pc3 androgen independent  (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: Chemistry (Weinheim an Der Bergstrasse, Germany)

Article Title: Solvent‐Free Process for Preparing Metal‐Organic Framework Composites Based on Carbon‐Based Quantum Dots and Their Derivatives as Drug Delivery Systems for Andrographolide

doi: 10.1002/chem.202500655

Figure Lengend Snippet: IC 50 curves for MIL‐53(Al) (blue line), ADG (red line) and ADGMIL‐53 (green line) using PC3 cell line at 48 h of treatment.

Article Snippet: Human androgen‐independent prostate cancer cells PC3 were purchased from American Type Culture Collection (ATCC), (Manassas, VA, USA), CRL‐1435.

Techniques:

Journal: International journal of molecular sciences

Article Title: Enhancing Radiation Therapy Response in Prostate Cancer Through Metabolic Modulation by Mito-Lonidamine: A 1 H and 31 P Magnetic Resonance Spectroscopy Study.

doi: 10.3390/ijms26020509

Figure Lengend Snippet: Figure 1. Glucose consumption and lactate production of PC3 and CWR22Rv1 prostate cancer cells under LND and Mito-LND treatment. PC3 and CWR22Rv1 cells were treated with either placebo (control), 200 µM LND, or 2 µM Mito-LND. Glucose (A), lactate (B), and the ratio of lactate produced/glucose consumed (C). Glucose and lactate were measured in the media at the 24- (red bars) and 48 h time points (blue bars) using a YSI 2300 STAT PLUS Biochemical Analyzer fitted with glucose or lactate membranes. Data in the figures are the net changes in the mean ± S.D. (n = 3). Asterisks indicate a statistically significant change (p < 0.01), comparing metabolite concentrations of either LND or Mito-LND vs. control using a Student’s t-test.

Article Snippet: Androgen-independent PC3 (Accession CVCL_0035) and androgen-dependent CWR22Rv1 (Accession CVCL_1045) prostate cancer cells were obtained from ATCC (Manassas, VA, USA).

Techniques: Control, Produced

Journal: International journal of molecular sciences

Article Title: Enhancing Radiation Therapy Response in Prostate Cancer Through Metabolic Modulation by Mito-Lonidamine: A 1 H and 31 P Magnetic Resonance Spectroscopy Study.

doi: 10.3390/ijms26020509

Figure Lengend Snippet: Figure 2. Seahorse mitochondrial stress test on PC3 and CWR22Rv1 (CWR) prostate cancer cells under LND and Mito-LND treatment. The figure shows the effect of 200 µM LND and 2 µM Mito- LND at 24 h on the mitochondrial function of PC3 (A,C) and CWR22Rv1 cells (B,D). The test was carried out using the following reagents: Oligomycin (O), FCCP (F), and Rotenone/Antimycin-A (R). Oxygen consumption rate (OCR) of PC3 (A) and CWR22Rv1 cells (B). Extracellular acidification rate (ECAR) of PC3 (C) and CWR22Rv1 cells (D). Data were gathered on a Seahorse XFe96 Extracellular Flux Analyzer and are shown as mean ± SD (n = 14).

Article Snippet: Androgen-independent PC3 (Accession CVCL_0035) and androgen-dependent CWR22Rv1 (Accession CVCL_1045) prostate cancer cells were obtained from ATCC (Manassas, VA, USA).

Techniques:

Journal: International journal of molecular sciences

Article Title: Enhancing Radiation Therapy Response in Prostate Cancer Through Metabolic Modulation by Mito-Lonidamine: A 1 H and 31 P Magnetic Resonance Spectroscopy Study.

doi: 10.3390/ijms26020509

Figure Lengend Snippet: Figure 3. Effect of 200 µM LND and 2 µM Mito-LND on the cell energy phenotypes of PC3 and CWR22Rv1 after 24 h of treatment. (A) Seahorse energy phenotype map plotting the basal oxygen consumption rates (OCRs) and basal extracellular acidification rates (ECARs) in the control, LND, and Mito-LND groups shown in Figure 2. (B) Quantified OCR and ECAR values of cells with standard deviation and p-values comparing controls to treated groups. Data were gathered via a Mito Stress Test Assay using the Seahorse XFe96 Extracellular Flux Analyzer and shown as mean ± SD (n = 14).

Article Snippet: Androgen-independent PC3 (Accession CVCL_0035) and androgen-dependent CWR22Rv1 (Accession CVCL_1045) prostate cancer cells were obtained from ATCC (Manassas, VA, USA).

