Quantitative PCR (qPCR)

Following gentamicin treatment, infected pMacs were removed from the tissue culture plate using CellStripper (Corning), followed by trypsin treatment (Gibco, Life Technologies). The pMacs were then washed in 5% FBS, transferred to buffer RLT with β‐mercaptoethanol (Sigma, M6250), and frozen at −80°C, per manufacturer's protocols (RNeasy Mini kit, Qiagen). The samples were then thawed and RNA was isolated from the cells per manufacturer's protocols (RNeasy Mini kit, Qiagen). cDNA was then generated according the manufacturer's protocol, (First‐Strand cDNA synthesis Using SuperScript II RT, Life Technologies; with DNase treatment). qPCR was performed per manufacturer's protocols (SsoAdvanced Universal SYBR Green Supermix, BIO‐RAD, 1725274) using the following primers, with Rn18s serving as the control: Hamp1 (hepcidin): 5′‐GCAGAAGAGAAGGAAGAGAGACACC‐3′ and 5′‐TGTAGAGAGGTCAGGATGTGGCTC‐3′;

Fpn1 (ferroportin): 5′‐TGGAACTCTATGGAAACAGCCT −3′ and 5′‐TGGCATTCTTATCCACCCAGT‐3′; Slc11a2 (Dmt1): 5′‐AACCAACAAGCAGGTGGTTGA‐3′ and 5′‐CCTTGTAGATGTCCACAGCCAGAGT‐3′; Tfr1: 5′‐GTGGAGTATCACTTCCTGTCGC‐3′ and 5′‐CCCCAGAAGATATGTCGGAAAGG‐3′; Lcn2: 5′‐TGGCCCTGAGTGTCATGTG‐3′ and 5′‐CTCTTGTAGCTCATAGATGGTGC‐3′; Il‐6: 5′‐CCAGAAACCGCTATGAAGTTCCT‐3′ and 5′‐CACCAGCATCAGTCCCAAGA‐3′; Rn18s (18S): 5′‐CGGCTACCACATCCAAGGAA‐3′ and 5′‐GCTGGAATTACCGCGGCT‐3′.