Katushka

Preparation of sections, immunohistochemistry, confocal microscopy, apoptosis TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) assay and image analysis were performed as previously described [29 (link)]. Lungs and spleens were immune-stained with anti-DTA (Cat#4701, ViroStat, Portland, ME, USA), cleaved Cas-3 (Cat# 9664S, Cell Signaling, MA, USA), P-53 (Cat# SC-6243, Santa Cruz Biotechnology, USA), Mad1 (Cat# 4682, Cell Signaling, MA, USA), E2F1 (Cat# MA5-14344, Thermo Fisher Scientific, Waltham, MA, USA), F4/80(Cat# ab6640, Abcam, Cambridge, MA, USA), Smad3 (Cat# 51-1500, Thermo Fisher Scientific, Waltham, MA, USA), Katushka (Cat# AB233, Evrogen, Moscow, Russia) antibodies at 1:200 dilution and with a TUNEL kit (MBL international, Woburn, MA, USA) according to manufacturer's instructions. Donkey anti-Goat IgG (H+L) secondary antibodies conjugated with Cy2 dye (Cat# 705-225-147, Jackson ImmunoResearch, PA, USA) was used for DTA detection. HiDef Detection HRP Polymer System (Cat# 954D-10, Cell Marque, CA, USA) was used for cleaved Cas-3, P53, Mad1, Smad3, E2F1 and Katushka detection. Pollnk-2 Plus HRP Detection Kit for Rat Primary Antibody (Cat# D46-6, GBI Labs, WA, USA) was used for F4/80 detection.

Plasmids expressing fluorescent proteins (Table 1) were obtained from the following commercial sources: tdTomato (Clontech: pCMV-tdTomato), mCherry (Clontech: pLVX-EF1α-IRES-mCherry), Katushka (Evrogen: pTurboFP635-C), tdKatushka2 (Addgene: 30181/ pTEC22) and firefly luciferase (Touchlight Genetics Ltd, Leatherhead, UK: proTLx Lux). Plasmids were maintained in Escherichia coli strain TOP10 and purified using Endo-Free giga kit (Qiagen, Manchester, UK).