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phosphorylated chk1 ser345 (Cell Signaling Technology Inc)

Name: phosphorylated chk1 ser345
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Cell Signaling Technology Inc phosphorylated chk1 ser345
Phosphorylated Chk1 Ser345, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phosphorylated chk1 ser345 - by Bioz Stars, 2026-03

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$Synergistic toxicity evoked by combined treatment with the CHK1 inhibitor PF477736 (PF) and the RAD51 inhibitor B02 in J82 CisPt . (A) The viability of J82 CisPt cells was analyzed after treatment with differently combined low and moderate toxic doses of CHK1 i PF477736 and RAD51 i B02 as indicated. Cell viability was measured after a 72 h treatment period using the AlamarBlue Assay. Based on the data obtained from three independent experiments each performed in quadruplicate, the combination indices (CIs) were calculated using CompuSyn software (CI < 0.9 indicating synergistic effects, CI ≈1 additive effects and CI > 1.2 antagonistic effects). (B) Protein expression and activation of different apoptosis‐related factors were examined via Western Blot analyses using protein extracts of J82 CisPt cells treated for 6 or 24 h with 10 μM B02, 1 μM PF477736 or both substances. (C) 10 μM B02 and 1 μM PF477736 ± the pan caspase inhibitor QVD were added 24 h after seeding and after 24, 48 and 72h treatment period the induction of SubG1 fraction was analyzed by propidium iodide staining followed by detection by flow cytometry. Data presented are the mean + SD from three independent experiments. *** p ≤ .001; * p ≤ .05.$

Journal: International Journal of Cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse

doi: 10.1002/ijc.35164

Figure Lengend Snippet: Synergistic toxicity evoked by combined treatment with the CHK1 inhibitor PF477736 (PF) and the RAD51 inhibitor B02 in J82 CisPt . (A) The viability of J82 CisPt cells was analyzed after treatment with differently combined low and moderate toxic doses of CHK1 i PF477736 and RAD51 i B02 as indicated. Cell viability was measured after a 72 h treatment period using the AlamarBlue Assay. Based on the data obtained from three independent experiments each performed in quadruplicate, the combination indices (CIs) were calculated using CompuSyn software (CI < 0.9 indicating synergistic effects, CI ≈1 additive effects and CI > 1.2 antagonistic effects). (B) Protein expression and activation of different apoptosis‐related factors were examined via Western Blot analyses using protein extracts of J82 CisPt cells treated for 6 or 24 h with 10 μM B02, 1 μM PF477736 or both substances. (C) 10 μM B02 and 1 μM PF477736 ± the pan caspase inhibitor QVD were added 24 h after seeding and after 24, 48 and 72h treatment period the induction of SubG1 fraction was analyzed by propidium iodide staining followed by detection by flow cytometry. Data presented are the mean + SD from three independent experiments. *** p ≤ .001; * p ≤ .05.

Article Snippet: The following antibodies were used: antibodies detecting Ser139 phosphorylated histone H2AX (γH2AX) (Millipore (Billerica, MA, USA) or Abcam [Cambridge, UK]), α‐tubulin, β‐actin (Santa Cruz Biotechnology [Santa Cruz, CA, USA]), Ser345 phosphorylated CHK1, CHK1, H2AX, cleaved caspase 7, caspase 7, PARP, Ser15 phosphorylated p53 (Cell Signaling [Danvers, MA, USA]), Ser4/8 phosphorylated RPA32, Ser824 phosphorylated KAP1 (Bethyl Laboratories [Montgomery, AL, USA]), Ser33 phosphorylated RPA32, RAD51, Pericentrin (Abcam [Cambridge, UK]), RPA32 (Millipore [Billerica, MA, USA]), rat anti‐BrdU (Abcam [Cambridge, UK]), mouse anti‐BrdU (BD [Franklin Lakes, NJ, USA]) and Ser10 phosphorylated histone 3 (pH 3) (Thermo Fisher [Waltham, MA, USA]).

Techniques: Alamar Blue Assay, Software, Expressing, Activation Assay, Western Blot, Staining, Flow Cytometry

Combining B02 or PF477736 with other CHK1‐ or RAD51‐inhibitors, respectively, likewise induces S‐phase arrest, replication stress, and DNA damage in J82 CisPt . J82 CisPt cells were co‐treated with 10 μM B02 + 1 μM LY2603618 (LY) (A) or 1 μM PF477736 (PF) + 30 μM RI(dl)2 (RI2) (B). Following 24 h treatment, propidium iodide‐based cell cycle analysis was performed by flow cytometry with emphasis on the proportion of cells in S‐phase. A total of 10,000 counts were measured for quantification. Induction of γH2AX and pRPA32 (S4, S8) was examined via Western Blot analyses with protein extracts of J82 CisPt cells treated for 6 or 24 h with the corresponding combination or mono‐treatments.

Journal: International Journal of Cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse

doi: 10.1002/ijc.35164

Figure Lengend Snippet: Combining B02 or PF477736 with other CHK1‐ or RAD51‐inhibitors, respectively, likewise induces S‐phase arrest, replication stress, and DNA damage in J82 CisPt . J82 CisPt cells were co‐treated with 10 μM B02 + 1 μM LY2603618 (LY) (A) or 1 μM PF477736 (PF) + 30 μM RI(dl)2 (RI2) (B). Following 24 h treatment, propidium iodide‐based cell cycle analysis was performed by flow cytometry with emphasis on the proportion of cells in S‐phase. A total of 10,000 counts were measured for quantification. Induction of γH2AX and pRPA32 (S4, S8) was examined via Western Blot analyses with protein extracts of J82 CisPt cells treated for 6 or 24 h with the corresponding combination or mono‐treatments.

Techniques: Cell Cycle Assay, Flow Cytometry, Western Blot

$A CTL or SSB plasmid was incubated with HSS at indicated concentrations for 30 min at room temperature. Extracts were examined via immunoblotting. B CTL or SSB plasmid was added to HSS at a final concentration of 40 ng/μL. After different time of incubation at room temperature, the extracts were examined via immunoblotting. C CTL or SSB plasmid was added to HSS at different concentrations as indicated. Extracts were examined via immunoblotting analysis. D CTL or SSB plasmid was added to HSS at a final concentration of 40 ng/μL. After different time of incubation, the extracts were examined via immunoblotting. E Lambda phosphatase was added to HSS supplemented with CTL or SSB plasmid. After a 15-minute incubation, the extracts were examined via immunoblotting. F ATM inhibitor KU55933 (1 mM), ATR inhibitor VE-822 (1 mM), or DNA-PK inhibitor NU7441 (25 μM) was added to HSS supplemented with CTL or SSB plasmid. After incubation, the extracts were examined via immunoblotting. G CTL or SSB plasmid was added to Mock- or Mre11-depleted HSS. After incubation, the extracts were examined via immunoblotting. H Quantification and statistical analysis of P-ATM/ATM shown in Panel G. Data are presented as mean values ± SD. ** p = 0.0011 (two-tailed, paired t-test). n = 3. a.u., arbitrary units. I CTL or SSB plasmid was added to HSS at a final concentration of 40 ng/μL. After different times of incubation, the extracts were examined via immunoblotting. J Quantification of intensity of protein bands from Fig. 1I& Fig. S1I, and ratios of P-ATM/ATM, and P-Chk1/Chk1 were shown. These samples derive from the same experiment and blots were processed in parallel. Data are presented as mean values ± SD. n = 3. K CTL or SSB plasmid was added to HSS at 40 ng/μL. After different incubation times, the extracts and DNA-bound fractions were examined via immunoblotting. The data presented in Panel A – G, I , and K are representative of three biological replicates.] in Panel C – G indicates mobility shifts of Mre11 and Nbs1.} indicates non-specific bands. Source data are provided as a Source Data file.$

