*3.5. Pathway Analysis of DEGs*

To find the pathways regulated by iron nanoparticles, the DEGs with over 1.5-fold change were annotated by the KEGG database. As a result, 106 DEGs in HF, 283 DEGs in HP, 199 DEGs in KF, and 152 DEGs in KP were enriched in 10, 25, 18, and 12 KEGG pathways, respectively (File S3 (Supplementary Materials)). The comparison of the top 10 pathways revealed that there was great difference between HF and KF, but some similarity between HP and KP (Figure 6A,B). Only the pathways in cancer were enriched in four treatments (Figure 6C).

In HF, the fatty acid metabolism was the most significantly enriched pathway, in which the ACACA, FADS1, and FADS2 genes play important roles in cell membrane formation and repair. The metabolic pathways mainly involved in lipid metabolism and glycerophospholipid metabolism were also enriched in HF. In contrast, the HIF-1 signaling way was most prominently enriched in KF, which consisted of CDKN1A, FLT1, HMOX1, IGF1R, PLCG2, TFRC, STAT3, LDHA, ENO1, PGK1, and ELOB genes (Figure S4). This signaling way can mediate adaptive responses to reduced oxygen availability; it was crucial for angiogenesis, vascular reactivity and remodeling, glucose and energy

metabolism, inflammation, tumor, and iron homeostasis [48]. The hypoxia and free radicals can activate the function of HIF-1 [49]. By producing ROS, the HIF-1 signaling way was activated by FeNPs in KG1a. PLCG2 was induced by ROS to affect the IP3/DAG pathways, the ubiquitination of HIF-1α was inhibited by the downregulated ELOB, and the activity of HIF-1α was enhanced by the upregulated receptor tyrosine kinase (RTK). The iron deprivation can stimulate TFRC transcription through HIF-1 [50]. LY6E, highly upregulated in HF and KF (Figure 3B), was identified as an activator of HIF-1 and functioned as a novel conductor of tumor growth through its modulation of the PTEN/PI3K/Akt/HIF-1 axis [35].

**Figure 6.** Comparative Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs (*p* < 0.05): (**A**) comparative top 10 enriched pathways of HL60 and KG1a treated with FeNPs and (**B**) comparative top 10 enriched pathways of HL60 and KG1a treated with PBNPs. The pathways in black and blue were HL60 and KG1a cells, respectively. (**C**) Four-way Venn analysis of all KEGG pathways in four groups. The detailed information of all KEGG pathways is shown in File S3 (Supplementary Materials). HF, FeNP-treated HL60 cells; HP, PBNP-treated HL60 cells; KF, FeNP-treated KG1a cells; and KP, PBNP-treated KG1a cells.

It is interesting that the Platinum drug resistance pathway was also highly enriched in KF, in which several genes involved in platinum drug resistance were upregulated, including BIRC3, CDKN1A, GSTM2, and BBC3 (File S1 (Supplementary Materials)). ROS increase plays a key role in both platinum and iron nanoparticle-induced cancer cell death. Therefore, the activation of the platinum drug

resistance pathway in KF may underline the resistance of KG1a cell to FeNP treatment (Figure 1C). Interestingly, CDKN1A and GSTM2 were also upregulated in HP and BIRC3 and CDKN1A were also upregulated in KP (File S1 (Supplementary Materials)). However, none of these genes were regulated in HF. This contributed to the decrease of cell viability of HF and no significant decrease of cell viability of HP, KF, and KP (Figure 1C).

When exposed to PBNPs, 40% top 10 pathways were identical between KG1a and HL60, in which the mineral absorption was most significantly enriched in two cells (Figure 6B). In HP and KP, Ferritin (FTH1 and FTL) and MTs were changed to regulate cellular metal ions concentration due to increased iron ions (File S3 and Figure S5 (Supplementary Materials)). In contrast, the mineral absorption pathway in KG1a was more complex (File S3 and Figure S5 (Supplementary Materials)), including the multi-responses of HMOX1 to Fe2<sup>+</sup>, SLC8A1(NCX1) to Na<sup>+</sup> and Ca2+, and SLC30A1(ZNT1) to Zn2<sup>+</sup> [7,51,52]. The activation of the mineral absorption pathway made the two cells survive under the treatment of PBNPs (Figure 1C); however, the significant change of lipid metabolism induced by FeNPs made HL60 easy to kill (Figure 1C) although FTL was upregulated and TFRC was downregulated (Figure 2).
