Journal: iScience

Article Title: SART1 modulates poly-(ADP-ribose) chain accumulation and PARP1 chromatin localization.

doi: 10.1016/j.isci.2024.111252

Figure Lengend Snippet: Figure 2. SART1 interaction with PARP1 and cellular localization are modified by DNA damage (A) SART1-PARP1 interaction is induced by DNA damage as measured by immunoprecipitation in the presence or absence of DNA damage (6 Gy IR). (B) Immunofluorescence analysis of HeLa cells untreated or treated with 4 Gy of IR. Cells were fixed on coverslips 4 h after treatment and blotted with anti-SART1 antibody, DAPI staining was performed to visualize the nuclei of cells. DAPI and SART1 signal were merged to discriminate cytoplasmic and nuclear localization of SART1. One hundred cells were counted and assessed for nuclear or cytoplasmic SART1 signal. Results of the counts are shown as histograms on the right part of the figure. Scale bar, 1 mm. Two independent experiments are shown (Repeat 1 and 2).

Article Snippet: UWB1.289 and UWB1.289+BRCA1 cell lines were used for clonogenic assays to assess sensitivity to DNA damage by ionizing irradiation and to three PARP inhibitors (olaparib (TMO-T3015, TargetMOL), rucaparib (TMO-T4463, TargetMOL) and talazoparib (TMO-T6253, TargetMOL) in SART1-proficient or -deficient (silenced) cells.

Techniques: Immunoprecipitation, Staining

Journal: iScience

Article Title: SART1 modulates poly-(ADP-ribose) chain accumulation and PARP1 chromatin localization.

doi: 10.1016/j.isci.2024.111252

Figure Lengend Snippet: Figure 4. SART1 fragment cellular localization, influence on PARP1 chromatin binding and PARP1 interaction (A) SART1 sequence analysis and fragment design. Left panel, multiple sequence analysis (ClustalW). Residues in yellow are conserved and red boxes indicate RG/RGG-rich motifs. Central panel, schema of the full length SART1 (WT), the three fragments created, the location of RG/RGG-rich motifs, and the cluster PARylated serine residues (F3). Right panel, western blot showing expression of each fragment in 293T cells. (B) SART1 fragments’ cellular localization. UWB cells were transfected with vectors expressing SART1 F1, F2, F3 or empty plasmid. Tubulin, lamin-B1, and histone H2AX have been used as cytoplasm, nuclear, and chromatin references, respectively. (C) Chromatin fraction in UWB cells after expression of SART1 fragments and then untreated or treated with 4 Gy of IR. UWB cells were transfected with vector expressing SART1 F1, F2, F3 or empty plasmid, the day after cells were mock-treated or irradiated with 4 Gy of IR and collected after 4 h. Change in PARP1 chromatin level between treated and untreated sample was measured with ImageJ and normalized on chromatin loading control. (D) SART1 fragments-PARP1 interaction in presence or absence of DNA damage. 293T cells were transfected with plasmids expressing SART1 fragments or empty vector and cells collected untreated or 4 h after 8 Gy irradiation.

Article Snippet: UWB1.289 and UWB1.289+BRCA1 cell lines were used for clonogenic assays to assess sensitivity to DNA damage by ionizing irradiation and to three PARP inhibitors (olaparib (TMO-T3015, TargetMOL), rucaparib (TMO-T4463, TargetMOL) and talazoparib (TMO-T6253, TargetMOL) in SART1-proficient or -deficient (silenced) cells.

Techniques: Binding Assay, Sequencing, Western Blot, Expressing, Transfection, Plasmid Preparation, Irradiation, Control

Journal: iScience

Article Title: SART1 modulates poly-(ADP-ribose) chain accumulation and PARP1 chromatin localization.

doi: 10.1016/j.isci.2024.111252

Figure Lengend Snippet: Figure 5. SART1 fragments effect on chromatin localization and PARylation activity (A) Experimental design to analyze SART1 fragments effect on PARylation. UWB cells silenced for SART1 with a shRNA targeting the 30UTR of the sequence were seeded on day 1. The next day cells were transfected with a plasmid expressing either a SART1 fragment, a silencing-resistant full-length protein, or with an empty plasmid. On day 3, cells were reseeded. On day 4, they were mock-treated or irradiated with 4 Gy IR and collected at 4 h and 8 h after IR. (B) PAR chains in samples transfected with empty plasmid, full-length SART1, or SART1 fragment in presence or absence of DNA damage. PAR levels were evaluated using an anti-PAR antibody and their intensity was measured using ImageJ software. Data are shown as mean G SE of three independent experiments and analyzed with Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. If not noted, the difference was not significant. (C) Mutagenesis of first RGG sequence in F1. (D and E) PAR chains in samples transfected with SART1 F1, mutated F1 (F1-ALA) and empty plasmid in presence or absence of DNA damage. UWB cells silenced for SART1 and transfected with SART1 F1, mutated F1 (F1-ALA) and empty plasmid were irradiated with 4 Gy of IR and collected at 4 h and 8 h after IR. PAR levels were evaluated using an anti-PAR antibody. Fragment expression has been evaluated with anti-FLAG antibody and anti-tubulin as loading control. An anti- gH2AX antibody has been used to visualize DNA damage and an anti-H2AX antibody has been used as loading control. Whole cell (D) and chromatin (E) extracts.

Article Snippet: UWB1.289 and UWB1.289+BRCA1 cell lines were used for clonogenic assays to assess sensitivity to DNA damage by ionizing irradiation and to three PARP inhibitors (olaparib (TMO-T3015, TargetMOL), rucaparib (TMO-T4463, TargetMOL) and talazoparib (TMO-T6253, TargetMOL) in SART1-proficient or -deficient (silenced) cells.

Techniques: Activity Assay, shRNA, Sequencing, Transfection, Plasmid Preparation, Expressing, Irradiation, Software, Mutagenesis, Control