Journal: Nature Communications

Article Title: Distinct regulation of ATM signaling by DNA single-strand breaks and APE1

doi: 10.1038/s41467-024-50836-6

Figure Lengend Snippet: A CTL or SSB plasmid was incubated with HSS at indicated concentrations for 30 min at room temperature. Extracts were examined via immunoblotting. B CTL or SSB plasmid was added to HSS at a final concentration of 40 ng/μL. After different time of incubation at room temperature, the extracts were examined via immunoblotting. C CTL or SSB plasmid was added to HSS at different concentrations as indicated. Extracts were examined via immunoblotting analysis. D CTL or SSB plasmid was added to HSS at a final concentration of 40 ng/μL. After different time of incubation, the extracts were examined via immunoblotting. E Lambda phosphatase was added to HSS supplemented with CTL or SSB plasmid. After a 15-minute incubation, the extracts were examined via immunoblotting. F ATM inhibitor KU55933 (1 mM), ATR inhibitor VE-822 (1 mM), or DNA-PK inhibitor NU7441 (25 μM) was added to HSS supplemented with CTL or SSB plasmid. After incubation, the extracts were examined via immunoblotting. G CTL or SSB plasmid was added to Mock- or Mre11-depleted HSS. After incubation, the extracts were examined via immunoblotting. H Quantification and statistical analysis of P-ATM/ATM shown in Panel G. Data are presented as mean values ± SD. ** p = 0.0011 (two-tailed, paired t-test). n = 3. a.u., arbitrary units. I CTL or SSB plasmid was added to HSS at a final concentration of 40 ng/μL. After different times of incubation, the extracts were examined via immunoblotting. J Quantification of intensity of protein bands from Fig. 1I& Fig. S1I, and ratios of P-ATM/ATM, and P-Chk1/Chk1 were shown. These samples derive from the same experiment and blots were processed in parallel. Data are presented as mean values ± SD. n = 3. K CTL or SSB plasmid was added to HSS at 40 ng/μL. After different incubation times, the extracts and DNA-bound fractions were examined via immunoblotting. The data presented in Panel A – G, I , and K are representative of three biological replicates.] in Panel C – G indicates mobility shifts of Mre11 and Nbs1.} indicates non-specific bands. Source data are provided as a Source Data file.

Article Snippet: Antibodies against ATM phosphorylation at Ser1981 (Rockland Immunochemicals, Cat#200-301-500), Chk1 (Santa Cruz Biotechnology, Cat#sc-8408), Chk1 phosphorylation at Ser345 (Cell Signaling Technology, Cat#2348), Chk2 (Santa Cruz Biotechnology, Cat#sc-9064), Chk2 phosphorylation at Thr68 (Santa Cruz Biotechnology, Cat#sc-16297-R), Flag (Thermo Fisher, Cat#MA1-91878), GST (Santa Cruz Biotechnology, Cat#sc-138), H2AX (Cell Signaling Technology, Cat#7631 S), H2AX phosphorylation at Ser139 (Cell Signaling Technology, Cat#2577 S), H3 (Abcam, Cat#ab1791), His (Santa Cruz Biotechnology, Cat#sc-8036), Myc (Santa Cruz Biotechnology, Cat#sc-40), RPA32 phosphorylation at Ser33 (Bethyl Laboratories, Cat#A300-246A), and PCNA (Santa Cruz Biotechnology, Cat#sc-56) were purchased from commercially available vendors.

Techniques: Plasmid Preparation, Incubation, Western Blot, Concentration Assay, Two Tailed Test

DNA damage-response defect and failure of checkpoint activation in transformed ADPKD cells. ( A , B ) Phosphorylation-dependent activation of ATM and ATR, but not CHK1 or CHK2, in response to DNA damage. One hour after exposure to ionizing radiation (IR, A) or ultraviolet radiation (UV, B), ADPKD cells exhibited phosphorylation-mediated activation of ATM at Ser1987 ( A ) and ATR at Ser428 ( B ). However, phosphorylation-dependent activation of CHK1 at Ser345 ( B ) and CHK2 at Tyr68 ( A ) was not observed in the same ADPKD cells. p84 was included as a loading control. ( C ) Failure of IR to induce ATM nuclear foci (IRIF) in ADPKD cells. One hour after IR, cells were fixed and immunostained with anti-phospho-S1987-ATM or anti-γH2AX antibodies. γH2AX nuclear foci, which indicate DNA damage sites , were detected in both HK2 and WT9-7 ADPKD cells. In contrast, ATM IRIF were observed in HK2 cells but not in WT9-7 cells. Representative microscopic fields and the mean percentages of cells (± SEM) in each condition containing nuclei with more than five IRIF are shown. Over 200 cells were examined and scored in three separate experiments. The presence of γH2AX nuclear foci in untreated WT9-7 cells is worth noting; it indicates spontaneous DNA damage in these ADPKD cells.

Journal: International Journal of Molecular Sciences

Article Title: The Link between Autosomal Dominant Polycystic Kidney Disease and Chromosomal Instability: Exploring the Relationship

doi: 10.3390/ijms25052936

Figure Lengend Snippet: DNA damage-response defect and failure of checkpoint activation in transformed ADPKD cells. ( A , B ) Phosphorylation-dependent activation of ATM and ATR, but not CHK1 or CHK2, in response to DNA damage. One hour after exposure to ionizing radiation (IR, A) or ultraviolet radiation (UV, B), ADPKD cells exhibited phosphorylation-mediated activation of ATM at Ser1987 ( A ) and ATR at Ser428 ( B ). However, phosphorylation-dependent activation of CHK1 at Ser345 ( B ) and CHK2 at Tyr68 ( A ) was not observed in the same ADPKD cells. p84 was included as a loading control. ( C ) Failure of IR to induce ATM nuclear foci (IRIF) in ADPKD cells. One hour after IR, cells were fixed and immunostained with anti-phospho-S1987-ATM or anti-γH2AX antibodies. γH2AX nuclear foci, which indicate DNA damage sites , were detected in both HK2 and WT9-7 ADPKD cells. In contrast, ATM IRIF were observed in HK2 cells but not in WT9-7 cells. Representative microscopic fields and the mean percentages of cells (± SEM) in each condition containing nuclei with more than five IRIF are shown. Over 200 cells were examined and scored in three separate experiments. The presence of γH2AX nuclear foci in untreated WT9-7 cells is worth noting; it indicates spontaneous DNA damage in these ADPKD cells.

Article Snippet: Antibodies against γ-H2AX, phosphorylated L-S/T-Q, phosphorylated Chk1 at serine 345, phosphorylated Chk2 at threonine 68, phosphorylated ATR at serine 428, histone H3, and phosphorylated serine 10 of histone H3 were also obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Transformation Assay, Phospho-proteomics, Control

DNA damage and defective response in ADPKD kidneys. Sections from formalin-fixed, paraffin-embedded ADPKD nephrectomy specimens were immunostained with anti-8OHdG, anti-γH2AX, anti-CHK2-T68P, anti-CHK1-S345P, and anti-ATM-S1987P antibodies (all shown), as well as with anti-ATR, anti-Mre11, anti-NFBD1/MDC1, anti-53BP1, and anti-pSQ antibodies. Representative microscopic fields are displayed. Punctate nuclear staining with 8-OHdG antibodies ( A ) and γH2AX antibodies ( B ) can be observed. However, there is minimal or no specific nuclear staining for phosphorylated CHK2-T68P ( C ) or cytosolic staining for phosphorylated ATM-S1987P ( D ), phosphorylated CHK1-S345P ( E ), or phosphorylated CHK2-T68P ( F ). Staining results are summarized in .

Journal: International Journal of Molecular Sciences

Article Title: The Link between Autosomal Dominant Polycystic Kidney Disease and Chromosomal Instability: Exploring the Relationship

doi: 10.3390/ijms25052936

Figure Lengend Snippet: DNA damage and defective response in ADPKD kidneys. Sections from formalin-fixed, paraffin-embedded ADPKD nephrectomy specimens were immunostained with anti-8OHdG, anti-γH2AX, anti-CHK2-T68P, anti-CHK1-S345P, and anti-ATM-S1987P antibodies (all shown), as well as with anti-ATR, anti-Mre11, anti-NFBD1/MDC1, anti-53BP1, and anti-pSQ antibodies. Representative microscopic fields are displayed. Punctate nuclear staining with 8-OHdG antibodies ( A ) and γH2AX antibodies ( B ) can be observed. However, there is minimal or no specific nuclear staining for phosphorylated CHK2-T68P ( C ) or cytosolic staining for phosphorylated ATM-S1987P ( D ), phosphorylated CHK1-S345P ( E ), or phosphorylated CHK2-T68P ( F ). Staining results are summarized in .

Techniques: Formalin-fixed Paraffin-Embedded, Staining

Journal: Cell reports

Article Title: MEF2A suppresses stress responses that trigger DDX41-dependent IFN production

doi: 10.1016/j.celrep.2023.112805

Figure Lengend Snippet:

Article Snippet: Rabbit anti phosphorylated CHK1 , Cell Signaling Technology , RRID:AB_331212.

Techniques: Virus, Recombinant, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Negative Control, Software

APE1 and its nuclease activity is important for ATR–Chk1 DDR pathway in mammalian cells. ( A ) siRNA-mediated APE1-knockdown impaired H 2 O 2 -induced ATR–Chk1 DDR pathway in MDA-MB-231 cells. Total cell lysates were extracted and analyzed via immunoblotting analysis as indicated. ( B, C ) The H 2 O 2 -induced Chk1 phosphorylation was compromised by AR03 or APE1i III at different doses (5, 10, or 15 μM for 2 h) in MDA-MB-231 cells. ( D ) The H 2 O 2 -induced Chk1 phosphorylation was reduced in APE1-KD PANC1 cells. ( E, F ) The H 2 O 2 -induced Chk1 phosphorylation was impaired by AR03 or APE1i III in a dose-dependent manner (5, 10 or 15 μM for 2 h) in PANC1 cells. (A–F) ‘Chk1-P-S345/Chk1’ was quantified by the intensity of Chk1-P-S345 versus total Chk1. Means ± SD, n = 3. ( G ) AR03 or APE1i III but not E3330 inhibited APE1’s endonuclease activity. Different doses of AR03, APE1i III, or E3330 (1, 2, or 5 mM) was added to endonuclease assays containing 0.185 μM APE1 protein and a 5’-FAM-labeled dsDNA-AP substrate. After incubation for 30 min, samples were analyzed via 20% TBE gel. ( H ) AR03 or Inhibitor III could inhibit APE1’s exo-nuclease activity. Different doses of AR03, APE1i III, or E3330 (1, 2 or 5 mM) was added to exonuclease assays containing 0.185 μM APE1 protein and 5’-FAM-labeled dsDNA-SSB substrate. After 30-min incubation, samples were examined via 20% TBE gel.

Journal: Nucleic Acids Research

Article Title: APE1 assembles biomolecular condensates to promote the ATR–Chk1 DNA damage response in nucleolus

doi: 10.1093/nar/gkac853

Figure Lengend Snippet: APE1 and its nuclease activity is important for ATR–Chk1 DDR pathway in mammalian cells. ( A ) siRNA-mediated APE1-knockdown impaired H 2 O 2 -induced ATR–Chk1 DDR pathway in MDA-MB-231 cells. Total cell lysates were extracted and analyzed via immunoblotting analysis as indicated. ( B, C ) The H 2 O 2 -induced Chk1 phosphorylation was compromised by AR03 or APE1i III at different doses (5, 10, or 15 μM for 2 h) in MDA-MB-231 cells. ( D ) The H 2 O 2 -induced Chk1 phosphorylation was reduced in APE1-KD PANC1 cells. ( E, F ) The H 2 O 2 -induced Chk1 phosphorylation was impaired by AR03 or APE1i III in a dose-dependent manner (5, 10 or 15 μM for 2 h) in PANC1 cells. (A–F) ‘Chk1-P-S345/Chk1’ was quantified by the intensity of Chk1-P-S345 versus total Chk1. Means ± SD, n = 3. ( G ) AR03 or APE1i III but not E3330 inhibited APE1’s endonuclease activity. Different doses of AR03, APE1i III, or E3330 (1, 2, or 5 mM) was added to endonuclease assays containing 0.185 μM APE1 protein and a 5’-FAM-labeled dsDNA-AP substrate. After incubation for 30 min, samples were analyzed via 20% TBE gel. ( H ) AR03 or Inhibitor III could inhibit APE1’s exo-nuclease activity. Different doses of AR03, APE1i III, or E3330 (1, 2 or 5 mM) was added to exonuclease assays containing 0.185 μM APE1 protein and 5’-FAM-labeled dsDNA-SSB substrate. After 30-min incubation, samples were examined via 20% TBE gel.

Article Snippet: Primary antibodies were purchased from respective vendors: APE1 (Santa Cruz Biotechnology Cat#sc-17774), ATR (Santa Cruz Biotechnology Cat#515173), ATM (GeneTex Cat#GTX70103), ATM phosphorylation at Ser1981 (Abcam Cat#ab81292), ATR phosphorylation at Thr1989 (Cell Signaling Technology Cat#D5K8W), ATRIP (Santa Cruz Biotechnology Cat#365383), Chk1 (Santa Cruz Biotechnology Cat#sc-8408), Chk1 phosphorylation at Ser345 (Cell Signaling Technology Cat#133D3), Chk1 phosphorylation at Ser317 (Cell Signaling Technology Cat#D12H3), Chk2 (Santa Cruz Biotechnology Cat#sc-9064), Chk2 phosphorylation at Thr68 (Santa Cruz Biotechnology Cat#sc-16297), eGFP (Life Technologies Corporation Cat#TA150041), ETAA1 (Abcam Cat#ab122245), H2AX (Cell Signaling Technology Cat#2D17A3), H2AX phosphorylation at Ser139 (Cell Signaling Technology Cat#2577s), NPM1 (Santa Cruz Biotechnology Cat#sc-47725), p53 phosphorylation at Ser15 (Cell Signaling Technology Cat#9284), p53 (Santa Cruz Biotechnology Cat#sc-126), PCNA (Santa Cruz Biotechnology Cat#sc-56), RPA32 (Thermo Fisher Scientific Cat#MA1-26418), RPA32 phosphorylation at Ser33 (Bethyl Laboratories Cat#A300-246A), TopBP1 (Santa Cruz Biotechnology Cat#271043), Tubulin (Santa Cruz Biotechnology Cat#sc-8035), YFP (BioVision Cat#3991–100).

