Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: (A) coIP of endogenous TIM and PARP1 in 293T cells released from G1/S block. (B) Left: PLA of PARP1 with EdU-labeled replication forks in U2OS WT versus PARP1 KO cells. Right: quantification of cells positive for PARP1:EdU PLA foci. Scale bar: 10 μm; **p < 0.01, Student’s t test. (C) Left: The PARP1:TIM PLA in U2OS cells. S phase cells were marked with EdU. Right: percentages of cells positive for PLA foci in EdU positive or negative cells. Scale bar: 10 μm; **p < 0.01, Student’s t test. (D) Schematic of TIM-PAB and the catalytic domain of PARP1. (E) Anti-FLAG coIP of TIM variants with endogenous PARP1 in 293T cells. EV, empty vector. (F) Anti-myc coIP of TIM-myc with FLAG-PARP1 variants. (G) Top: schematic of the Flp-In T-REx system to express siRNA-resistant FLAG-TIM WT or EQ/EQ/TD by doxycycline (dox) mediated derepression of the promoter. Bottom: verification of U2OS Flp-In cell lines reconstituting TIM WT or EQ/EQ/TD in response to increasing doses of dox, following transfection of TIM siRNA. (H) Anti-FLAG coIP of endogenous PARP1 by TIM WT or EQ/EQ/TD from the Flp-In cells. (I) Dot plot of the DNA fiber iodo-deoxyuridine (IdU) track length from Flp-In cells. Red bars indicate the median from at least 150 tracks. ****p < 0.0001, n.s., not significant; two-way ANOVA with post hoc test and false discovery rate (FDR) adjustment. (J) Dot plot of the IdU/chloro-deoxyuridine (CldU) ratios from Flp-In cells treated with 4 mM HU for 4 h. ****p < 0.0001; two-way ANOVA. (B and C) Mean ± SD from 3 biological replicates. (I and J) Representative plot from 3 biological replicates. See also .

Article Snippet: PARP1 , Bethyl Laboratories , Cat#A301-376A; RRID:AB_937941.

Techniques: Blocking Assay, Labeling, Plasmid Preparation, Transfection

Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: (A) Schematic of two conserved PBMs within TIM with the relative positions of binding partners TIPIN and PARP1. (B) Top: schematic of the glutathione S-transferase-tagged recombinant TIM fragment containing amino acids with PBM2 WT or K949A/K950A (KA). Bottom: slot blot loaded with corresponding recombinant PBM2 and incubated with biotinylated PAR chains (pADPr). (C) Top: schematic of synthesized PBM1 26 amino acid-peptides, either WT or with 4 mutations in basic residues. Bottom: slot blot of corresponding peptides incubated with pADPr. (D) In vitro auto-PARylation assay with recombinant PARP1, activating dsDNA, and 50 μM NAD + , as well as 10 μM olaparib, where indicated. (E) Anti-FLAG IP of IVTT FLAG-TIM WT reacted with recombinant PARP1 in the presence of 25 μM of biotinylated NAD + . (F) Anti-FLAG IP of IVTT FLAG-TIM reacted with recombinant PARP1 in the presence of 25 μM of biotinylated NAD + . (G) Quantification of (F). Mean ± SD from three biological replicates. *p < 0.05; ***p < 0.001; ****p < 0.0001; one-way ANOVA. (H) Model for the recognition of auto-PARylated PARP1 by TIM (1) and subsequent PARP1-dependent TIM PARylation (2). (I and J) Anti-FLAG IP of FLAG-TIM in denaturing conditions followed by anti-PAR western blot (WB) from 293T cells treated with 1 μM CPT and 10 μM PARGi (PDD00017273) with or without 10 mM olaparib pretreatment for 1 h. (K) coIP of endogenous PARP1 and PARylated PARP1 by anti-FLAG TIM IP in chromatin-enriched fractions of U2OS cells treated with 1 μM CPT and 10 μM PARGi for 1 h. (L) In vivo PARylation of FLAG-TIM in cells transfected with 2 independent siRNAs against HPF1 and treated with CPT/PARGi. See also .

Article Snippet: PARP1 , Bethyl Laboratories , Cat#A301-376A; RRID:AB_937941.

