Journal: Nature

Article Title: Transcription–replication conflicts underlie sensitivity to PARP inhibitors

doi: 10.1038/s41586-024-07217-2

Figure Lengend Snippet: a , Efficiency of siRNA-mediated depletion of TIMELESS and TIPIN in HeLa cells by immunoblotting. PCNA and α-tubulin served as loading controls. b-c , Inhibition of transcription elongation by DRB. b , Outline of the experiment. c , Representative images of HeLa cells indicating inhibition of EU incorporation by DRB; the nuclei were counterstained with Hoechst 33342. Scale bar: 10 μm. d , Induction of a DNA damage response in U2OS and hTERT-RPE1 cells transfected with control siRNA or siRNAs targeting TIMELESS or TIPIN . γH2AX levels were determined by flow cytometry; bars indicate means ± 1 s.d.; n = 3 replicates; ANOVA. e-h , Transcription inhibitors cordycepin (CORD) and triptolide (TLP) suppress the DNA damage response induced by depletion of TIMELESS or TIPIN. e , Outline of the experiment. f , Flow cytometry profiles for EdU incorporation and DNA content. g , Quantification of EU incorporation; plots show medians and value ranges of 25-75% and 10-90%, filled circles indicate the individual cells in the top and bottom deciles; n = 2 replicates; >2624 cells per group (range: 2624-2899); ANOVA. h , Quantification of γH2AX mean intensity and number of 53BP1 foci per cell; plots show medians and value ranges of 25–75% and 10–90%, filled circles indicate the individual cells in the top and bottom deciles; n = 2 replicates; >66 cells per group (range: 66–194); ANOVA. i-k , Ongoing DNA replication is required for induction of a DNA damage response by depletion of TIMELESS or TIPIN. i , Outline of the experiment. j , γH2AX levels and DNA content ascertained by flow cytometry. k , Quantification of γH2AX positive cells determined separately for the EdU-positive and EdU-negative cells; bars indicate means ± 1 s.d.; n = 3 replicates; ANOVA. CTRL, control; TIM, TIMELESS; TIP, TIPIN; transf., transfection; Thy, thymidine; IF, immunofluorescence; PI, propidium iodide; r.u., relative units; NS, not significant.

Article Snippet: Drugs and chemical compounds used in this study were purchased from the following sources: thymidine (Sigma-Aldrich catalogue no. T1895), EdU (Thermo Fisher Scientific, catalogue no. A10044), 5-ethynyl uridine (EU) (Jena Biosciences, catalogue no. CLK-N002-10), camptothecin (Sigma-Aldrich, catalogue no. C9911), 5,6-dichlorbenzimidazol 1-β- d -ribofuranosid (DRB; Sigma-Aldrich, catalogue no. D1916), cordycepin (Tocris, catalogue no. 2294), triptolide (Tocris, catalogue no. 3253), olaparib (Selleckchem, catalogue no. S1060), talazoparib (Selleckchem, catalogue no. S7048), veliparib (Selleckchem, catalogue no. S1004), saruparib (Selleckchem, catalogue no. S9875), hydrogen peroxide (Sigma-Aldrich, catalogue no. H3410), RO-3306 (Sigma-Aldrich, catalogue no. SML0569), TMZ (Sigma-Aldrich, catalogue no. T2577), nocodazole (Tocris, catalogue no. 1228) and PARGi (Tocris, catalogue no. 7006).

Techniques: Western Blot, Inhibition, Transfection, Control, Flow Cytometry, Immunofluorescence

Journal: Nature

Article Title: Transcription–replication conflicts underlie sensitivity to PARP inhibitors