Techniques: Activity Assay, Knockdown, Western Blot, Phospho-proteomics, Labeling, Incubation

APE1 overexpression leads to the activation of ATR–Chk1 DDR pathway independent of its nuclease and redox function in mammalian cells. ( A ) ATR–Chk1 DDR pathway was activated by the overexpression of YFP-APE1 but not YFP nor CTL transfection (no vector transfection) in MDA-MB-231 cells. After different times of incubation (i.e. 2, 3, 4 or 7 days), total cell lysates were extracted and examined via immunoblotting analysis as indicated. ( B ) Chk1 phosphorylation induced by YFP-APE1 was dependent on ATR, but not ATM nor DNA-PKcs. After 4-d overexpression of YFP-APE1 or YFP, different DDR kinase inhibitors (5 μM of VE-822, KU55933 or NU7441) were added to MDA-MB-231 cells for 2 h, followed by total cell lysate extraction and immunoblotting analysis as indicated. ( C ) APE1 inhibitors almost had no noticeable effect on Chk1 phosphorylation by YFP-APE1 overexpression. After 2-day overexpression of YFP-APE1 or YFP, different APE1 inhibitors (1 μM of AR03, APE1i III or E3330) were added to MDA-MB-231 cells for 2 days, followed by total cell lysate extraction and immunoblotting analysis. ( D ) The endonuclease activity of WT or mutant (i.e. D308A, C64A, E96Q, ED) His-APE1 was examined on 20% TBE gel. ( E ) The exonuclease activity of WT or mutant (i.e. D308A, C64A, E96Q, ED) His-APE1 was examined on 20% TBE gel. ( F ) Chk1 phosphorylation was triggered by WT/mutant YFP-APE1 (i.e. D308A, C64A, E96Q, ED) overexpression in MDA-MB-231 cells. After 4-day overexpression of WT/mutant YFP-APE1 or YFP in MDA-MB-231, total cell lysates were extracted and analyzed via immunoblotting analysis. The Chk1-P-S345/Chk1 data are presented as means ± SD, n = 3.

Journal: Nucleic Acids Research

Article Title: APE1 assembles biomolecular condensates to promote the ATR–Chk1 DNA damage response in nucleolus

doi: 10.1093/nar/gkac853

Figure Lengend Snippet: APE1 overexpression leads to the activation of ATR–Chk1 DDR pathway independent of its nuclease and redox function in mammalian cells. ( A ) ATR–Chk1 DDR pathway was activated by the overexpression of YFP-APE1 but not YFP nor CTL transfection (no vector transfection) in MDA-MB-231 cells. After different times of incubation (i.e. 2, 3, 4 or 7 days), total cell lysates were extracted and examined via immunoblotting analysis as indicated. ( B ) Chk1 phosphorylation induced by YFP-APE1 was dependent on ATR, but not ATM nor DNA-PKcs. After 4-d overexpression of YFP-APE1 or YFP, different DDR kinase inhibitors (5 μM of VE-822, KU55933 or NU7441) were added to MDA-MB-231 cells for 2 h, followed by total cell lysate extraction and immunoblotting analysis as indicated. ( C ) APE1 inhibitors almost had no noticeable effect on Chk1 phosphorylation by YFP-APE1 overexpression. After 2-day overexpression of YFP-APE1 or YFP, different APE1 inhibitors (1 μM of AR03, APE1i III or E3330) were added to MDA-MB-231 cells for 2 days, followed by total cell lysate extraction and immunoblotting analysis. ( D ) The endonuclease activity of WT or mutant (i.e. D308A, C64A, E96Q, ED) His-APE1 was examined on 20% TBE gel. ( E ) The exonuclease activity of WT or mutant (i.e. D308A, C64A, E96Q, ED) His-APE1 was examined on 20% TBE gel. ( F ) Chk1 phosphorylation was triggered by WT/mutant YFP-APE1 (i.e. D308A, C64A, E96Q, ED) overexpression in MDA-MB-231 cells. After 4-day overexpression of WT/mutant YFP-APE1 or YFP in MDA-MB-231, total cell lysates were extracted and analyzed via immunoblotting analysis. The Chk1-P-S345/Chk1 data are presented as means ± SD, n = 3.

Techniques: Over Expression, Activation Assay, Transfection, Plasmid Preparation, Incubation, Western Blot, Phospho-proteomics, Extraction, Activity Assay, Mutagenesis

The APE1 N33 motif is required for ATR–Chk1 DDR pathway in vivo and in vitro . ( A ) Overexpression of WT but not △N33 tGFP-APE1 nor tGFP activated Chk1 phosphorylation in PANC1 cells. APE1 or N33-depletion over-expression plasmid was transfected to PANC1 cells. After 1-day incubation of various overexpression plasmid transfection, different doses of doxycycline (0, 10 or 50 μg/ml) were added to PANC1 cells for another 2 days. Total cell lysates were then extracted and analyzed via immunoblotting analysis. ( B ) △N33 tGFP-APE1 was deficient in nuclear localization in PANC1 cells. tGFP, WT or △N33 tGFP-APE1 overexpression plasmid was transfected to PANC1 cells. After 1-day incubation of overexpression plasmid transfection, 50 μg/ml doxycycline was added to PANC1 cells for another 2 days, followed with fluorescence microscopy analysis. Scale bars = 10 μm. ( C ) Chk1 phosphorylation was triggered by the excess addition of recombinant His-APE1 protein in a dose-dependent manner in PANC1 nuclear extracts. Different doses of His-APE1 protein were added to PANC1 nuclear extracts for incubation for 30 min at room temperature. The samples were analyzed via immunoblotting analysis as indicated. ( D ) Chk1 phosphorylation induced by excess addition of recombinant His-APE1 protein in PANC1 nuclear extracts was independent of its nuclease activity and redox function. WT or mutant (i.e. D308A, C65A, E96Q, ED) His-APE1 was added to PANC1 nuclear extracts for 30-min incubation at room temperature, followed by immunoblotting analysis. ( E ) Pulldown assays suggest that WT/mutant His-APE1 associated with ATR, ATRIP and RPA32 in PANC1 nuclear extracts. ‘Input’ or ‘Pulldown’ samples were examined via immunoblotting analysis as indicated. ( F ) Chk1 phosphorylation induced by WT His-APE1-YFP protein in PANC1 nuclear extracts was inhibited by VE-822 but not KU55933 nor NU7441 (1 mM). ( G ) Chk1 phosphorylation was activated by excess addition of WT His-APE1-YFP but not △N33 His-APE1-YFP protein in PANC1 nuclear extracts. ( H ) Pulldown assays suggest that WT but not △N33 His-APE1-APE1 associated with ATR, ATRIP and RPA32 in PANC1 nuclear extracts pre-treated with DNase I (2 mg/ml). ‘Input’ or ‘Pulldown’ samples were examined via immunoblotting analysis as indicated. (A, C, D, G) Chk1-P-S345/Chk1 was quantified and examined as means ± SD ( n = 3).

Journal: Nucleic Acids Research

Article Title: APE1 assembles biomolecular condensates to promote the ATR–Chk1 DNA damage response in nucleolus

doi: 10.1093/nar/gkac853

Figure Lengend Snippet: The APE1 N33 motif is required for ATR–Chk1 DDR pathway in vivo and in vitro . ( A ) Overexpression of WT but not △N33 tGFP-APE1 nor tGFP activated Chk1 phosphorylation in PANC1 cells. APE1 or N33-depletion over-expression plasmid was transfected to PANC1 cells. After 1-day incubation of various overexpression plasmid transfection, different doses of doxycycline (0, 10 or 50 μg/ml) were added to PANC1 cells for another 2 days. Total cell lysates were then extracted and analyzed via immunoblotting analysis. ( B ) △N33 tGFP-APE1 was deficient in nuclear localization in PANC1 cells. tGFP, WT or △N33 tGFP-APE1 overexpression plasmid was transfected to PANC1 cells. After 1-day incubation of overexpression plasmid transfection, 50 μg/ml doxycycline was added to PANC1 cells for another 2 days, followed with fluorescence microscopy analysis. Scale bars = 10 μm. ( C ) Chk1 phosphorylation was triggered by the excess addition of recombinant His-APE1 protein in a dose-dependent manner in PANC1 nuclear extracts. Different doses of His-APE1 protein were added to PANC1 nuclear extracts for incubation for 30 min at room temperature. The samples were analyzed via immunoblotting analysis as indicated. ( D ) Chk1 phosphorylation induced by excess addition of recombinant His-APE1 protein in PANC1 nuclear extracts was independent of its nuclease activity and redox function. WT or mutant (i.e. D308A, C65A, E96Q, ED) His-APE1 was added to PANC1 nuclear extracts for 30-min incubation at room temperature, followed by immunoblotting analysis. ( E ) Pulldown assays suggest that WT/mutant His-APE1 associated with ATR, ATRIP and RPA32 in PANC1 nuclear extracts. ‘Input’ or ‘Pulldown’ samples were examined via immunoblotting analysis as indicated. ( F ) Chk1 phosphorylation induced by WT His-APE1-YFP protein in PANC1 nuclear extracts was inhibited by VE-822 but not KU55933 nor NU7441 (1 mM). ( G ) Chk1 phosphorylation was activated by excess addition of WT His-APE1-YFP but not △N33 His-APE1-YFP protein in PANC1 nuclear extracts. ( H ) Pulldown assays suggest that WT but not △N33 His-APE1-APE1 associated with ATR, ATRIP and RPA32 in PANC1 nuclear extracts pre-treated with DNase I (2 mg/ml). ‘Input’ or ‘Pulldown’ samples were examined via immunoblotting analysis as indicated. (A, C, D, G) Chk1-P-S345/Chk1 was quantified and examined as means ± SD ( n = 3).

Techniques: In Vivo, In Vitro, Over Expression, Phospho-proteomics, Plasmid Preparation, Transfection, Incubation, Western Blot, Fluorescence, Microscopy, Recombinant, Activity Assay, Mutagenesis

APE1 assembles biomolecular condensates in vitro to promote ATR DDR pathway. ( A ) WT but not △N33 His-APE1-YFP formed phase separation in PANC1 nuclear extracts. Recombinant YFP, WT or △N33 His-APE1-YFP protein (0.55 mM) was added to PANC1 nuclear extract and incubated for 15 min at room temperature, followed by fluorescence microscopy analysis. ( B ) DNase I and RNase A had almost no effect on the APE1-assembled phase separation in nuclear extracts. DNase I and RNase A (2 mg/ml each) was added to PANC1 nuclear extracts and incubated for 30 min at 37°C. Then WT His-APE1-YFP protein (0.55 mM) was added and incubated for 10 min at room temperature, followed by fluorescence microscopy analysis. ( C ) APE1 inhibitors had no noticeable effect on phase separation formed by APE1 in nuclear extracts. WT His-APE1-YFP protein (0.55 mM) was added to PANC1 supplemented with DMSO or APE1 inhibitors (AR03, APE1i III, and E3330). After 15-min incubation at room temperature, reaction mixtures were examined via fluorescence microscopy analysis. ( D ) APE1 inhibitors had no effect on the Chk1 phosphorylation induced by the excess addition of APE1 protein in PANC1 nuclear extracts. APE1 inhibitor (AR03, APE1i III or E3330) was added to PANC1 nuclear extracts and incubated for 10 min at room temperature, which was supplemented with WT His-APE1-YFP (0.55 mM) and incubated for another 30 min at room temperature. The samples were then analyzed via immunoblotting analysis as indicated. ( E ) WT but not △N33 His-APE1-YFP associated with ATR, TopBP1 and ETAA1 in PANC1 nuclear extracts. ‘Input’ and ‘Pulldown’ samples from pulldown experiments were examined via immunoblotting analysis as indicated. AR03 or E3330 was added to 1 mM. ( F ) WT but not △N33 His-APE1-YFP formed phase separation in a LLPS buffer. WT but not △N33 His-APE1-YFP (0.55 mM) was added to a LLPS buffer and incubated for 15 min at room temperature, followed by fluorescence microscopy analysis. ( G ) Phase separation formed by recombinant WT His-APE1-YFP protein was fused into large biomolecular condensates in a LLPS buffer. WT His-APE1-YFP (0.55 mM) was added to a buffer and incubated for 2 h before fluorescence microscopy analysis. BF, bright field. ( H ) Phase separation assembled by APE1 in a LLPS buffer was not affected by the APE1 inhibitors. WT His-APE1-YFP protein (0.55 mM) was added to LLPS buffer supplemented with DMSO or APE1 inhibitors (AR03, APE1i III and E3330). After 15-min incubation at room temperature, reaction mixtures were examined via fluorescence microscopy analysis. ( I–M ) The requirement of NT33 motif within APE1 for phase separation in a LLPS buffer was bypassed with the presence of different DNA or RNA. WT but not △N33 His-APE1-YFP (0.55 mM) was added to a buffer supplemented with 0.5 μM of different DNA/RNA structures (dsDNA (I), dsDNA with AP site (J), ssDNA (K), ssDNA with AP site (L) and ssRNA (M)). Reaction mixtures were incubated for 15 min at room temperature before fluorescence microscopy analysis. All scale bars = 50 μm.

Journal: Nucleic Acids Research

Article Title: APE1 assembles biomolecular condensates to promote the ATR–Chk1 DNA damage response in nucleolus

doi: 10.1093/nar/gkac853

Figure Lengend Snippet: APE1 assembles biomolecular condensates in vitro to promote ATR DDR pathway. ( A ) WT but not △N33 His-APE1-YFP formed phase separation in PANC1 nuclear extracts. Recombinant YFP, WT or △N33 His-APE1-YFP protein (0.55 mM) was added to PANC1 nuclear extract and incubated for 15 min at room temperature, followed by fluorescence microscopy analysis. ( B ) DNase I and RNase A had almost no effect on the APE1-assembled phase separation in nuclear extracts. DNase I and RNase A (2 mg/ml each) was added to PANC1 nuclear extracts and incubated for 30 min at 37°C. Then WT His-APE1-YFP protein (0.55 mM) was added and incubated for 10 min at room temperature, followed by fluorescence microscopy analysis. ( C ) APE1 inhibitors had no noticeable effect on phase separation formed by APE1 in nuclear extracts. WT His-APE1-YFP protein (0.55 mM) was added to PANC1 supplemented with DMSO or APE1 inhibitors (AR03, APE1i III, and E3330). After 15-min incubation at room temperature, reaction mixtures were examined via fluorescence microscopy analysis. ( D ) APE1 inhibitors had no effect on the Chk1 phosphorylation induced by the excess addition of APE1 protein in PANC1 nuclear extracts. APE1 inhibitor (AR03, APE1i III or E3330) was added to PANC1 nuclear extracts and incubated for 10 min at room temperature, which was supplemented with WT His-APE1-YFP (0.55 mM) and incubated for another 30 min at room temperature. The samples were then analyzed via immunoblotting analysis as indicated. ( E ) WT but not △N33 His-APE1-YFP associated with ATR, TopBP1 and ETAA1 in PANC1 nuclear extracts. ‘Input’ and ‘Pulldown’ samples from pulldown experiments were examined via immunoblotting analysis as indicated. AR03 or E3330 was added to 1 mM. ( F ) WT but not △N33 His-APE1-YFP formed phase separation in a LLPS buffer. WT but not △N33 His-APE1-YFP (0.55 mM) was added to a LLPS buffer and incubated for 15 min at room temperature, followed by fluorescence microscopy analysis. ( G ) Phase separation formed by recombinant WT His-APE1-YFP protein was fused into large biomolecular condensates in a LLPS buffer. WT His-APE1-YFP (0.55 mM) was added to a buffer and incubated for 2 h before fluorescence microscopy analysis. BF, bright field. ( H ) Phase separation assembled by APE1 in a LLPS buffer was not affected by the APE1 inhibitors. WT His-APE1-YFP protein (0.55 mM) was added to LLPS buffer supplemented with DMSO or APE1 inhibitors (AR03, APE1i III and E3330). After 15-min incubation at room temperature, reaction mixtures were examined via fluorescence microscopy analysis. ( I–M ) The requirement of NT33 motif within APE1 for phase separation in a LLPS buffer was bypassed with the presence of different DNA or RNA. WT but not △N33 His-APE1-YFP (0.55 mM) was added to a buffer supplemented with 0.5 μM of different DNA/RNA structures (dsDNA (I), dsDNA with AP site (J), ssDNA (K), ssDNA with AP site (L) and ssRNA (M)). Reaction mixtures were incubated for 15 min at room temperature before fluorescence microscopy analysis. All scale bars = 50 μm.

Techniques: In Vitro, Recombinant, Incubation, Fluorescence, Microscopy, Phospho-proteomics, Western Blot

APE1 forms biomolecular condensates in nucleoli to recruit ATR, TopBP1, and ETAA1 for ATR activation in a NPM1-independent fashion in cancer cells but not unmalignant cells. ( A ) Overexpressed YFP-APE1 but not YFP colocalized with NPM1 in nucleoli in PANC1 cells. After overexpression of YFP or YFP-APE1 for 3 days, PANC1 cells were fixed and incubated with anti-NPM1-AF647 fluorescence antibody for overnight at 4°C. ( B–D ) ATR and its activators TopBP1 and ETAA1 were colocalized with YFP-APE1 in nucleoli in PANC1 cells. YFP or YFP-APE1 overexpression plasmid was added to PANC1 cells. After overexpression of YFP or YFP-APE1 for 3 days, PANC1 cells were fixed and incubated with anti-ATR-AF647 (B), anti-TopBP1-AF647 (C), or anti-ETAA1-AF647 (D) for overnight at 4°C. ( E ) After overexpression of YFP or YFP-APE1 for 3 days, PANC1 cells were fixed and incubated with anti-Chk1-P-S345 antibody for overnight at 4°C, followed by incubation with anti-Rabbit secondary antibody conjugated with AF594 at 4°C for 4 h. ( F ) After overexpression of mCherry or mCherry-APE1 for 3 days, PANC1 cells were fixed and incubated with anti-γH2AX-AF488 at 4°C overnight. ( G ) Overexpressed YFP-APE1 was not colocalized with NPM1 in nucleoli in HPDE cells. Similar experiment to Panel (A) was performed in HPDE cells. ( H ) Nucleolar localization of NPM1 was not affected when endogenous APE1 was knocked down via siRNA. Control (CTL) siRNA or APE1 siRNA was added and transfected to PANC1 cells for 3 days. The cells were then fixed and incubated with anti-APE1-AF488 and anti-NPM1-AF647 for overnight at 4°C. ( I ) YFP-APE1 still assembled condensates when endogenous NPM1 was knocked down. CTL siRNA or NPM1 siRNA was transfected to PANC1 cells and incubated for 1 day. Then YFP or YFP-APE1 overexpression plasmid was transfected and incubated for 3 days. The cells were fixed and incubated with anti-NPM1-AF647 at 4°C overnight. (A–I) All cells were examined by fluorescence microscopy analysis. All scale bars = 10 μm.

Journal: Nucleic Acids Research

Article Title: APE1 assembles biomolecular condensates to promote the ATR–Chk1 DNA damage response in nucleolus

doi: 10.1093/nar/gkac853

Figure Lengend Snippet: APE1 forms biomolecular condensates in nucleoli to recruit ATR, TopBP1, and ETAA1 for ATR activation in a NPM1-independent fashion in cancer cells but not unmalignant cells. ( A ) Overexpressed YFP-APE1 but not YFP colocalized with NPM1 in nucleoli in PANC1 cells. After overexpression of YFP or YFP-APE1 for 3 days, PANC1 cells were fixed and incubated with anti-NPM1-AF647 fluorescence antibody for overnight at 4°C. ( B–D ) ATR and its activators TopBP1 and ETAA1 were colocalized with YFP-APE1 in nucleoli in PANC1 cells. YFP or YFP-APE1 overexpression plasmid was added to PANC1 cells. After overexpression of YFP or YFP-APE1 for 3 days, PANC1 cells were fixed and incubated with anti-ATR-AF647 (B), anti-TopBP1-AF647 (C), or anti-ETAA1-AF647 (D) for overnight at 4°C. ( E ) After overexpression of YFP or YFP-APE1 for 3 days, PANC1 cells were fixed and incubated with anti-Chk1-P-S345 antibody for overnight at 4°C, followed by incubation with anti-Rabbit secondary antibody conjugated with AF594 at 4°C for 4 h. ( F ) After overexpression of mCherry or mCherry-APE1 for 3 days, PANC1 cells were fixed and incubated with anti-γH2AX-AF488 at 4°C overnight. ( G ) Overexpressed YFP-APE1 was not colocalized with NPM1 in nucleoli in HPDE cells. Similar experiment to Panel (A) was performed in HPDE cells. ( H ) Nucleolar localization of NPM1 was not affected when endogenous APE1 was knocked down via siRNA. Control (CTL) siRNA or APE1 siRNA was added and transfected to PANC1 cells for 3 days. The cells were then fixed and incubated with anti-APE1-AF488 and anti-NPM1-AF647 for overnight at 4°C. ( I ) YFP-APE1 still assembled condensates when endogenous NPM1 was knocked down. CTL siRNA or NPM1 siRNA was transfected to PANC1 cells and incubated for 1 day. Then YFP or YFP-APE1 overexpression plasmid was transfected and incubated for 3 days. The cells were fixed and incubated with anti-NPM1-AF647 at 4°C overnight. (A–I) All cells were examined by fluorescence microscopy analysis. All scale bars = 10 μm.

