Random hexamer primers

RNA from sorted cells was extracted from frozen cell pellets using the NucleoSpin RNA XS kit (Macherey Nagel) according to the manufacturer’s protocol. cDNA was prepared with random hexamer primers (Carl Roth), dithiothreitol (Sigma-Aldrich), RiboLock (ThermoFisher Scientific) and M-MLV reverse transcriptase, (Promega, Madison, WI, USA). qPCR was performed in a Qiagen rotor gene using Sybr-green master mix (Biorad, Hercules, CA, USA). All qPCRs were controlled for technical quality by excluding measurements, in which the technical replicates varied with SD > 0.5. CT-Values of genes of interest (GOI) were calibrated to the housekeeper gene Rps6, by calculating the ratio 2^{(CTRps6 – CTGOI)}. Genes with CTs over 30 were assumed to be not detected. The limit of detection (lod) of most qPCRs was between the ratios 0.0005 and 0.0025 and was set to 0.0015 in the graphs, if not stated otherwise, to provide a reference for the comparison of expression levels between genes. Primer sequences are shown in Supplementary Materials.

RNA was isolated after lysis by PeqGold TriFast (Peqlab) followed by chloroform-phenol extraction. Reverse transcription was performed with M-MLV reverse transcriptase (Thermo Fisher) and random hexamer primers (Carl Roth) as described previously (8 (link)). Quantitative real-time PCR was performed with SoFast Eva Green master mix (BioRad) and primers, the sequences of which are listed in Supplemental Table 6. Hprt (hypoxanthine guanine phosphoribosyl transferase, Entrez Gene ID 15452) served as the house-keeping gene for expression normalization. Relative gene expression was calculated with the ΔCt method and presented as log₂ expression, where log₂ expression_gene = Ct_gene — Ct_Hprt.
Protein extraction and Western blotting were performed exactly as described previously (61 (link)). We used the following antibodies: rabbit anti-mouse FTH1 (1:1000; Cell Signaling, 3998), rabbit anti-mouse Ferritin light chain (1:1000; Abcam, ab69090), mouse anti-human Transferrin receptor (1:1000; Invitrogen, PA1-84854), rabbit anti-human FPN1 (1:2000; Eurogentec, NRU 451443), and rabbit anti-human β-actin (1:500; Sigma-Aldrich, A2066).