Trifast reagent

RNA was extracted using Trifast reagent (Euroclone), according to the manufacturer’s instructions. 1 μg of RNA was reverse-transcribed into cDNA using random hexamers and SuperScript II Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA). cDNA was used to determine transcript levels by qRT-PCR in a StepOne RT-PCR machine (Applied Biosystems, Foster city, CA, USA) using SYBR® Select Master Mix (Life Technologies). Primers used are listed in Supplementary Table S3. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as endogenous control was amplified with primers 5’-GAG AAG GCT GGG GCT CAT TT (forward) and 5’-AGT GAT GGC ATG GAC TGT GG (reverse). Data were analyzed using the ΔΔCT method. ΔC_t was calculated subtracting the average C_t value of GAPDH as control to the average C_t value of each transcript for PC3 and LnCaP. ΔΔC_t is the difference between the ΔC_t for each transcript for PC3 and the ΔC_t of each transcript for LnCaP as control. The reported fold expression, expressed as RQ (relative quantity), was calculated by 2^−ΔΔCt. The analysis was repeated three times in triplicate. The mean ± standard deviation (SD) of a representative experiment is reported (* p < 0.05).

Total RNA was isolated using Trifast reagent (EuroClone) according to the manufacturer instructions from 3 × 10⁵ cells/well treated with increasing concentrations of 1d and 1h for 48 h and 72 h. Complementary DNA (cDNA) was obtained using GoTaq^® 2 Step RT-qPCR Kit (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions. RT-qPCR was carried out using SYBR Green (Promega) on the Quant Studio 7 Pro Real-Time PCR System (ThermoFisher, Waltham, MA, USA). The results were normalized to glyceraldehyde 3-phoshate dehydrogenase (GAPDH) for HUVECs and HGFs and to β-actin (βACT) for HOBs using the 2^−ΔΔCt method. Primer sequences are reported in Table 2.

RT-PCR was performed with the objective of evaluating the gene expression of different markers in a certain stage of osteoblastic cells (ALP and OCN) and constitutive markers of fibroblast activity (COL1, FN1, MMP8). Total RNA was isolated using the Trifast reagent (EuroClone, Pero (MI), Italy). RNA was quantified on a Nanophotometer NP80 spectrophotometer (Implen NanoPhotometer, Westlake Village, CA, USA) for analysis of RNA integrity, purity, and concentration. Afterward, the GoTaq^®2 Step RT-qPCR Kit (Promega, Madison, WI, USA) was used to obtain complementary DNA (cDNA) according to the manufacturer. SYBR Green (GoTaq^® 2 Step RT-qPCR Kit, Promega) was used to perform RT-qPCR. An amount of 10 µL of mixes, composed of 1 µL of cDNA, 0.2 µL of primer mixture, and 5 µL of master mix, was plated in a 96-well plate, and gene expression was determined using Quant Studio 7 Pro Real-Time PCR System (ThermoFisher, Waltham, MA, USA). The results were normalized to β-actin (β-ACT) for HOB and to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) using the 2 ^−ΔΔCt method. Primer sequences are reported in Table 1.

For the splicing assay, 5×10⁵ Flag-TDP-43-WT, Flag-TDP-43-12×Q/N and AggIn cells were seeded in 6-well plates and induced with tetracycline for 72 h. Uninduced cells were used as a control. After induction, cells were collected and RNA was extracted using Trifast reagent (Euroclone, Milan, Italy) according to the manufacturer's instruction. Reverse transcription was performed using M-MLV Reverse Transcriptase (Gibco-BRL) following the manufacturer's protocol. A PCR with TAQ DNA Polymerase (Roche Diagnostics, Mannheim, Germany) was performed for 35 amplification cycles (95°C for 30 s, 55°C for 30 s, 72°C for 30 s) to amplify POLDIP3, BIM and MADD cDNAs. The primers used to test the splicing pattern of POLDIP3, BIM and MADD endogenous genes were: POLDIP3 forward (5′-GCTTAATGCCAGACCGGGAGTTGGA-3′); POLDIP3 reverse (5′-TCATCTTCATCCAGGTCATATAAATT-3′); BIM forward (5′-TCTGAGTGTGACCGAGAAGG-3′); BIM reverse (5′-TCTTGGGCGATCCATATCTC-3′); MADD forward (5′-GACCTGAATTGGGTGGCGAGTTCCCT-3′); MADD reverse (5′-CATTGGTGTCTTGTACTTGTGGCTC-3′).

Total RNA was extracted using TriFast reagent (EuroClone) and reverse transcription was achieved using LunaScript^® RT SuperMix Kits (New England Biolabs) following the manufacturer’s protocol. Template complementary DNA (cDNA) was subjected to qRT-PCR using FluoCycle II SYBR Master Mix (Euroclone) according to the manufacturer’s protocol (48 (link)). Primers were published or designed with Primer-BLAST software https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (Table S2). Reactions were run in duplicate on a LightCycler 480 II (Roche), using the following setup: denaturation at 95°C for 5 minutes; amplification at 95°C for 15 seconds, and 60°C for 60 second (45 cycles). Target expression was normalized to GAPDH by the cycle threshold (Ct) method and expressed using the 2^−ΔΔCt calculation as fold over control.

RNA was extracted using Trifast reagent (Euroclone), according to the manufacturer’s instructions. 1 μg of RNA was reverse-transcribed into cDNA using random hexamers and SuperScript II Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA). cDNA was used to determine transcript levels by qRT-PCR in a StepOne RT-PCR machine (Applied Biosystems, USA) using SYBR^® Select Master Mix (Life Technologies). Primers used are listed in Supplementary Table 1. GADPH used as endogenous control was amplified with primers 5′-GAG AAG GCT GGG GCT CAT TT (forward) and 5′-AGT GAT GGC ATG GAC TGT GG (reverse). Data were analysed using the ΔΔCt method. ΔCt was calculated subtracting the average Ct value of GADPH as control to the average Ct value of each transcripts for Mel501 and Mel 501ac. ΔΔCt is the difference between the ΔCt for each transcript for Mel501ac and the ΔCt of each transcript for Mel501 as control. The reported fold expression, expressed as RQ (Relative Quantity), was calculated by 2-ΔΔCt. The analysis was repeated three times in triplicate. The mean ± SD of a representative experiment is reported (*p < 0.05).