Techniques: Activation Assay, Over Expression, Incubation, Fluorescence, Plasmid Preparation, Control, Transfection, Microscopy

While TopBP1 is dispensable for YFP-APE1 nucleolar localization, the W119 residue within APE1’s putative ATR activation domain is required for its biomolecular condensate formation in nucleoli and nucleolar ATR activation. ( A ) YFP-APE1 still assembled condensates and activated ATR-chk1 pathway when endogenous TopBP1 was knocked down via siRNA. After 1-d incubation of CTL or TopBP1 siRNA in PANC1 cells, YFP or YFP-APE1 overexpression plasmid was added and incubation for another 3 d. The cells were fixed and incubated with anti-TopBP1-AF647 for fluorescence microscopy analysis. ( B ). After 1-d incubation of CTL, TopBP1 and/or ETAA1 siRNA in PANC1 cells, YFP or YFP-APE1 overexpression plasmid was added and incubated for another 3 d. The cells were extracted and examined by immunoblotting analysis as indicated. ( C ) Schematic diagrams of functional domains of hAPE1, hTopBP1 and hETAA1 as well as sequence alignment of the N-terminal domain and putative ATR activation domain (AAD) within APE1. hAPE1 (human APE1, NCBI#: P27695.1), mAPE1 (mouse APE1, NCBI#: NP_033817.1), xAPE1 ( Xenopus laevis APE1, NCBI#: AAH72056.1), hTopBP1 (human TopBP1, NCBI#: Q92547.3), and hETAA1 (human ETAA1, NCBI#: NP_061875.2). * identical;: highly conserved;. low conservation. In the AAD alignment, highlighted green indicates aa with hydrophobic side chains, highlighted yellow indicates aa with polar uncharged side chains, highlighted pink indicates aa with positive charged sides chains, highlighted turquoise indicates aa with negative charged sides chains, and highlighted gray indicates the conserved WxxP/N peptide. ( D ) The K6, K7 and W119 residues within APE1 are important for Chk1 phosphorylation induced by YFP-APE1 in PANC1 cells. After YFP, WT or mutant (i.e. K6R/K7R, K24R/K25R, K31R/K32R, W119R) YFP-APE1 overexpression plasmid was added to PANC1 cells and incubation for 3 d, total cell lysates were extracted and examined via immunoblotting analysis as indicated. ( E ) APE1 forms biomolecular condensates in nucleoli dependent on its W119 residue. After WT or mutant (i.e. K6R/K7R, K24R/K25R, K31R/K32R, W119R) YFP-APE1 overexpression plasmid was added to PANC1 cells and incubation for 3 d, the cells were fixed and incubated with anti-NPM1-AF647 for overnight at 4°C, followed by fluorescence microscopy analysis. ( F ) WT but not W119R YFP-APE1 directly activates ATR to phosphorylate Chk1 phosphorylaton at S345 in vitro . In in vitro kinase assays, equal amount of His-YFP, WT or W119R His-APE1-YFP was added to the kinase assays (purified Flag-ATR as kinase and purified His-Chk1 as substrate). Beads bound with lysates from HEK293 cells without Flag-ATR transfection were used as a negative control (‘Control’). ‘No addition’ indicate the kinase assays without the addition of His-YFP or WT/W119R His-APE1-YFP. The samples were examined via immunoblotting analysis as indicated. (A, E) All scale bars = 10 μm.

Journal: Nucleic Acids Research

Article Title: APE1 assembles biomolecular condensates to promote the ATR–Chk1 DNA damage response in nucleolus

doi: 10.1093/nar/gkac853

Figure Lengend Snippet: While TopBP1 is dispensable for YFP-APE1 nucleolar localization, the W119 residue within APE1’s putative ATR activation domain is required for its biomolecular condensate formation in nucleoli and nucleolar ATR activation. ( A ) YFP-APE1 still assembled condensates and activated ATR-chk1 pathway when endogenous TopBP1 was knocked down via siRNA. After 1-d incubation of CTL or TopBP1 siRNA in PANC1 cells, YFP or YFP-APE1 overexpression plasmid was added and incubation for another 3 d. The cells were fixed and incubated with anti-TopBP1-AF647 for fluorescence microscopy analysis. ( B ). After 1-d incubation of CTL, TopBP1 and/or ETAA1 siRNA in PANC1 cells, YFP or YFP-APE1 overexpression plasmid was added and incubated for another 3 d. The cells were extracted and examined by immunoblotting analysis as indicated. ( C ) Schematic diagrams of functional domains of hAPE1, hTopBP1 and hETAA1 as well as sequence alignment of the N-terminal domain and putative ATR activation domain (AAD) within APE1. hAPE1 (human APE1, NCBI#: P27695.1), mAPE1 (mouse APE1, NCBI#: NP_033817.1), xAPE1 ( Xenopus laevis APE1, NCBI#: AAH72056.1), hTopBP1 (human TopBP1, NCBI#: Q92547.3), and hETAA1 (human ETAA1, NCBI#: NP_061875.2). * identical;: highly conserved;. low conservation. In the AAD alignment, highlighted green indicates aa with hydrophobic side chains, highlighted yellow indicates aa with polar uncharged side chains, highlighted pink indicates aa with positive charged sides chains, highlighted turquoise indicates aa with negative charged sides chains, and highlighted gray indicates the conserved WxxP/N peptide. ( D ) The K6, K7 and W119 residues within APE1 are important for Chk1 phosphorylation induced by YFP-APE1 in PANC1 cells. After YFP, WT or mutant (i.e. K6R/K7R, K24R/K25R, K31R/K32R, W119R) YFP-APE1 overexpression plasmid was added to PANC1 cells and incubation for 3 d, total cell lysates were extracted and examined via immunoblotting analysis as indicated. ( E ) APE1 forms biomolecular condensates in nucleoli dependent on its W119 residue. After WT or mutant (i.e. K6R/K7R, K24R/K25R, K31R/K32R, W119R) YFP-APE1 overexpression plasmid was added to PANC1 cells and incubation for 3 d, the cells were fixed and incubated with anti-NPM1-AF647 for overnight at 4°C, followed by fluorescence microscopy analysis. ( F ) WT but not W119R YFP-APE1 directly activates ATR to phosphorylate Chk1 phosphorylaton at S345 in vitro . In in vitro kinase assays, equal amount of His-YFP, WT or W119R His-APE1-YFP was added to the kinase assays (purified Flag-ATR as kinase and purified His-Chk1 as substrate). Beads bound with lysates from HEK293 cells without Flag-ATR transfection were used as a negative control (‘Control’). ‘No addition’ indicate the kinase assays without the addition of His-YFP or WT/W119R His-APE1-YFP. The samples were examined via immunoblotting analysis as indicated. (A, E) All scale bars = 10 μm.