Techniques: Binding Assay, Recombinant, Dot Blot, Incubation, Synthesized, In Vitro, Western Blot, In Vivo, Transfection

Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: (A) Left: TIM-myc and FLAG-PARP1 were transiently expressed in U2OS or PARP1 KO cells and analyzed by WB. Where indicated, 10 mM MG132 was treated for 4 h. (B) Quantification of TIM with each MG132 condition normalized to its corresponding untreated condition. *p < 0.05; **p < 0.01; Student’s t test. (C) WB analysis of FLAG-TIM WT or PBM1/2 levels in U2OS or PARP1 KO cells. (D) Expression of TIM variants in U2OS cells versus IVTT expression. (E) Anti-FLAG IP of FLAG-TIM in denaturing conditions followed by anti-PAR WB in 293T cells treated with 10 μM PARGi with or without 1 μM CPT for 1 h. (F) Ubiquitination assay of TIM-myc WT and PBM1/2 overexpressed in 293T cells with His-Ub and treated with 1 μM CPT and 10 μM MG132. (G) Analysis of pRPA32 S4/S8 in U2OS cells transfected with increasing amounts of FLAG-TIM WT or PBM1/2. (H) Structure depicting the MCM PI of TIM that includes anchor and DBM, and their conservation throughout multiple species. Adapted from PDB: 7PFO and created using Chimera. (I) coIP of FLAG-TIM PBM1/2 or PBM1/2 ∆PI (amino acid D273–281 and amino acid ∆316–322) by endogenous MCM6 in 293T cells. (J) Analysis of pRPA32 S4/S8 in U2OS cells induced by FLAG-TIM WT, PBM1/2, or PBM1/2 ∆PI. (K) The PLA between TIM and PARP1. A total of 10 μM olaparib was treated for 24 h, and 10 μM EdU was added for the last 30 min before fixation. Scale bar: 10 μm ****p < 0.0001; two-way ANOVA. (B and K) Mean ± SD from 3 biological replicates. See also .

Article Snippet: PARP1 , Bethyl Laboratories , Cat#A301-376A; RRID:AB_937941.

Techniques: Expressing, Ubiquitin Proteomics, Transfection

Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PARP1 , Bethyl Laboratories , Cat#A301-376A; RRID:AB_937941.

Techniques: Control, Virus, Recombinant, TNKS1 Histone Ribosylation Assay, Transfection, Protease Inhibitor, In Situ, DNA Extraction, Mutagenesis, Cell Viability Assay, Staining, Negative Control, Software

Journal: Insects

Article Title: Insecticides for Suppression of Nylanderia fulva

doi: 10.3390/insects8030093

Figure Lengend Snippet: Insecticide baits tested for acceptance and efficacy against Nylanderia fulva in laboratory bioassays.

Article Snippet: Advance ® 375A Select , Abamectin , Granular , BASF, Research Triangle Park, NC, USA.

Techniques:

Journal: Insects

Article Title: Insecticides for Suppression of Nylanderia fulva

doi: 10.3390/insects8030093

Figure Lengend Snippet: Nylanderia fulva acceptance of 15 commercially available baits in no-choice laboratory assays over three seasons (2009–2010) (n = 10 replications for each bait per season).

Article Snippet: Advance ® 375A Select , Abamectin , Granular , BASF, Research Triangle Park, NC, USA.

Techniques:

Journal: Insects

Article Title: Insecticides for Suppression of Nylanderia fulva

doi: 10.3390/insects8030093

Figure Lengend Snippet: Mean percent mortality rank-score of Nylanderia fulva at 3 days after continuous exposure to 15 commercially available baits in no-choice laboratory assays over three seasons (2009–2010) (n = 10 replications for each bait per season).

Article Snippet: Advance ® 375A Select , Abamectin , Granular , BASF, Research Triangle Park, NC, USA.

Techniques:

Journal: Insects

Article Title: Insecticides for Suppression of Nylanderia fulva

doi: 10.3390/insects8030093

Figure Lengend Snippet: Mean ranked days until 100% mortality of Nylanderia fulva after continuous exposure to 15 commercially available baits in no-choice laboratory assays over three seasons (2009–2010) (n = 10 replications for each bait per season).

Article Snippet: Advance ® 375A Select , Abamectin , Granular , BASF, Research Triangle Park, NC, USA.

Techniques: