pfm39  (MedChemExpress)

Journal: Cell reports

Article Title: Break-induced replication is activated to repair R-loop-associated double-strand breaks in SETX-deficient cells

doi: 10.1016/j.celrep.2025.116386

Figure Lengend Snippet: (A, B, and E) U2OS WT and SETX -KO EGFP-BIR reporter cells were infected with lentiviruses expressing MRE11 shRNA, CtIP shRNA, or vector. Three days were given to allow shRNA expression to achieve effective knockdown of each gene. For the end-resection assay (A), shRNA-treated cells were infected with I-SceI-expressing lentiviruses to induce DSBs. Two days after I-SceI infection, cells were harvested for the end-resection assay. To examine the pRPA2 level (B), shRNA-treated cells were treated with 10 Gy IR. Cell lysates were collected 1 h after IR, and Western blotting was performed using the indicated antibodies. For BIR efficiency analysis (E), shRNA-treated cells were infected with I-SceI-expressing lentiviruses to induce DSBs. The percentage of EGFP-positive cells was quantified by FACS analysis 5 days after I-SceI infection. Data are shown as mean ± SD of n = 3 biological replicates. (C) U2OS WT and SETX -KO EGFP-BIR reporter cells were infected with lentiviruses expressing XPF shRNA or vector. Three days later, cells were infected with I-SceI-expressing lentiviruses to induce DSBs. Inhibitor PFM01 (100 μM) or DMSO was applied to cells together with I-SceI lentiviruses. Two days after I-SceI infection, cells were harvested for the end-resection assay. Data are shown as mean ± SD of n = 3 biological replicates. (D) U2OS WT and SETX -KO EGFP-BIR reporter cells were infected with I-SceI-expressing lentiviruses to induce DSBs, and simultaneously treated with DMSO or inhibitor PFM39 (100 μM). Two days after I-SceI induction, cells were harvested for end resection assay, and the data are shown as mean ± SD of n = 3 biological replicates. (F and G) U2OS WT and SETX -KO EGFP-BIR reporter cells were infected with lentiviruses encoding BLM shRNA, DNA2 shRNA, EXO1 shRNA, or vector. Three days later, cells were then infected with I-SceI-expressing lentiviruses to induce DSBs for end resection assay (F), or treated with 10 Gy IR to examine the pRPA2 level (G). Data are shown as mean ± SD of n = 3 biological replicates. For the end resection assay shown in (A), (C), (D), and (F), ssDNA% was normalized to the I-SceI cleavage efficiency as indicated by γH2AX ChIP values.

Article Snippet: PFM39 , TargetMol Chemicals , T38709.

Techniques: Infection, Expressing, shRNA, Plasmid Preparation, Knockdown, Resection Assay, Western Blot

Journal: Genes & Development

Article Title: MRN–CtIP, EXO1, and DNA2–WRN/BLM act bidirectionally to process DNA gaps in PARPi-treated cells without strand cleavage

doi: 10.1101/gad.352421.124

Figure Lengend Snippet: MRE11 inhibition rescues ssDNA gap repair without affecting ssDNA gap formation (see also ). ( A , top ) Schematic of the DNA fiber spreading assay with the S1 nuclease in the presence and absence of Mirin. ( Bottom ) Representative images of DNA fibers in SUM149PT treated with S1 ± 10 μM olaparib for 1 h and ±50 μM Mirin. Scale bar, 10 μm. ( B ) Dot plot and median of IdU tract lengths in SUM149PT and SUM149PT + BRCA1 cells treated with S1 ± 10 μM olaparib and ±50 μM Mirin for 1 h. The S1 nuclease was added immediately after (time 0) and 30 min (time 30) after olaparib removal ( n = 3). At least 180 tracts were scored for each sample. Statistics: Kruskal–Wallis followed by Dunn's multiple comparisons test. (ns) Nonsignificant, (*) P < 0.0332, (**) P < 0.0021, (***) P < 0.0002, (****) P < 0.0001. ( C , left ) Schematic of electron microscopy experiment in the presence and absence of Mirin and REV1i (JH-RE-06). ( Right ) Percentage of replication forks with daughter strand gaps in SUM149PT + BRCA1 and SUM149PT cells treated with 10 μM olaparib ± 50 μM Mirin and ±2 μM REV1i (JH-RE-06) for 1 h. Cells were collected immediately after PARPi removal (T0). The first and fifth columns are repeated data from D used for easier comparison between samples in different figures. “# RI” indicates the number of analyzed replication intermediates. ( n = 3). Columns indicate mean ± SD. Mean values are shown above each data set. Statistics: unpaired t -test. (ns) Nonsignificant, (*) P < 0.0332, (**) P < 0.0021, (***) P < 0.0002. ( D ) Length of daughter strand ssDNA gaps in nucleotides in SUM149PT and SUM149PT + BRCA1 cells treated as in C . The first and fifth columns are repeated data from D used for easier comparison between samples in different figures. “# RI” indicates the number of analyzed replication intermediates. ( n = 3). Statistics: unpaired t -test with Welch correction. (ns) Nonsignificant, (****) P < 0.0001. Horizontal bars indicate median. Median values are shown above each data set. ( E , top ) Schematic of the DNA fiber assay performed by using the spreading technique with the S1 nuclease in the presence and absence of PRIMPOL. ( Bottom ) Dot plot and median of IdU tract lengths in SUM149PT and SUM149PT + BRCA1 cells treated with S1 ± 10 μM olaparib and ±siPRIMPOL. The S1 nuclease was added immediately after olaparib removal (time 0) ( n = 3). ( F , top ) Schematic of the DNA fiber assay performed by using the combing technique with the S1 nuclease in the presence and absence of PRIMPOL. ( Bottom ) Dot plot and median of IdU tract lengths in SUM149PT and SUM149PT + BRCA1 cells treated with S1 ± 10 μM olaparib and ±siPRIMPOL. The S1 nuclease was added immediately after olaparib removal (time 0) ( n = 3). At least 130 tracts were scored for each sample in E and F . Statistics in E and F : Kruskal–Wallis followed by Dunn's multiple comparisons test. (ns) Nonsignificant, (**) P < 0.0021, (****) P < 0.0001.

