4 bromoanisole

NRCFs were conditioned and lysed in RNAZol RT solution (Merck KGaA, Burlington, MA, USA). Total RNA isolation, cDNA (complementary deoxyribonucleic acid) synthesis, and qPCR measurement was performed as described earlier [44 (link)]. In brief, DNA and proteins were precipitated by nuclease-free water (Acros Organics, Geel, Belgium) and 4-bromoanisole (Sigma-Aldrich, St. Louis, MO, USA). Then, RNA was precipitated with isopropanol (Sigma-Aldrich, St. Louis, MO, USA) and pellets were washed with ethanol (VWR International, Fontenay-sous-Bois, France) before resuspension in nuclease-free water. cDNA synthesis was performed by Sensifast cDNA synthesis kit (Bioline; London, UK) according to the manufacturer’s protocol. Measurements were performed on a LightCycler^® 480 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland) using LightCycler^® RNA Master SYBR Green I reagent (Roche Diagnostics, Basel, Switzerland) with primers presented in Supplementary Table S1. For control, Vimentin was used as a common fibroblast marker. Fold change was calculated using the ΔΔCt calculation method according to Schmittgen and Livak 2008 [45 (link)].

For RNA isolation, 10 transformants were grown in the same way as for the transposition assay. RNA isolation was then performed in a pool containing 1 mL of each culture. Hot acidic phenol extraction protocol was used for RNA isolation [23 (link)], but with the RNA being precipitated by LiCl and repurified using RNAzol^® RT (Molecular Research Center, Inc.Cincinnati, OH, USA) with 4-bromoanisole (Sigma Aldrich, St. Louis, MO, USA). This protocol produced RNA with only a very small amount of DNA contamination that was then completely removed by TURBO DNA-free™ Kit (Invitrogen, Carlsbad, CA, USA). Reverse transcription was done using High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems, Foster City, CA, USA). RT-qPCR was performed in triplicate with Rotor-Gene Q (Qiagen, Hilden, Germany) and SensiFAST™ 2X HRM kit (Bioline, London, UK), 10 ng of cDNA and 5 µM of each primer (Table S1). Data analysis and processing was done in LinReg software (Heart Failure Research Center, Amsterdam, NL; [24 (link)]). Statistics were performed in R Studio, V.1.2.1335 (R Development Core Team; Boston, MA, USA; [25 ]), using the powerTransform function for transformation followed by Univariate analysis of variance (ANOVA) with GLM procedure to decipher factor interactions and post hoc Tukey’s HSD test (p ≤ 0.05) to carry out multiple comparisons.