expression in prostate cancer by RT-qPCR assay, showing high expression of miR-183 in RWPE-1, LNCAP, and PC3 cells. * p < 0.05 vs. RWPE-1 cells, # p < 0.05 vs. LNCAP cells. b Determination of miR-183 expression following overexpression and inhibition of miR-183 in LNCAP and PC3 cells by RT-qPCR assay. c Cell viability examined by MTT assay. d , e Transwell assay for the detection of the effect of miR-183 on cell migration, blue indicates the migrated cells. f , g Transwell assay for the detection of the effect of miR-183 on cell invasion, blue indicates the invaded cells * p < 0.05 vs. mimic-NC group, # p < 0.05 vs. inhibitor-NC group. The miR-183-mimic group, miR-183-inhibitor group, mimic-NC group, and inhibitor-NC group referring to LNCaP or PC3 cells respectively transfected with miR-183 mimic, miR-183 inhibitor, mimic-NC, inhibitor-NC. Cell experiments were repeated three times

Article Snippet: Normal prostate epithelial cell line RWPE-1(ATCC ® CRL-11609TM), androgen-dependent (LNCaP) and androgen-independent (PC3) human prostate cancer cell lines (ATCC ® CRL-1740TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Inhibition, MTT Assay, Transwell Assay, Migration, Transfection

Journal: Cancer Cell International

Article Title: Inhibition of cancer cell-derived exosomal microRNA-183 suppresses cell growth and metastasis in prostate cancer by upregulating TPM1

doi: 10.1186/s12935-020-01686-x

Figure Lengend Snippet: miR-183 targets and negatively regulates the expression of TPM1 in prostate cancer. a Prediction of miR-183 downstream target genes, blue, red, and green indicates prediction results of miRDB, mirDIP and miRWalk database respectively, yellow indicates the top 100 significantly downregulated genes in GSE30994 profile, the region of number 5 referring the intersection of 4 data sets. b Expression of TPM1 in prostate cancer and normal tissues in GEPIA website. c The binding sites of miRNA and TPM1-3′UTR predicted by the software. d The interaction between miR-183 and TPM1 assessed by dual-luciferase reporter gene assay. e The expression of TPM1 in RWPE-1, LNCaP and PC3 cell lines measured by Western blot analysis. * p < 0.05 vs.. RWPE-1 cells, # p < 0.05 vs. LNCAP cells. f The expression of TPM1 measured by Western blot analysis. * p < 0.05 vs. mimic-NC, # p < 0.05 vs. inhibitor-NC group. The miR-183-mimic group, miR-183-inhibitor group, mimic-NC group, and inhibitor-NC group refer to LNCaP cells respectively transfected with miR-183 mimic, miR-183 inhibitor, mimic-NC, inhibitor-NC, respectively. Cell experiment was repeated three times

Article Snippet: Normal prostate epithelial cell line RWPE-1(ATCC ® CRL-11609TM), androgen-dependent (LNCaP) and androgen-independent (PC3) human prostate cancer cell lines (ATCC ® CRL-1740TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Expressing, Binding Assay, Software, Luciferase, Reporter Gene Assay, Western Blot, Transfection

Journal: Cancer Cell International

Article Title: Inhibition of cancer cell-derived exosomal microRNA-183 suppresses cell growth and metastasis in prostate cancer by upregulating TPM1

doi: 10.1186/s12935-020-01686-x

Figure Lengend Snippet: Exosomes derived from PC3 cells can be taken up by LNCaP cells. a Exosomes derived from PC3 cells were observed under a transmission electron microscope (× 5000). The size and morphology of vesicles were not uniform. b Detection of exosome diameter by DLS. c The expression of HSP70 and CD63 measured by Western blot analysis, the content of both proteins in exosomes higher than that in supernatant, lane 1 indicates exosome and lane 2 indicates cell. d The content of CD63 examined by flow cytometry. e The image of laser confocal microscopy. Exosomes derived from PC3 cells labeled by CFSE (green fluorescence) could be delivered into LNCAaP cells (× 400). Measurement data were expressed as mean ± standard deviation. Comparison between two groups was analyzed by unpaired t -test. Each experiment was repeated three times

Article Snippet: Normal prostate epithelial cell line RWPE-1(ATCC ® CRL-11609TM), androgen-dependent (LNCaP) and androgen-independent (PC3) human prostate cancer cell lines (ATCC ® CRL-1740TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Derivative Assay, Transmission Assay, Microscopy, Expressing, Western Blot, Flow Cytometry, Confocal Microscopy, Labeling, Fluorescence, Standard Deviation, Comparison

Journal: Cancer Cell International

Article Title: Inhibition of cancer cell-derived exosomal microRNA-183 suppresses cell growth and metastasis in prostate cancer by upregulating TPM1

doi: 10.1186/s12935-020-01686-x

Figure Lengend Snippet: PC3 cells-derived exosomal miR-183 downregulates TPM1. a The secretion of exosomes observed under an electron microscope (× 5000). b Quantification of HSP70 and CD63 in exosomes detected by Western blot. c The expression of f miR-183 and TPM1 in PC3 cells detected by RT-qPCR assay. d The expression of f miR-183 analyzed by RT-qPCR assay. e The expression of TPM1 in LNCaP cells evaluated by RT-qPCR assay. f The expression of TPM1 in LNCaP cells evaluated by Western blot assay. * p < 0.05 vs. exo-mimic-NC group, # p < 0.05 vs. exo-inhibitor-NC group. The exo-mimic-NC group, exo-miR-183-mimic group, exo-inhibitor-NC group, and miR-183-inhibitor group refer to exosome derived from PC3 cells respectively transfected with mimic-NC, exo-miR-183-mimic, inhibitor-NC and miR-183-inhibitor. The exosomes derived from the transfected PC3 cells were co-cultured with LNCAP cells Measurement data were expressed as mean ± standard deviation. Comparison between two groups was analyzed using independent sample t -test. Cell experiment was repeated three times

Article Snippet: Normal prostate epithelial cell line RWPE-1(ATCC ® CRL-11609TM), androgen-dependent (LNCaP) and androgen-independent (PC3) human prostate cancer cell lines (ATCC ® CRL-1740TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Derivative Assay, Microscopy, Western Blot, Expressing, Quantitative RT-PCR, Transfection, Cell Culture, Standard Deviation, Comparison

Journal: Cancer Cell International

Article Title: Inhibition of cancer cell-derived exosomal microRNA-183 suppresses cell growth and metastasis in prostate cancer by upregulating TPM1

doi: 10.1186/s12935-020-01686-x

Figure Lengend Snippet: PC3 cells-derived exosomal miR-183 promotes the development of prostate cancer through downregulation of TPM1. a Expression of TPM1 in LNCaP cells detected by Western blot analysis. b Detection for proliferation in LNCaP cells by CCK-8 assay. c , d Detection for migration and invasion in LNCaP cells by Transwell assay (× 200). * p < 0.05 vs. si-NC group, # p < 0.05 vs. oe-NC group. The oe-TPM1 group, si-TPM1 group, oe-NC group, and si-NC group refer to LNCaP cells transfected with oe-TPM1, si-TPM1 and their negative controls. The exo-miR-183-inhibitor + si-TPM1 group and exo-miR-183-inhibitor + si-NC group refer to si-TPM1 transfected-LNCaP cells co-cultured with exosome derived from PC3 cells transfected with miR-183-inhibitor and si-TPM1, The exo-miR-183-inhibitor + si-NC group refers to si-NC transfected-LNCaP cells co-cultured with exosome derived from PC3 cells transfected with miR-183-inhibitor. Measurement data were expressed as mean ± standard deviation. Comparisons among multiple groups were conducted by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Statistical analysis in relation to time-based measurements within each group was realized using repeated measurement ANOVA, followed by a Bonferroni’s post-hoc test. n = 6

Article Snippet: Normal prostate epithelial cell line RWPE-1(ATCC ® CRL-11609TM), androgen-dependent (LNCaP) and androgen-independent (PC3) human prostate cancer cell lines (ATCC ® CRL-1740TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Derivative Assay, Expressing, Western Blot, CCK-8 Assay, Migration, Transwell Assay, Transfection, Cell Culture, Standard Deviation

Journal: Cancer Cell International

Article Title: Inhibition of cancer cell-derived exosomal microRNA-183 suppresses cell growth and metastasis in prostate cancer by upregulating TPM1

doi: 10.1186/s12935-020-01686-x

Figure Lengend Snippet: The mechanism graph of the regulatory function of exosomal miR-183 in prostate cancer. Exosomal miR-183 derived from PC3 cells facilitated prostate cancer LNCaP cell proliferation, migration, and invasion via downregulating TPM1

Article Snippet: Normal prostate epithelial cell line RWPE-1(ATCC ® CRL-11609TM), androgen-dependent (LNCaP) and androgen-independent (PC3) human prostate cancer cell lines (ATCC ® CRL-1740TM) were obtained from ATCC (Manassas, VA, USA).

Techniques: Derivative Assay, Migration

Journal: Cancer Science

Article Title: Cyclohexanone curcumin analogs inhibit the progression of castration‐resistant prostate cancer in vitro and in vivo

doi: 10.1111/cas.13897

Figure Lengend Snippet: Cytotoxicity of curcumin and its cyclohexanone analogs on PC3 cells

Article Snippet: An androgen‐independent human prostate cancer cell line (PC3) was purchased from ATCC.

Techniques:

Journal: Cancer Science

Article Title: Cyclohexanone curcumin analogs inhibit the progression of castration‐resistant prostate cancer in vitro and in vivo

doi: 10.1111/cas.13897

Figure Lengend Snippet: Anti‐invasion effect of curcumin and its cyclohexanone analogs on PC3 cells. For invasion assay, PC3 cells were treated with a non‐toxic concentration of curcumin, or its cyclohexanone analogs (A) or various concentrations of analogs 2A (B) or 2F (C). For migration assay, PC3 cells were treated with analogs 2A (D) and 2F (E). The invading or migrating cells were photographed under phase‐contrast microscopy and quantified by IMAGE J software. Data are represented as mean ± SD of three independent experiments. ** P < .01 and *** P < .001 vs control

Article Snippet: An androgen‐independent human prostate cancer cell line (PC3) was purchased from ATCC.

Techniques: Invasion Assay, Concentration Assay, Migration, Microscopy, Software, Control

Journal: Cancer Science

Article Title: Cyclohexanone curcumin analogs inhibit the progression of castration‐resistant prostate cancer in vitro and in vivo

doi: 10.1111/cas.13897

Figure Lengend Snippet: Effect of analogs 2A and 2F on secretion (A,B) and activity (C,D) of MMP‐2 and MMP‐9 in PC3 cells. PC3 cells were incubated with increasing concentrations of analogs 2A (A) or 2F (B) for 24 h. MMP secretion in the culture medium was then determined by gelatin‐zymography. For MMP activity, slab gels containing culture medium of PC3 cells were directly incubated with various concentrations of analogs 2A (C) and 2F (B) for 16 h. Level of MMP secretion and activity was quantified by IMAGE J software. Data are represented as mean ± SD of three independent experiments. * P < 0.05, ** P <0.01 and *** P < .001 vs control

Article Snippet: An androgen‐independent human prostate cancer cell line (PC3) was purchased from ATCC.

Techniques: Activity Assay, Incubation, Zymography, Software, Control

Journal: PLoS ONE

Article Title: Arg-Gly-Asp (RGD)-Modified E1A/E1B Double Mutant Adenovirus Enhances Antitumor Activity in Prostate Cancer Cells In Vitro and in Mice

doi: 10.1371/journal.pone.0147173

Figure Lengend Snippet: Prostate cancer PC3 cells were subcutaneously injected into nude mice. After tumor lesions reached approximately 6–7 mm in size virus was injected into the tumor lesion once a day for three days and the mice were sacrificed at day 28. Tumor volume was measured after animals were treated with viruses (n = 5 per group). Data are presented as mean tumor volume ± standard deviation. **P < 0.005.

Article Snippet: Human androgen-dependent prostate cancer cell line LNCaP (metastasis to the lymph node), human androgen-independent prostate cancer cell lines PC3 (metastasis to the bone) and DU145 (metastasis to the brain), and normal adult prostate epithelial cells infected with a single copy of human papilloma virus 18 (named RWPE-1) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Injection, Virus, Standard Deviation

Journal: Diabetes

Article Title: Exendin-4, a GLP-1 receptor agonist, attenuates prostate cancer growth.

doi: 10.2337/db13-1169

Figure Lengend Snippet: Figure 2—Ex-4 inhibits prostate cancer cell proliferation via the GLP-1R. LNCap cells (A), PC3 cells (B), ALVA-41 cells (C), and DU145 cells (D) were maintained in the recommended media supplemented with 10% FBS with or without Ex-4 (0.1–10 nmol/L). After 0, 24, 48, 72, and 96 h, the cells were harvested, and cell proliferation was analyzed by cell counting using a hemocytometer. Control (nontreated), black circles with solid line; Ex-4 (0.1 nmol/L), black squares with dotted line; Ex-4 (1 nmol/L), white circles with solid line; Ex-4 (10 nmol/L), white squares with dotted line. One-way ANOVA was performed to calculate statistical significance: *P < 0.05 vs. control; **P < 0.01 vs. control.

Article Snippet: The LNCap human androgen-sensitive prostate cancer cell line, and the PC3 andDU145 human androgen-independent prostate cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA).

Techniques: Cell Counting, Control