RNA was isolated from primary AMs using TRIzol (Life Technologies, Carlsbad, CA, USA). For first-strand cDNA synthesis, a total of 50 ng of DNA-free RNA was prepared and performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Primers for quantitative real-time PCR are shown in Table 1 . The qRT-PCR assay was manipulated using iTag Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) in a CFX Connect Real-Time PCR Detection System (Bio-Rad), and the results were calculated through the 22DDCt threshold methodology following normalization with GAPDH [61 (link)].
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