doi: 10.1038/s41586-024-07217-2

Figure Lengend Snippet: a-b , Transcription inhibitors cordycepin (CORD) and triptolide (TLP) suppress the DNA damage response induced by PARP inhibitors. a , Outline of the experiment. b , Quantification of γH2AX mean intensity; plots show medians and value ranges of 25–75% and 10–90%, filled circles indicate the individual cells in the top and bottom deciles; n = 2 replicates; >1668 cells per group (range: 1668–2282); ANOVA. c-d , The induction of a DNA damage response by PARP inhibitors depends on whether cells are exposed to these inhibitors in early, mid or late S phase. c , Outline of the experiment. Cells were exposed to PARP inhibitors 0−3.5, 3.5–7 or 7–10.5 h after release from a thymidine block, corresponding to early, mid or late S phase, respectively. d , Quantification of the percentage of γH2AX positive cells by flow cytometry; bars indicate means ± 1 s.d.; n = 3 replicates; ANOVA. e , Distribution of human genes according to replication timing (early, mid or late S phase) and level of nascent transcription (High Tx, upper tertile of all expressed genes; Mid Tx, middle tertile; Low Tx, lower tertile; No Tx, non-expressed genes). Nascent transcription was determined by EUseq analysis of HeLa cells. f-h , Induction of MiDAS in prometaphase cells following treatment of cells with PARP inhibitors in early or late S phase (0–3.5 or 7–10.5 h after release from a thymidine block). f , Outline of the experiment; the Cdk1 inhibitor RO-3306 inhibited entry into mitosis; nocodazole (Noco) prevented exit from mitosis. g , Representative images of prometaphase cells with MiDAS; the DNA was counterstained with DAPI. Scale bar: 5 μm. h , Quantification of the percentage of prometaphase cells with >3 EdU foci; bars indicate means ± 1 s.d.; n = 3 replicates; >127 prometaphase cells per group (range: 127–400); ANOVA. i-j , Induction of R-loops in cells treated with PARP inhibitors. i , Outline of the experiment. The U2OS cells used in this experiment express GFP-RNaseH1 D210N in a doxycycline (DOX)-dependent manner. j , Quantification of the number of GFP-RNaseH1 D210N foci following treatment with PARP inhibitors; plots show medians and value ranges of 25–75% and 10–90%, filled circles indicate the individual cells in the top and bottom deciles; n = 2 replicates; >192 cells per group; ANOVA. k-l , The DNA damage response induced by PARP inhibitors is suppressed by expression of RNase H1. k , Outline of the experiment. The HeLa cells used in this experiment express FLAG-RNaseH1 in a DOX-dependent manner. l , Quantification of the number of 53BP1 foci per cell and of γH2AX mean intensity; plots show medians and value ranges of 25–75% and 10–90%, filled circles indicate the individual cells in the top and bottom deciles; n = 2 replicates; >141 cells per group (range: 141–206); ANOVA. Thy, thymidine; PARPi, PARP inhibitor; r.u., relative units; Olap, olaparib (10 μΜ); Tal, talazoparib (100 nM); NS, not significant.

Article Snippet: Drugs and chemical compounds used in this study were purchased from the following sources: thymidine (Sigma-Aldrich catalogue no. T1895), EdU (Thermo Fisher Scientific, catalogue no. A10044), 5-ethynyl uridine (EU) (Jena Biosciences, catalogue no. CLK-N002-10), camptothecin (Sigma-Aldrich, catalogue no. C9911), 5,6-dichlorbenzimidazol 1-β- d -ribofuranosid (DRB; Sigma-Aldrich, catalogue no. D1916), cordycepin (Tocris, catalogue no. 2294), triptolide (Tocris, catalogue no. 3253), olaparib (Selleckchem, catalogue no. S1060), talazoparib (Selleckchem, catalogue no. S7048), veliparib (Selleckchem, catalogue no. S1004), saruparib (Selleckchem, catalogue no. S9875), hydrogen peroxide (Sigma-Aldrich, catalogue no. H3410), RO-3306 (Sigma-Aldrich, catalogue no. SML0569), TMZ (Sigma-Aldrich, catalogue no. T2577), nocodazole (Tocris, catalogue no. 1228) and PARGi (Tocris, catalogue no. 7006).

Techniques: Blocking Assay, Flow Cytometry, Expressing

Journal: Journal of Biological Chemistry

Article Title: The Requirement of c-Jun N-terminal Kinase 2 in Regulation of Hypoxia-inducing Factor-1α mRNA Stability

doi: 10.1074/jbc.m112.365882

Figure Lengend Snippet: FIGURE 3. JNK2 regulated hif-1 mRNA stability. A and B, WT (A) or WT and JNK2/ MEFs (B) (1 106/well) were seeded into 10-mm dish and exposed to 0.5 mM nickel for 12 h and 24 h (A), or 24 h (B). The whole cell lysate was incubated with phosphatase for 40 min at 30 °C. Proteins were resolved in SDS-PAGE and revealed by Western blotting assay. C, WT(Vector), JNK2/ (Vector), and JNK2/ (HA-JNK) MEFs were treated with nickel (0.5 mM) for 12 h in complete medium. Cells were then accommodated in methionine- and cysteine-free DMEM for 1 h. 35S-labeled methionine and cysteine was then added for the indicated times for pulse assay. Cell extracts were immunoprecipitated with anti-HIF-1 antibody or control IgG, and subjected to SDS-PAGE. Autoradiography was used to visualize 35S-labeled HIF-1. D, hif-1 mRNA levels in the individual cells were determined by real-time PCR. The asterisk (*) indicates a significant decrease as compared with those in WT(Vector) and JNK/ (HA-JNK2) MEFs or non-silencing control cells (p 0.05). E, basal levels of hif-1 promoter activity were evaluated by transfecting the indicated cells with a construct containing hif-1 promoter-driven luciferase. pRL-TK vector was used as an internal control. The results are expressed as the ratios of firefly to Renilla lucifease activity, as means S.D. (n 3). The asterisk (*) indicates a significant increase as compared with that of WT(Vector) cells or JNK/ (HA-JNK2) cells (p 0.05). F and G, mRNA degradation rate of hif-1 was detected following treatment with actinomycin D (5 M) for the indicated time. The PCR products were separated over 2% agarose gels, stained with ethidium bromide. The densitometric analyses of the product bands were conducted using the software of ImageQuant 5.2 (GE Healthcare). The results were shown as means S.D. (n 3). H, indicated cells were treated with cordycepin (5 M) for 4 h. Real-time PCR was conducted to detect the hif-1 mRNA expression. The asterisk (*) indicates a significant decrease as compared with that in WT cells under the same treatment (p 0.05).

