Col4a1

Total RNA was isolated using the PureLink™ RNA Mini Kit (Invitrogen, 12183025). Reverse transcription was performed with 2 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, 4368814). qRT-PCR assays were carried out in quintuplicates using the Real-Time PCR System (BioRad CFX Opus 96) and the following TaqMan probes: COL4A1 (Hs00266237, PN4453320), GAPDH (Hs99999905, PN 4453320), EGFR (Hs01076090, PN 4453320), MALAT1 (Hs01910177, PN 4448892), RPRM (Hs04189060, PN 4448892) and WNT16 (Hs00365138, PN 4453320) (Thermo Fisher Scientific, Carlsbad, CA, USA). Data were normalized with GAPDH as reference gene. Relative expression was calculated using the 2ΔΔCt method [102 (link)].

Samples were sectioned at a thickness of 5 µm. All the sections were incubated for 1 hour with primary antibody directed against IL1B (Cell Signaling Technology, #2022, 1:200), COL2A1 (Santa Cruz, sc-52658, 1:200), COL4A1 (Thermo Fisher Scientific, PA5-85634, 1:500), LGALS1 (Cell Signaling Technology, #5418, 1:200), MMP3 (Abcam, ab223666, 1:500) after blocking endogenous peroxidase using 3% hydrogen peroxide for 5 minutes at room temperature. After rinsing, the sections were incubated for 1 hour with biotinylated horseradish peroxidase-conjugated goat antirabbit IgG. Diaminobenzidine was used to develop peroxidase staining.

Total RNA was extracted from the MSC and MSC_LIF cell lines using the TRIZOL^® extraction reagent (Thermo Fisher Scientific). The quantification of RNA was performed on a NanoDrop^TM 1000 spectrophotometer (Thermo Fisher Scientific). The purity of the samples obtained by the ratio A260 nm: A280 nm demonstrated a proportion between 1.8 and 2.0. Aliquots of 1 μg of high quality RNA were used for cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen Waltham, MA, United States) after treatment with DNAse I. To analyze the expression of proangiogenic factors in MSC and MSC_LIF cells, we used the following primer sets purchased from Integrated DNA Technologies^TM (Coralville, IA, United States): ACTA2, TAGLN, COL4A1, CCL2, CXCL2, ANG, VEGF, and NG2 (Table 1), and Sybr Green (Thermo Fisher Scientific). The reactions were done in triplicate, and the average cycle thresholds (Ct) values were used to calculate the expression of each gene using the B2M gene as a normalizer. The PCR amplification was performed on an ABI7500 real-time PCR system (Thermo Fisher Scientific), under standard thermal cycle conditions.