Nanodrop spectrophotometer

Total RNA was extracted from cells by Trizol reagent (Invitrogen) according to the manufacturer’s instructions (33 (link)). RNA yield and purity were checked using a Nanodrop spectrophotometer (EuroClone), and total RNA from each sample was reverse transcribed into cDNA using Moloney Murine Leukemia Virus Reverse Transcriptase (Sigma-Aldrich). Real-time PCR was performed on a StepOne™ Real-Time PCR System (Thermo Fisher) using SensiFAST SYBR Hi-Rox (Bioline). The comparative Ct method (ΔΔCt) was used to quantify gene expression, and the relative quantification was calculated as 2^−ΔΔCt. The presence of non-specific amplification products was excluded by melting curve analysis. Statistical analyses on real-time PCR data were performed using the Relative Expression Software Tool (REST) (34 (link)). The forward and reverse primer sequences were reported in Table 1.

Total RNA was extracted from frozen renal tissues using TRIzol reagent according to the manufacturer's instructions. RNA yield and purity were checked using a NanoDrop spectrophotometer (EuroClone), and total RNA from each sample was reverse transcribed into cDNA using SuperScript II Reverse Transcriptase. Real-time PCR was performed on an ABI Prism 7500 device using Power SYBR Green Master Mix 2× (Thermo Fisher Scientific). The comparative Ct method (ΔΔCt) was used to quantify gene expression, and the relative quantification was calculated as 2^−ΔΔCt. The presence of nonspecific amplification products was excluded by melting curve analysis (primers are listed in Table 1). Data were normalized to GAPDH expression.

Breast cancer cells were seeded in Boiden chambers on 8.0 μm pore size Millipore filters and in filters coated with 50 μL of collagen type I solution (0.2 μg/μL). Total mRNA was extracted using the Trizol reagent (Invitrogen), following the manufacturer’s instructions. Quantity and quality of RNA were checked using the Nanodrop spectrophotometer (EuroClone). Total RNA was reverse-transcribed into cDNA using the reverse transcriptase SuperScript II (Invitrogen). Real-time qPCR was performed with the ABI-Prism 7500 using Power SYBR Green Master Mix 2 (Applied Biosystems) and specific primers for alpha-smooth muscle actin (α-SMA), E-cadherin, fibronectin, vimentin, MMP-2, MMP-9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 1). The comparative Ct method (ΔΔCt) was used to quantify gene expression, and the relative quantification was calculated as 2^-ΔΔCt. GAPDH gene amplification was used as a reference standard to normalize the target signal. Amplification specificity was controlled by melting curve analysis.

Cell cultures were lysed in RL lysis buffer (Norgen Biotek Corp., Thorold, ON, Canada). RNA was isolated from cells by using a Total RNA Purification kit (Norgen Biotek Corp., Thorold, ON, Canada). The quantification of the isolated RNA was determined by NanoDrop spectrophotometer (ND-1000, EuroClone, Milan, Italy). Reverse transcription was conducted with SuperScript III (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. qRT-PCR was performed with the use of the iQTM SYBR Green Super Mix (Bio-Rad Laboratories, Hercules, CA, United States) and specific primers (reported in Table 1). All reactions were performed in a 96-well format with the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, MA, United States). The relative quantities of specific mRNA were obtained with the use of the comparative Ct method and were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Maione et al., 2021 (link); Zuccolini et al., 2022 (link)). The expression of each target gene was assessed in triplicate (Ferrera et al., 2021 (link); Maione et al., 2021 (link)).

Cell cultures were lysed in RL lysis buffer (Norgen Biotek corp., Thorold, Canada). RNA was isolated from cells by using a Total RNA Purification kit (Norgen Biotek corp., Thorold, Canada). The quantification of the isolated RNA was determined by NanoDrop spectrophotometer (ND-1000, EuroClone, Milan, Italy). Reverse transcription was conducted with SuperScript III (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. qRT-PCR was performed with the use of the iQTM SYBR Green Super Mix (Bio-Rad Laboratories, Hercules, CA, USA) and specific primers (reported in Additional file 1: Table S3). All reactions were performed in a 96-well format with the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Massachusetts, USA). The relative quantities of specific mRNA were obtained with the use of the comparative Ct method and were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Cell cultures were lysed in RL lysis buffer (Norgen Biotek corp., Thorold, Canada). RNA was isolated from cells by using a Total RNA Purification kit (Norgen Biotek corp., Thorold, Canada). The quantification of the isolated RNA was determined by NanoDrop spectrophotometer (ND-1000, EuroClone, Milan, Italy). Reverse transcription was conducted with SuperScript III (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. qRT-PCR was performed with the use of the iQTM SYBR Green Super Mix (Bio-Rad Laboratories, Hercules, CA, USA) and specific primers (reported in Table S5). All reactions were performed in a 96-well format with the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, MA, USA). The relative quantities of specific mRNA were obtained with the use of the comparative Ct method and were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).