Article Snippet: Cordycepin was purchased from Santa Cruz Biotechnology Inc. Nickel chloride was purchased from SigmaAldrich.

Techniques: Incubation, SDS Page, Western Blot, Plasmid Preparation, Labeling, Immunoprecipitation, Control, Autoradiography, Real-time Polymerase Chain Reaction, Activity Assay, Construct, Luciferase, Staining, Software, Expressing

Journal: Nutrients

Article Title: Cordycepin, an Active Constituent of Nutrient Powerhouse and Potential Medicinal Mushroom Cordyceps militaris Linn., Ameliorates Age-Related Testicular Dysfunction in Rats

doi: 10.3390/nu11040906

Figure Lengend Snippet: Chemical structure, histological analysis, Johnsen’s scores, and seminiferous tubule size in aged rat testes. ( A ) Cordycepin structure. ( B ) Representative images of tubular cross sections of testis: ( a ) young control rats YC, ( b ) vehicle-treated aged rats AC, ( c ) COR 5 mg/kg treated aged rats, ( d ) COR 10 mg/kg treated aged rats, and ( e ) COR 20 mg/kg treated aged rats. Sections were stained with hematoxylin and eosin (H&E). The images are typical of those obtained in five independent experiments. Scale bar = 45 μM and magnification = 200x. ( C ) Johnsen’s score. ( D ) Tubular size (µM). The results are expressed as mean ± SEM ( n = 10). # p < 0.05 compared with YC group and * p < 0.05 compared with AC group. COR, cordycepin; LC, Leydig cell; ST, Sertoli cell; SG, spermatogonia; SC, spermatocyte; SM, spermatid; SP, spermatozoa.

Article Snippet: The cordycepin-rich fraction was recrystallized in absolute ethanol to obtain pure cordycepin (hereafter referred to as COR) identified by 1 H (500 MHz) and 13 C (125 MHz) nuclear magnetic resonance (JEOL, Tokyo, Japan).

Techniques: Control, Staining

Journal: Nutrients

Article Title: Cordycepin, an Active Constituent of Nutrient Powerhouse and Potential Medicinal Mushroom Cordyceps militaris Linn., Ameliorates Age-Related Testicular Dysfunction in Rats

doi: 10.3390/nu11040906

Figure Lengend Snippet: Effect of cordycepin on sperm kinematics.

Article Snippet: The cordycepin-rich fraction was recrystallized in absolute ethanol to obtain pure cordycepin (hereafter referred to as COR) identified by 1 H (500 MHz) and 13 C (125 MHz) nuclear magnetic resonance (JEOL, Tokyo, Japan).

Techniques:

Journal: Nutrients

Article Title: Cordycepin, an Active Constituent of Nutrient Powerhouse and Potential Medicinal Mushroom Cordyceps militaris Linn., Ameliorates Age-Related Testicular Dysfunction in Rats

doi: 10.3390/nu11040906

Figure Lengend Snippet: Effect of COR on the spermatogenesis parameters in aged rats. ( A ) Percentage of tubules with sperm, ( B ) sperm count per tubule, ( C ) Sertoli number, ( D ) germ cell count, and ( E ) Sertoli cell index. # p < 0.05 compared with YC group and * p < 0.05 compared with AC group. YC, young rats; AC, aged rats; COR, cordycepin.

Article Snippet: The cordycepin-rich fraction was recrystallized in absolute ethanol to obtain pure cordycepin (hereafter referred to as COR) identified by 1 H (500 MHz) and 13 C (125 MHz) nuclear magnetic resonance (JEOL, Tokyo, Japan).

Techniques: Cell Counting

Journal: Nutrients

Article Title: Cordycepin, an Active Constituent of Nutrient Powerhouse and Potential Medicinal Mushroom Cordyceps militaris Linn., Ameliorates Age-Related Testicular Dysfunction in Rats

doi: 10.3390/nu11040906

Figure Lengend Snippet: Effect of COR on the expression levels of sex hormone receptors. ( A ) Protein expression of AR, FSHR, and LHR. ( B ) Relative expression levels (fold) in three independent experiments normalized to β-actin. ( C ) The mRNA expression of AR, FSHR, and LHR. ( D ) Relative expression levels (fold) in three independent experiments normalized to that of GAPDH. # p < 0.05 compared with YC group and * p < 0.05 compared with the AC group. AR, androgen receptor; LHR, luteinizing hormone receptor; FSHR, follicle-stimulating hormone receptor; YC, young rat group; AC, aged rat group; COR-20, cordycepin (20 mg/kg) treated AC group; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The cordycepin-rich fraction was recrystallized in absolute ethanol to obtain pure cordycepin (hereafter referred to as COR) identified by 1 H (500 MHz) and 13 C (125 MHz) nuclear magnetic resonance (JEOL, Tokyo, Japan).

Techniques: Expressing

Journal: Nutrients

Article Title: Cordycepin, an Active Constituent of Nutrient Powerhouse and Potential Medicinal Mushroom Cordyceps militaris Linn., Ameliorates Age-Related Testicular Dysfunction in Rats

doi: 10.3390/nu11040906

Figure Lengend Snippet: Effect of COR on expression levels of antioxidant enzymes in rat testes. ( A ) Protein expression of GPx4, GSTm5, and PRx4. ( B ) Relative expression levels (fold) in three independent experiments normalized to β-actin. ( C ) The mRNA expression of GPx4, GSTm5, and PRx4. ( D ) Relative expression levels (fold) in three independent experiments normalized to that of GAPDH. # p < 0.05 compared with YC group and * p < 0.05 compared with AC group. YC, young rat group; AC, aged group; COR-20, cordycepin (20 mg/kg) treated AC group; PRx4, peroxiredoxin-4; GSTm5, glutathione S-transferase mu 5; GPx4, glutathione peroxidase 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The cordycepin-rich fraction was recrystallized in absolute ethanol to obtain pure cordycepin (hereafter referred to as COR) identified by 1 H (500 MHz) and 13 C (125 MHz) nuclear magnetic resonance (JEOL, Tokyo, Japan).

Techniques: Expressing

Journal: Nutrients

Article Title: Cordycepin, an Active Constituent of Nutrient Powerhouse and Potential Medicinal Mushroom Cordyceps militaris Linn., Ameliorates Age-Related Testicular Dysfunction in Rats

doi: 10.3390/nu11040906

Figure Lengend Snippet: Effect of COR on expression of key biomolecules involved in spermatogenesis. ( A ) Protein expression of CREB-1, nectin-2, and inhibin-α. ( B ) Relative expression levels (fold) in three independent experiments normalized to β-actin. ( C ) The mRNA expression of CREB-1, nectin-2, and inhibin-α. ( D ) Relative expression levels (fold) in three independent experiments normalized to that of GAPDH. # p < 0.05 compared with YC group and * p < 0.05 compared with the AC group. YC, young rat group; AC, aged rat group; COR-20, cordycepin (20 mg/kg) treated AC group; CREB-1, cyclic adenosine monophosphate (cAMP) responsive element binding protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The cordycepin-rich fraction was recrystallized in absolute ethanol to obtain pure cordycepin (hereafter referred to as COR) identified by 1 H (500 MHz) and 13 C (125 MHz) nuclear magnetic resonance (JEOL, Tokyo, Japan).

Techniques: Expressing, Binding Assay

Journal: Nutrients

Article Title: Cordycepin, an Active Constituent of Nutrient Powerhouse and Potential Medicinal Mushroom Cordyceps militaris Linn., Ameliorates Age-Related Testicular Dysfunction in Rats

doi: 10.3390/nu11040906

Figure Lengend Snippet: Effect of COR on protein expression levels of mTOR and SIRT1in testis tissue. ( A ) Protein expression of mTOR and SIRT1. ( B ) Relative expression levels (fold) in three independent experiments normalized to β-actin. ( C ) The mRNA expression of mTOR and SIRT1. ( D ) Relative expression levels (fold) in three independent experiments normalized to GAPDH. # p < 0.05, compared with YC group and * p < 0.05 compared with the AC group. YC, young rat group; AC, aged rat group; COR-20, cordycepin (20 mg/kg) treated AC group; mTOR, growth-related mammalian target of rapamycin; SIRT1, silent mating type information regulation 2 homolog; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The cordycepin-rich fraction was recrystallized in absolute ethanol to obtain pure cordycepin (hereafter referred to as COR) identified by 1 H (500 MHz) and 13 C (125 MHz) nuclear magnetic resonance (JEOL, Tokyo, Japan).

Techniques: Expressing