Article Snippet: For the experiments with the MRE11, REV1, and DNA2 inhibitors, 10 μM olaparib was added during the 1 h of IdU treatment concomitantly with 50 μM MRE11 inhibitor Mirin (Millipore Sigma M9948), 2 μM REV1 inhibitor (AOBIOUS Laboratories AOB13138) , 100 μM MRE11 exonuclease activity inhibitor PFM39 (Sigma-Aldrich SML1839) , 25 or 75 μM MRE11 endonuclease activity inhibitor PFM03 (MedChemExpress HY-148078) , or 30 μM DNA2 inhibitor C5 (AOBIOUS Laboratories AOB9082).

Techniques: Inhibition, Electron Microscopy, Comparison

Journal: Genes & Development

Article Title: MRN–CtIP, EXO1, and DNA2–WRN/BLM act bidirectionally to process DNA gaps in PARPi-treated cells without strand cleavage

doi: 10.1101/gad.352421.124

Figure Lengend Snippet: The MRE11 complex extends DNA gaps through CtIP-stimulated exonuclease activity (see also ). ( A ) Schematic of the plasmid-based DNA substrate with a 10 nt long DNA gap, indicating various degradation scenarios. See the text for details. ( B ) Nuclease assays with a 10 nt long gapped DNA substrate with MRX and increasing concentrations of phosphorylated Sae2 (pSae2). (Lane 1 ) The linearized substrate is included for reference. Shown is a representative gel from three independent experiments. ( C ) Nuclease assays with a 68 nt long gapped DNA substrate with MRN, phosphorylated CtIP (pCtIP), and RPA. (Lane 8 ) The linearized substrate is included for reference. Shown is a representative gel from two independent experiments. ( D ) Annealing resection assays with a 10 nt long gapped DNA substrate and MRX and pSae2, as indicated. ( Top ) Zoomed-in view of the ssDNA gap, indicating the positions of the probes used to detect DNA resection. ( Middle ) Quantitation of resection efficiency measured with the 3′-specific probe (red; left ) or the 5′-specific probe (blue; right ). Averages are shown; n ≧ 3; error bars indicate SEM. ( Bottom ) Representative gels from at least three independent experiments. ( E ) Annealing resection assays with a linearized DNA substrate (otherwise identical to D ) and MRX and pSae2, as indicated. ( Top ) Zoomed-in view of the DSB, indicating the positions of the probes used to detect DNA resection. ( Middle ) Quantitation of resection efficiency measured with the 3′-specific probe (red; left ) or the 5′-specific probe (blue; right ). Averages are shown; n ≧ 3; error bars indicate SEM. ( Bottom ) Representative gels from at least three independent experiments. ( F ) Annealing resection assays of the gapped plasmid-based DNA substrate with the T7 exonuclease. Shown is a representative gel from three independent experiments showing that both probes exhibit a similar annealing efficacy. ( G ) Exonuclease assays with the indicated oligonucleotide-based DNA substrates and increasing concentrations of MRN, as indicated. ( Top ) Cartoons of the various DNA substrates. The red asterisk represents the position of the 32 P label. ( Bottom ) Representative gels from three independent experiments. ( H ) Quantitation of experiments shown in G . Averages are shown; n = 3; error bars indicate SEM. ( I ) Exonuclease assays with 1 nt long gapped DNA substrate and MRN, as indicated. ( Top ) A cartoon of the substrate. The red asterisk represents the position of the 32 P label. ( Bottom ) Representative gel from three independent experiments. ( J ) Exonuclease assays as in I but with a substrate containing eight phosphorothioate bonds at the 3′ side of the gap, represented by the orange line in the cartoon. ( Top ) A cartoon of the substrate. The red asterisk represents the position of the 32 P label. ( Bottom ) Representative gel from three independent experiments. ( K ) Exonuclease assays with a 1 nt long gapped DNA substrate, MRN, and increasing concentrations of pCtIP, as indicated. ( Top ) A cartoon of the substrate. The red asterisk represents the position of the 32 P label. ( Middle ) Quantitation of DNA degradation. Averages are shown; n = 3; error bars indicate SEM. ( Bottom ) Representative gel from three independent experiments. ( L ) Exonuclease assays with a 1 nt long gapped DNA substrate, MRE11, and increasing concentrations of pCtIP, as indicated. ( Top ) A cartoon of the substrate. The red asterisk represents the position of the 32 P label. ( Middle ) Quantitation of DNA degradation. Averages are shown; n = 3; error bars indicate SEM. ( Bottom ) Representative gel from three independent experiments. ( M ) Quantitation of exonuclease assays with substrates having gaps of different lengths, with MRN, in the absence or presence of human RPA, as shown in . Averages are shown; n = 3; error bars indicate SEM.

