Smad3

ON TARGETplus SMARTpool siRNA (Horizon), Silencer Select siRNA (Thermo Fisher Scientific), and Gene Silencer (Santa Cruz Biotechnology) (see list below) were transfected into PAECs by the 4D Nucleofector Core unit using p5 Primary Cell Solution according to manufacturer’s recommendations (Lonza). The knockdown efficiency was determined by qPCR and immunoblotting. PAECs transfected with siRNA were incubated for 48 hours, and then treated with 10 μg/mL of HERV-K dUTPase for 72 hours.
siRNAs used for transfection: TLR4 (Ambion, catalog s14194); TLR2 (Ambion, catalog s162); TLR6 (Ambion, catalog s20215); MYD88 (Ambion, catalog s9134); NRP1 (Dharmacon, catalog L019484); ENG (Dharmacon, catalog L011026); MCAM (Ambion, catalog s8572); RELA (Ambion, catalog s11915); SMAD3 (Ambion, catalog s535079); STAT1 (Ambion, catalog s279); ATF2 (Santa Cruz, catalog sc29205); Negative Control No.1 (Ambion, catalog 4390844); Nontargeting Control (Dharmacon, catalog D001810); and Control siRNA-A (Santa Cruz, catalog Sc37007).

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.

Immunoblotting from cell lysates was performed as previously described (Papaspyropoulos et al., 2022 (link)). The following primary antibodies were used at a concentration of 1:1000; HES1 (AB5702), GAPDH (Cell Signaling #5174), SMAD3 (ThermoFisher #51–1500), pSMAD3 (Cell Signaling #9520), VEGF (Santa Cruz; sc‐507) and HRP‐linked anti‐rabbit secondary antibodies were used at a concentration of 1:1000 (Cell Signaling #7074).

NHDF or tHF cells seeded in 12 well plates were transfected with 40uM siRNAs (SMAD3 and IRF7; Thermo Fisher Scientific) or miRNA mimics (custom designed; IDT) per well using Lipofectamine RNAiMax (Thermo Fisher Scientific) according to the manufacturer’s instructions. 33ug/mL ISD90 (IDT) and 16.5ug/mL polyI:C (Invivogen) was transfected into cells using Lipofectamine 3000 according to the manufacturer’s instructions.

Total RNA was isolated from transfected or infected cells using the Trizol RNA isolation method. cDNA was prepared using 1000ng of total RNA and random hexamer primers. Samples were incubated at 16°C for 30 minutes, 42°C for 30 minutes and 85°C for 5 minutes. Real-time PCR (Taqman) was used to analyze cDNA levels in transfected or infected samples. An ABI StepOnePlus Real Time PCR machine was used with the following program for 40 cycles: 95°C for 15 sec and 60°C for one minute. Relative expression was determined using the ΔΔCt method using 18S as the standard control. JunB, SERPINE, SMAD3, IFNA1, IFNB1, RSAD2, IRF7 and 18S primer/probe sets were obtained from Thermo Fisher Scientific.