Techniques: Residue, Activation Assay, Incubation, Over Expression, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, Functional Assay, Sequencing, Phospho-proteomics, Mutagenesis, In Vitro, Purification, Transfection, Negative Control, Control

APE1-induced ATR–Chk1 nDDR leads to rRNA transcription suppression and cell viability reduction. ( A ) APE1 overexpression led to pre-rRNA transcription suppression in an ATR-dependent fashion. YFP or YFP-APE1 overexpression plasmid was added to PANC1 cells and incubated for 2 days. DMSO or VE-822 (1 μM) was then added and incubated for another day. The total RNA was extracted and analyzed via qRT-PCR, and pre-rRNA synthesis was normalized to beta-Actin. n = 3. ( B ) WT but not K6R/K7R nor W119R YFP-APE1 suppressed rRNA transcription. YFP or WT or mutant YFP-APE1 overexpression plasmid was added to PANC1 cells and incubated for 3 days, followed by total RNA extraction and qRT-PCR analysis. n = 3. ( C ) Overexpression of YFP-APE1 but not YFP led to p53 Ser15 phosphorylation, which was sensitive to VE-822. After YFP or YFP-APE1 overexpression plasmid was added to PANC1 cells and incubated for 3 days, DMSO or VE-822 (1 μM) was added and incubated for another 2 h, followed by total cell lysate extraction and immunoblotting analysis as indicated. ( D ) YFP-APE1 overexpression led to cell viability reduction. After YFP or YFP-APE1 overexpression plasmid was added to PANC1 cells and incubated for 2 days, DMSO or VE-822 (1 μM) was added and incubated for another day. The cells were analyzed via MTT assays. n = 6. ( E ) Overexpression of YFP-APE1 led to cell cycle arrests at S and G2/M phases in PANC1 cells. YFP or WT YFP-APE1 overexpression plasmid was added to PANC1 cells. After incubation for 4 days, the cells were collected and examined via FACS analysis. The data are presented as means ± SD, n = 3. ( F–H ) Overexpression APE1 led to more endogenous DNA damage in PANC1 cells. YFP or WT YFP-APE1 overexpression plasmid was added to PANC1 cells. After incubation for 4 days, the cells were collected and analyzed via comet assays under alkaline or neutral conditions. Scale bar = 100 μm. The data are presented as Scatter dot plot and the lines indicate means, n > 50.

Journal: Nucleic Acids Research

Article Title: APE1 assembles biomolecular condensates to promote the ATR–Chk1 DNA damage response in nucleolus

doi: 10.1093/nar/gkac853

Figure Lengend Snippet: APE1-induced ATR–Chk1 nDDR leads to rRNA transcription suppression and cell viability reduction. ( A ) APE1 overexpression led to pre-rRNA transcription suppression in an ATR-dependent fashion. YFP or YFP-APE1 overexpression plasmid was added to PANC1 cells and incubated for 2 days. DMSO or VE-822 (1 μM) was then added and incubated for another day. The total RNA was extracted and analyzed via qRT-PCR, and pre-rRNA synthesis was normalized to beta-Actin. n = 3. ( B ) WT but not K6R/K7R nor W119R YFP-APE1 suppressed rRNA transcription. YFP or WT or mutant YFP-APE1 overexpression plasmid was added to PANC1 cells and incubated for 3 days, followed by total RNA extraction and qRT-PCR analysis. n = 3. ( C ) Overexpression of YFP-APE1 but not YFP led to p53 Ser15 phosphorylation, which was sensitive to VE-822. After YFP or YFP-APE1 overexpression plasmid was added to PANC1 cells and incubated for 3 days, DMSO or VE-822 (1 μM) was added and incubated for another 2 h, followed by total cell lysate extraction and immunoblotting analysis as indicated. ( D ) YFP-APE1 overexpression led to cell viability reduction. After YFP or YFP-APE1 overexpression plasmid was added to PANC1 cells and incubated for 2 days, DMSO or VE-822 (1 μM) was added and incubated for another day. The cells were analyzed via MTT assays. n = 6. ( E ) Overexpression of YFP-APE1 led to cell cycle arrests at S and G2/M phases in PANC1 cells. YFP or WT YFP-APE1 overexpression plasmid was added to PANC1 cells. After incubation for 4 days, the cells were collected and examined via FACS analysis. The data are presented as means ± SD, n = 3. ( F–H ) Overexpression APE1 led to more endogenous DNA damage in PANC1 cells. YFP or WT YFP-APE1 overexpression plasmid was added to PANC1 cells. After incubation for 4 days, the cells were collected and analyzed via comet assays under alkaline or neutral conditions. Scale bar = 100 μm. The data are presented as Scatter dot plot and the lines indicate means, n > 50.

Techniques: Over Expression, Plasmid Preparation, Incubation, Quantitative RT-PCR, Mutagenesis, RNA Extraction, Phospho-proteomics, Extraction, Western Blot

A working model of distinct mechanisms of APE1 in the ATR DNA damage response. ( A ) Stress conditions (canonical function): APE1 promotes the ATR–Chk1 DDR pathway activation via its AP endonuclease and exonuclease activities under stress conditions, which is considered as canonical function of APE1. ( B ) Unperturbed conditions (non-canonical function): under unperturbed conditions, APE1 overexpression leads to biomolecular condensates (liquid–liquid phase separation) in nucleoli in vivo and in nuclear extracts in vitro via its N33 motif and/or W119 residue in AAD domain. ATR/ATRIP, TopBP1, and ETAA1 are recruited to the APE1-induced condensates for ATR activation. APE1-induced nucleolar ATR DDR suppresses rRNA transcription, arrests cell cycle, elevates DNA damage, and reduces cell viability. See text for more details.

Journal: Nucleic Acids Research

Article Title: APE1 assembles biomolecular condensates to promote the ATR–Chk1 DNA damage response in nucleolus

doi: 10.1093/nar/gkac853

Figure Lengend Snippet: A working model of distinct mechanisms of APE1 in the ATR DNA damage response. ( A ) Stress conditions (canonical function): APE1 promotes the ATR–Chk1 DDR pathway activation via its AP endonuclease and exonuclease activities under stress conditions, which is considered as canonical function of APE1. ( B ) Unperturbed conditions (non-canonical function): under unperturbed conditions, APE1 overexpression leads to biomolecular condensates (liquid–liquid phase separation) in nucleoli in vivo and in nuclear extracts in vitro via its N33 motif and/or W119 residue in AAD domain. ATR/ATRIP, TopBP1, and ETAA1 are recruited to the APE1-induced condensates for ATR activation. APE1-induced nucleolar ATR DDR suppresses rRNA transcription, arrests cell cycle, elevates DNA damage, and reduces cell viability. See text for more details.

Techniques: Activation Assay, Over Expression, In Vivo, In Vitro, Residue

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