Journal: Aging Cell

Article Title: Non‐enzymatic function of WRN RECQL helicase regulates removal of topoisomerase‐I‐DNA covalent complexes and triggers NF‐κB signaling in cancer

doi: 10.1111/acel.13625

Figure Lengend Snippet: WRN‐mediated NF‐κB activation requires CHK1 and PARP1. (a) U2‐OS Cells were treated with CPT (50 nM) for indicated time periods, and activation of DDR proteins was assessed by Western blotting. (b) NEMO‐knockdown (NEMO‐KD) U2‐OS cells were generated by lentivirus mediated shRNA expression system. Expression of NEMO protein in NEMO‐WT (control shRNA) and NEMO‐KD cells was assessed by Western blotting. (c) NEMO‐WT and NEMO‐KD cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB activation was assessed in terms of IκBα degradation by Western blotting. (d) ATM‐knockdown (ATM‐KD) U2‐OS cells were generated by lentivirus mediated shRNA expression system. Expression of ATM protein in ATM‐WT (control shRNA) and ATM‐KD cells was assessed by Western blotting. (e) ATM‐WT and ATM‐KD cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB activation was assessed in term of IκBα degradation by Western blotting. (f) WRN‐WT cells expressing NF‐κB driven luciferase reporter were treated with CPT (50 nM) in the absence or presence of CHK1i or PARP1i for indicated time periods and NF‐κB activation was assessed in terms of fold increase in luciferase activity, which is normalized by renilla expression. All the values indicated are mean ± SD ( n = 3 for a, c, e) or mean ± SEM ( n = 4 for f). ** p < 0.01 with respect to vehicle treatment at respective time points

Article Snippet: Anti‐phospho‐345‐CHK1 was from Epitomics (Cambridge, UK).

Techniques: Activation Assay, Western Blot, Generated, shRNA, Expressing, Luciferase, Activity Assay

Journal: Aging Cell

Article Title: Non‐enzymatic function of WRN RECQL helicase regulates removal of topoisomerase‐I‐DNA covalent complexes and triggers NF‐κB signaling in cancer

doi: 10.1111/acel.13625

Figure Lengend Snippet: Non‐enzymatic role of WRN in ssDNA generation and activation of CHK1 and NF‐κB. (a) WRN‐KO cells were transfected for ectopic expression of EV (empty vector), WRN WT , WRN E84A , WRN K577M , and WRN E84A‐K577M . Cells were treated with CPT (50 nM) for indicated time periods, and phosphorylation of RPA2 and CHK1 was assessed by Western blotting. (b) WRN‐KO cells were transfected for ectopic expression of EV (empty vector), WRN WT , WRN E84A , WRN K577M , and WRN E84A‐K577M . Cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB promoter driven luciferase expression was assessed. All the values indicated are mean ± SEM ( n = 4). * p < 0.05 with respect to respective cell types at 0 h

Article Snippet: Anti‐phospho‐345‐CHK1 was from Epitomics (Cambridge, UK).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Luciferase

Journal: Aging Cell

Article Title: Non‐enzymatic function of WRN RECQL helicase regulates removal of topoisomerase‐I‐DNA covalent complexes and triggers NF‐κB signaling in cancer

doi: 10.1111/acel.13625

Figure Lengend Snippet: Role of WRN in ssDNA generation and nuclear translocation of p65 and its therapeutic implications. (a) Schematic representation for WRN‐mediated removal of TOP1cc, generation of ssDNA and CHK1 phosphorylation and NF‐κB activation. (b, c) WRN‐WT and WRN‐KO cells were treated with BrdU for 36 h for uniform labeling of BrdU on both the DNA strands. Further cells were exposed to CPT for indicated time periods for TOP1cc removal mediated ssDNA generation and nuclear translocation of NF‐κB (p65). BrdU exposed in ssDNA (red) and p65 (green) was assessed in non‐denaturing native condition by immunofluorescence microscopy (Bar: 5 μm). % Nuclei with more than threshold fluorescence were considered positive for p65 and/or BrdU and plotted in C. All the values indicated are mean ± SEM ( n = 3). * p < 0.05 with respect to respective untreated cell types. (d,e) Mice bearing WRN‐WT and WRN‐KO melanoma tumors were treated with vehicle or CPT (1 mg/kg, once on 1st, 3rd, and 5th day per week for 4 weeks). Tumor volume was measured once every alternate day. After 30 days, mice were sacrificed and tumors were removed and analyzed. Data represent the mean ± SD , n = 6 per group. * p < 0.01 w.r.t corresponding vehicle‐treated tumor. (f) Survival of patients with WRN high and low expression in cancer using data available at cBioportal. The EXP < −1 denotes mRNA expression is <1 standard deviations ( SD ) below the mean, and EXP > 1 denotes mRNA expression is >1 SD above the mean. (g) Schematic model for WRN‐mediated TOP1cc removal and NF‐κB activation. In response to TOP1cc, WRN plays key role in removal of TOP1cc, leading to generation of RPA coated ssDNA and activation of CHK1 and PARP1. Subsequently, CHK1 and PARP1 may facilitate NEMO translocation to cytoplasm and activation of NF‐κB. [Correction added on 01 June 2022, after first online publication: Figure labels, 6(e) and 6(f) are misplaced and have been corrected in this version].

Article Snippet: Anti‐phospho‐345‐CHK1 was from Epitomics (Cambridge, UK).

Techniques: Translocation Assay, Activation Assay, Labeling, Immunofluorescence, Microscopy, Fluorescence, Expressing