Primer sets

Cells were disseminated onto 24-well plates at 1.0 × 10⁵ cells/well in 500 µL of CnT-Prime for 24 h. The medium was then changed to either fresh medium containing various concentrations of NaB or fresh medium only (as a control), and the cells were incubated for 6 h at 37 °C in 5% CO₂. After this treatment, total RNA was extracted from the cells using an RNeasy Mini Kit (Qiagen, Copenhagen, Denmark) in accordance with the supplied manufacturer’s instructions. The cDNA was synthesized with PrimeScript RT Master Mix (TaKaRa Bio., Shiga, Japan), and a real-time quantitative reverse-transcription polymerase chain reaction (qPCR) assay was performed using a Thermal Cycler Dice Real Time System (TaKaRa) following the manufacturer’s instructions. TB Green Premix Ex Taq II (TaKaRa) was used for the qPCR reaction. Primer sets, which were based on sequences for ICAM-1, integrin α6, integrin β4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were purchased from TaKaRa. The primers for Gapdh were used as an endogenous control. The cDNA amplification conditions were as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Dissociation was performed to confirm the specificity of primers.

qPCR was performed as previously described [48 (link)]. Briefly, we used the StepOne Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Primer sets for tumor necrosis factor α, IL-1β, IL-6, IL-8, IL-11 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from TaKaRa Bio (Shiga, Japan). GAPDH was utilized as an endogenous control. Cycle parameters were 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Dissociation was performed each time to confirm the specificity of the primers.