Fig. 4

Telomerase depletion accelerates age-associated increased apoptosis, decreased autophagy and decreased immune activation of zebrafish gut mpeg + cells. The mpeg1.1: mcherry caax transgenic zebrafish line was used to assess A numbers (A1) and percentage of proliferation (A2) of gut mpeg⁺ cells, using double immunofluorescence staining against RFP to detect mpeg1.1: mcherry caax (red) and anti-PCNA (white) to detect proliferating cells. Yellow arrows in A) are pointing to examples of mpeg⁺ cells (red) that are also PCNA⁺ (white). Using the same fish (mpeg1.1: mcherry caax), we further analysed: B DNA damage response (DDR), using double immunofluorescence against RFP to detect mpeg1.1: mcherry caax (red) and gH2AX (white) to detect DDR. Yellow arrows in B) are pointing to examples of mpeg⁺ cells (red) that are also gH2AX ⁺ (white). B1 Quantification of the % of mpeg⁺ gH2AX⁺ cells; C Apoptosis, using double immunofluorescence against RFP to detect mpeg1.1: mcherry caax (red) and anti-cleaved caspase 3 (white) to detect apoptotic cells. Yellow arrows in C) are pointing to examples of mpeg⁺ cells (red) that are also cleaved-caspase 3⁺ (white). C1 Quantification of the % of mpeg⁺ cleaved-caspase 3⁺ cells; D Autophagy, using double immunofluorescence against RFP to detect mpeg1.1: mcherry caax (red) and LC3B (white) to detect autophagosomes. Yellow arrows in D) are pointing to examples of mpeg⁺ cells (red) that are also LC3B puncta⁺ (white). D1 Quantification of the % of mpeg⁺ LC3B ⁺ cells. tnfa: eGFP-F/mpeg1.1: mcherry caax fish were used to assess immune activation, E using double immunofluorescence against RFP to detect mpeg1.1:mcherry (red) and anti-GFP (white) to detect tnfa expressing cells. Yellow arrows in E) are pointing to examples of mpeg⁺ cells (red) that are also tnfa⁺ (white). E1 Quantification of the % of mpeg⁺ tnfa ⁺ cells; F using double immunofluorescence against RFP to detect mpeg1.1: mcherry caax (red) and anti-IL-6 (white) to detect IL-6 expressing cells. Yellow arrows in F) are pointing to examples of mpeg⁺ cells (red) that are also IL-6⁺ (white). F1 Quantification of the % of mpeg⁺ IL-6.⁺ cells. Nuclei are counterstained with DAPI (blue) and all scale bars represent 50 µm unless otherwise stated in the figure