To further characterise the effects of TAK-242 in mitigating the
response of LPS, the expression of pro-inflammatory, matrix degradation and
ECM-related genes was examined at the end of the 24 h treatment period. Total
RNA was extracted from treated NP cells using the QIAGEN RNeasy kit following
manufacturer’s instructions. Concentration and purity were measured by
NanoDrop, with 260/280 ratios between 1.8 and 2.0 for all samples. 200 ng of
total RNA were converted to cDNA using the iScript cDNA Synthesis kit (BioRad).
Primers listed inTable 2 were designed
using the Integrated DNA Technologies PrimerQuest Tool (Integrated DNA
Technologies). Quantitative PCR was performed using QuantiStudio 6 Flex system
(Applied Biosystems, ThermoFisher Scientific) and iTaq Universal SYBR Green kit
(BioRad) with the following amplification protocol: 95 °C for 30 s
followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
Analysis of gene expression was performed by QuantiStudio Real-Time PCR Software
v1.3 software (Applied Biosystems) using the 2−
(ΔΔCt) method to calculate fold change relative to the
untreated group.
response of LPS, the expression of pro-inflammatory, matrix degradation and
ECM-related genes was examined at the end of the 24 h treatment period. Total
RNA was extracted from treated NP cells using the QIAGEN RNeasy kit following
manufacturer’s instructions. Concentration and purity were measured by
NanoDrop, with 260/280 ratios between 1.8 and 2.0 for all samples. 200 ng of
total RNA were converted to cDNA using the iScript cDNA Synthesis kit (BioRad).
Primers listed in
using the Integrated DNA Technologies PrimerQuest Tool (Integrated DNA
Technologies). Quantitative PCR was performed using QuantiStudio 6 Flex system
(Applied Biosystems, ThermoFisher Scientific) and iTaq Universal SYBR Green kit
(BioRad) with the following amplification protocol: 95 °C for 30 s
followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
Analysis of gene expression was performed by QuantiStudio Real-Time PCR Software
v1.3 software (Applied Biosystems) using the 2−
(ΔΔCt) method to calculate fold change relative to the
untreated group.