Article Snippet: For the experiments with the MRE11, REV1, and DNA2 inhibitors, 10 μM olaparib was added during the 1 h of IdU treatment concomitantly with 50 μM MRE11 inhibitor Mirin (Millipore Sigma M9948), 2 μM REV1 inhibitor (AOBIOUS Laboratories AOB13138) , 100 μM MRE11 exonuclease activity inhibitor PFM39 (Sigma-Aldrich SML1839) , 25 or 75 μM MRE11 endonuclease activity inhibitor PFM03 (MedChemExpress HY-148078) , or 30 μM DNA2 inhibitor C5 (AOBIOUS Laboratories AOB9082).

Techniques: Activity Assay, Plasmid Preparation, Quantitation Assay

Journal: Genes & Development

Article Title: MRN–CtIP, EXO1, and DNA2–WRN/BLM act bidirectionally to process DNA gaps in PARPi-treated cells without strand cleavage

doi: 10.1101/gad.352421.124

Figure Lengend Snippet: Inhibition of MRE11 3′–5′ exonuclease activity and loss of CtIP rescue ssDNA gap repair in PARPi-treated cells (see also ). ( A , top ) Schematic of the DNA fiber spreading assay with the S1 nuclease in the presence and absence of PFM39. ( Bottom ) Dot plot and median of IdU tract lengths in SUM149PT and SUM149PT + BRCA1 cells treated with S1 ± 10 μM olaparib for 1 h and ±100 μM PFM39. The S1 nuclease was added immediately after (time 0) and 30 min (time 30) after olaparib removal ( n = 3). ( B , top ) Schematic of the DNA fiber spreading assay with the S1 nuclease in the presence and absence of CtIP. ( Bottom ) Dot plot and median of IdU tract lengths in SUM149PT and SUM149PT + BRCA1 cells treated with the S1 nuclease ± 10 μM olaparib for 1 h and ±siCtIP. The S1 nuclease was added immediately after (time 0) and 30 min (time 30) after olaparib removal ( n = 3). At least 170 tracts were scored for each sample in A and B . Statistics in A and B : Kruskal–Wallis followed by Dunn's multiple comparisons test. (ns) Nonsignificant, (*) P < 0.0332, (***) P < 0.0002, (****) P < 0.0001.

Article Snippet: For the experiments with the MRE11, REV1, and DNA2 inhibitors, 10 μM olaparib was added during the 1 h of IdU treatment concomitantly with 50 μM MRE11 inhibitor Mirin (Millipore Sigma M9948), 2 μM REV1 inhibitor (AOBIOUS Laboratories AOB13138) , 100 μM MRE11 exonuclease activity inhibitor PFM39 (Sigma-Aldrich SML1839) , 25 or 75 μM MRE11 endonuclease activity inhibitor PFM03 (MedChemExpress HY-148078) , or 30 μM DNA2 inhibitor C5 (AOBIOUS Laboratories AOB9082).

Techniques: Inhibition, Activity Assay