Figure 5: Differential modulation of iron-regulating transcripts and iron metabolism in susceptible HLCs.

(a) Heatmap of expression of genes in HLCs identified by SAM as being significantly different in HT- and NHT-HLCs after 24 hours treatment with 100 μM PZ. For visualization, the individual log2 fold-changes (log FC) of the top 40 genes was calculated by normalizing expression in PZ-treated samples to the respective vehicle control (DMSO) to yield a baseline-corrected expression represented by Δ. Transcripts highlighted in red TFRC and SPINK1 are related to iron metabolism. (b) Expression of TRFC mRNA in HLCs showing strong induction of TFRC in HT-HLCs (HT1, HT2, HT3) treated with 100 μM PZ for 24 hours. HFE mRNA in HLCs with inverse pattern of expression as for TFRC with increase in expression seen only for NHT1 and NHT2-HLCs following exposure to PZ. (c) Quantification of intracellular iron content in HLCs treated with 100 μM PZ or DMSO for 24 hours. An increase in the Fe²⁺/Fe³⁺ ratio relative to control indicates an accumulation of the redox-active form of iron (Fe²⁺) in cells treated with PZ. Data represents mean ± SE from three independent experiments; **p < 0.001 comparing HT-HLCs to NHT-HLCs treated with PZ. (d) GSEA for a custom gene set corresponding iron metabolism genes. (e) GSEA for Hallmark gene set for reactive oxygen species from MsigDB. In (d and e) coordinated upregulation of member genes in gene sets in HT-PZ relative to NHT-PZ samples, is shown. The ES, normalized enrichment score (NES), p-value (for custom gene set) and false discovery rate-adjusted q value (FDR) are indicated for each gene set.