Journal: Nucleic Acids Research

Article Title: TIRR regulates mRNA export and association with P-bodies in response to DNA damage

doi: 10.1093/nar/gkae688

Figure Lengend Snippet: TIRR associates with and regulates P-body formation in response to DNA damage. ( A ) Left: Representative confocal images showing IF of TIRR in cells with or without ETO treatment. TIRR expression is visualized in the left panels, P-bodies are visualized with anti-LSM14A antibody in the middle panels, and a merge is provided in the right panels. White arrows point to P-bodies. Right: Quantification of TIRR and LSM14A co-localization signal in cytoplasm using Pearson’s coefficient ( n > 50 cells); significance was determined using a t -test (* P ≤ 0.05). ( B ) Representative confocal images showing live cells expressing PA-TIRR-GFP (cytoplasmic and nuclear) and LSM14A-mScarlet (foci in cytoplasm) as a marker for P-bodies. Photoactivation was induced using a 413 nm laser, and the location of activation is indicated with a white arrow. Photoactivation was followed by time-lapse microscopy; time points at 60 and 108 s are shown. See also . ( C ) Top: PLA using anti-TIRR and anti-LSM14A antibodies in non-damage (−ETO) or damage (+ETO) conditions in HeLa cells. DAPI was used to label nuclei. PLA signals are visible as foci. Single antibodies were used as negative controls. Bottom: Quantification of top. Error bar = mean ± standard deviation; significance was determined using a non-parametric Mann–Whitney test (** P ≤ 0.01). ( D ) Top: IF analysis showing LSM14A staining in non-damage (−ETO) and damage (+ETO) conditions in cells expressing either shGFP (control) or shRNA targeting TIRR (shTIRR). DAPI was used to stain nuclei. Bottom: Quantification of TIRR signal in cytoplasm ( n > 50 cells); significance was determined using a t -test (** P ≤ 0.01, * P ≤ 0.05). ( E ) Top: IF analysis showing LSM14A staining in non-damage (−ETO) and damage (+ETO) conditions and with or without LMB treatment. DAPI was used to stain nuclei. Bottom: Quantification of TIRR signal in cytoplasm ( n > 50 cells); significance was determined using a t -test (*** P ≤ 0.001, ** P ≤ 0.01). ( F ) Left: IF of LSM14A in TIRR KD with siTIRR and rescue with GFP, TIRR-GFP WT or TIRR-GFP RBM. Right: Quantification of LSM14A foci ( n > 10 cells); significance was determined using a t -test (*** P ≤ 0.001, ** P ≤ 0.01).

Article Snippet: Primary antibodies used were as follows: GFP Monoclonal antibody (3H9, Chromotek) 1:8000–10 000, Rb pAb to beta-tubulin (Abcam) 1:2000–4000, GAPDH Monoclonal antibody (Proteintech) 1:2000–4000, Rabbit anti-53BP1 pAb (Novus Biologicals) 1:500, Anti-phospho-Histone H2A.X (Ser139) Antibody clone JBW301 (Millipore) 1:1000, Anti-NUDT16L1 (Atlas Antibodies) 1:500, Anti-LSM14A Purified MaxPab antibody (Novus Biologicals) 1:250, CRM1 C-1 antibody (Santa Cruz) 1:250, mCherry antibody 1:1000 (GeneTex), Rb pAb to LaminB1 (Abcam) 1:1000, Anti-FLAG antibody produced in Rabbit (Sigma) 1:1000 and Histone-H3 Polyclonal Antibody (Proteintech) 1:8000–1:10 000.

Techniques: Expressing, Marker, Activation Assay, Time-lapse Microscopy, Standard Deviation, MANN-WHITNEY, Staining, Control, shRNA

Journal: Nucleic Acids Research

Article Title: TIRR regulates mRNA export and association with P-bodies in response to DNA damage

doi: 10.1093/nar/gkae688

Figure Lengend Snippet: TIRR depletion leads to accumulation of mRNA in nucleus. ( A ) Representative confocal images showing co-localization of RNA FISH signals (left panel, merged with PB foci on right) for ZNF600 with LSM14A (PB foci, middle panel) in shGFP or shTIRR cells after ETO treatment ( n > 50 cells). White rectangles mark zoomed-in images on the right. White arrows point to P-bodies with FISH signal. ( B ) As in panel (A), for PELI2 mRNA. ( C ) Quantification of FISH and LSM14A co-localization signal in ZNF600 , PELI2 , SPEN and Tubulin by calling of PB foci with FISH signal overlap ( n > 50 cells); significance was determined using a t -test (* P ≤ 0.05, ** P ≤ 0.01).

Article Snippet: Primary antibodies used were as follows: GFP Monoclonal antibody (3H9, Chromotek) 1:8000–10 000, Rb pAb to beta-tubulin (Abcam) 1:2000–4000, GAPDH Monoclonal antibody (Proteintech) 1:2000–4000, Rabbit anti-53BP1 pAb (Novus Biologicals) 1:500, Anti-phospho-Histone H2A.X (Ser139) Antibody clone JBW301 (Millipore) 1:1000, Anti-NUDT16L1 (Atlas Antibodies) 1:500, Anti-LSM14A Purified MaxPab antibody (Novus Biologicals) 1:250, CRM1 C-1 antibody (Santa Cruz) 1:250, mCherry antibody 1:1000 (GeneTex), Rb pAb to LaminB1 (Abcam) 1:1000, Anti-FLAG antibody produced in Rabbit (Sigma) 1:1000 and Histone-H3 Polyclonal Antibody (Proteintech) 1:8000–1:10 000.

Techniques: