P0450

For histological analysis, mice were anesthetized by intraperitoneal injection of ketamine (150 mg/kg) and xylazine (10 mg/kg) and then transcardially perfused with PBS followed by 4% of paraformaldehyde (PFA). Afterwards, brains were dissected, post-fixed overnight in 4% PFA at 4 °C and embedded in paraffin. Paraffin-embedded brain sections were stained with Hematoxylin/Eosin (H&E) and cresyl violet in order to analyse brain morphology. Immunostainings were performed for 1–2 h at RT using antibodies against amyloid-β 4G8 (Covance, SIG-39320, 1/50), Iba1 (Wako, 019–19741, 1:2000), GFAP (Dako, Z0334, 1:800), Ki67 (Novocastra, Ki67P-CE, 1:1000). The secondary antibodies were HRP conjugated anti-rabbit (ImmunoLogic, DPVR110HRP, RTU), anti-mouse (Dako, P0447; 1:100), anti-goat (Dako, P0449; 1:80) and anti-rat (Dako, P0450; 1:75) and were all incubated for 30–45 min at RT. Signals were visualized with DAB (3,3-diaminobenzidine), using hematoxilin as a counterstaining.
Brain pictures were acquired using an inverted Leica DM4000B microscope and analysed using Leica application suite software. The number and type of amyloid plaques in the hippocampus were manually defined and quantified. Dense plaques were identified by the presence of dense core whereas diffuse plaques were identified by the presence of diffuse corona, as illustrated in Fig. 3d.

The extirpated left lung was inflated to 20 cm H₂O with OCT compound (Sakura Finetek Japan, Tokyo, Japan) and frozen in isopentane cooled in liquid nitrogen. Sections of 10 μm were fixed with cold acetone and blocked with Protein Block Serum-Free^® (Dako, Glostrup, Denmark). They were then incubated with the first antibodies: rabbit antimyeloperoxidase antibodies (1:200, RB-373; Thermo Fisher Scientific, Waltham, Massachusetts) for neutrophil staining, rat antimouse Mac-3 antibodies (1:500; BioLegend, San Diego, California) for macrophage staining, rabbit anti-CD3 polyclonal antibodies (1:100; ab5690, Abcam, Cambridgeshire, UK) and rat anti-CD19 monoclonal antibodies (1:500; ab25232, Abcam, Cambridgeshire, UK) for lymphocyte staining. The EnVision^® + system–HRP labeled polymer antirabbit (K4003; DAKO, Glostrup, Denmark) and polyclonal rabbit antirat immunoglobulins (P0450; DAKO, Glostrup, Denmark) were used as the second antibodies. Diaminobenzidine (DAKO, Glostrup, Denmark) was used to visualize the sections, and haematoxylin was used for counterstaining.

Cell lysates, after boiling for 5 minutes at 95°C and centrifugation for 10 minutes at 20800 g, were subjected to SDS-PAGE. Proteins were detected by Ponceau S or immunoblotting, using the following primary antibodies: α-LANA (18-210-100, ABI); α-Rad50 (GTX70228, GeneTex); α-Mre11 (ab33125, Abcam); α-NBS1 (NB100-143, Novus Biologicals); α-GAPDH (14C10, Cell Signalling); α-KbZIP (F33P1, Santa Cruz Biotechnology); α-HHV-8 ORF45 (2D4A5, Santa Cruz Biotechnology); α-HHV-8 ORF57 (LS-C60137, LSBio); α-CARD9 (Cell Signalling, 12416S); α-p65 (sc-109, Santa Cruz); α-p-p65 (S536, Cell Signalling); α-Brd4 (A301-985A100, Bethyl); α-LaminA/C (sc-6215, Santa Cruz); α-IRF3 (sc-9082, Santa Cruz); α-p-IRF3 (4947S, Cell Signalling); α-p-TBK1 (3504S, Cell Signalling). Subsequently, membranes were incubated with the following secondary antibodies: α-mouse (P0260, Dako); α-rabbit (P0488, Dako); α-rat (P0450, Dako). Phospho-p65 levels were digitally quantified using ImageJ and normalized to the corresponding total p65 protein levels and the sample used as negative control.

Paraffin-embedded tumor sections were stained with hematoxylin and eosin (H&E) and analyzed by pathologists in blinded fashion for tumor grading. Immunostainings were performed using antibodies against CD-56 (Abcam #ab8233; 1:50, overnight 4ºC), BrdU (BD Biosciences #347580; 1:100, 1 h RT), Ki67 (Novocastra, #Ki67P-CE, 1:1000, 1 h RT) and cleaved caspase-3 (Cell Signaling #9661; 1:200, 1 h RT). The secondary antibodies used were HRP conjugated anti-rabbit (ImmunoLogic #DPVR110HRP, 45 min at RT), anti-mouse (Dako #P0447; 1:100, 30 min at RT) and anti-rat (Dako #P0450; 1:75, 30 min at RT). Signals were visualized with DAB (3,3-diaminobenzidine), using hematoxylin as a counterstaining.
Periodic acid-Schiff (PAS) reagent was used to detect mucus-secreting cells. Slides were incubated with an aqueous solution of 1% periodic acid for 10 min at RT, followed by incubation with Schiff's reagent (Merck, HX383284) for 20 min. Hematoxylin staining was used as a counterstain.

All histological analyses were performed on fixed paraffin-embedded LV sections (5 μm). Cardiac interstitial fibrosis was assessed by picrosirius red staining (0.1 % w/v), excluding coronary vessels and perivascular regions. Data were quantified by digital image analysis (NIS-Elements, Nikon) with the observer blinded to sample identity. Immunocytochemistry for CD45 and F4/80 was performed using rat polyclonal (553076, 1:200; BD Bioscience) and rat monoclonal (MCA497GA, 1:200; AbD Serotec) antibodies, respectively, followed by secondary rabbit anti-rat IgG (P0450, 1:100; Dako) staining, using diaminobenzidine as the chromogen and nuclear counterstaining with haematoxylin.

Antibodies used included a mouse monoclonal against CHOP (B3, Santa Cruz, USA) used at a 1:200 dilution for confocal microscopy and Western blotting, a goat monoclonal against total eIF2α (K17, Santa Cruz) and a rabbit monoclonal against phosphorylated eIF2α (E90, Abcam, UK) used at 1:1000 and 1:500 for Western blotting respectively. The rat anti-HA antibody was from Roche (clone 3F10) and was used at a 1:500 dilution for confocal microscopy, and 1:1000 for Western blot. The goat polyclonal antibody against PP1 was acquired from Santa Cruz, USA (clone C19) and used at a dilution of 1:1000 for Western blot. The γ tubulin antibody (Sigma-Aldrich, T6557) was used at a 1:25,000 dilution for Western blot.
The following horseradish peroxidase (HRP) conjugated secondary antibodies were used for Western blotting at a 1:1000 dilution and were acquired from Dako, UK; rabbit anti-rat HRP (P0450), rabbit anti-mouse HRP (P0260), goat anti-rabbit HRP (P0448). In addition, the bovine anti-goat HRP antibody (Santa Cruz, USA sc-2384) was used at a 1:5000 dilution. The HA-HRP conjugated antibody (Roche, 3F10) was used at a 1:1000 dilution. Alexa Fluor antibodies goat anti-rat 488 and rabbit anti-mouse 568 were used at a 1:500 dilution for confocal microscopy.