Techniques: Control, Standard Deviation

Journal: International journal of molecular sciences

Article Title: Enhancing Radiation Therapy Response in Prostate Cancer Through Metabolic Modulation by Mito-Lonidamine: A 1 H and 31 P Magnetic Resonance Spectroscopy Study.

doi: 10.3390/ijms26020509

Figure Lengend Snippet: Figure 5. Lactate and alanine assessment by in vivo 1H MRS. (A,C) show representative 1H MRS spectra localized in tumors of PC3 and CWR22Rv1 cells in mice xenografts, respectively. They were acquired using the HDMD-Sel-MQC pulse sequence. The area integration of the lactate and alanine spectral signals, normalized to the water signal, are shown in (B) for PC3 and in (D) for CWR22Rv1 xenografts. These measurements were carried out in controls (vehicle) and animals treated with LND or Mito-LND at an oral dose of 7.5 µmol/kg once daily for 14 days. An asterisk (*) indicates a statistical significance (p < 0.05) when comparing the control (n = 5) and treated groups (n = 5) on Day 14.

Article Snippet: Androgen-independent PC3 (Accession CVCL_0035) and androgen-dependent CWR22Rv1 (Accession CVCL_1045) prostate cancer cells were obtained from ATCC (Manassas, VA, USA).

Techniques: In Vivo, Sequencing, Control

Journal: International journal of molecular sciences

Article Title: Enhancing Radiation Therapy Response in Prostate Cancer Through Metabolic Modulation by Mito-Lonidamine: A 1 H and 31 P Magnetic Resonance Spectroscopy Study.

doi: 10.3390/ijms26020509

Figure Lengend Snippet: Figure 6. In vivo measurement of intracellular (pHi), extracellular pH (pHe), and bioenergetics (βNTP/Pi) by 31P MRS. At the top, representative in vivo localized 31P MRS spectra of PC3 (A) and CWR22Rv1 (B) prostate cancer xenografts are shown. Below are the measurements of pHi, pHe, and βNTP/Pi measured by the analysis of 31P MRS in untreated (controls) and LND- and Mito-LND- treated PC3 (C) and CWR22Rv1 xenografts (D). LND and Mito-LND were administered orally at a dose of 57.5 µmole/kg once daily for 14 days. Peak assignments: 3-APP, 3-aminopropylphosphonat; PME, phosphomonoesters; Pi, inorganic phosphate; PDE, phosphodiesters; γ-, α-, and β-NTP, the γ-, α-, and β-moieties of nucleoside triphosphates. An asterisk (*) indicates a statistical significance (p < 0.05) between the control group (n = 5; red bars) and the treated group (n = 5; green bars) during the follow-up period.

Article Snippet: Androgen-independent PC3 (Accession CVCL_0035) and androgen-dependent CWR22Rv1 (Accession CVCL_1045) prostate cancer cells were obtained from ATCC (Manassas, VA, USA).

Techniques: In Vivo, Control

Journal: Scientific Reports

Article Title: Dissecting the novel molecular interactions of solute carrier family 4 member 4 (SLC4A4) for prostate cancer (PCa) progression

doi: 10.1038/s41598-024-72408-w

Figure Lengend Snippet: Upregulation of SLC4A4 in prostate cancer. ( A , B ) SLC4A4 transcript is highly expressed in several cancers, including prostate adenocarcinoma (PRAD), based on the TCGA study cohort. ( C , D ) A subset of PRAD showed higher SLC4A4 expression in Gleason score 7, 8, and 9 tumors and this was associated with the overall survival of patients. ( E , F ) Immunohistochemical (IHC) analysis of primary prostate tumor samples showed higher SLC4A4 protein expression compared to non-tumor tissues. ( G , H ) Compared to RWPE-1 normal prostate epithelial cells, the SLC4A4 mRNA level was higher in DU145 androgen receptor (AR)-negative prostate cancer (PCa) cells. In AR-positive PCa cells, SLC4A4 mRNA level was elevated in C4-2, followed by VCAP (* P < 0.05). SLC4A4 overexpression was also detected by Western blotting, with PC3 cells having higher SLC4A4 protein level in AR-negative PCa cells. In AR-positive PCa cells, both C4-2 and LNCAP showed higher SLC4A4 protein expression. The results are presented as means ± standard error of three independent experiments.

Article Snippet: Androgen-dependent 22RV1, LNCAP, VCAP, C4-2 and androgen-independent DU145and PC3 human tumor prostate cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Immunohistochemical staining, Over Expression, Western Blot