Fig 2: Effect of S100A9 silencing using S100A9-siRNA on cell viability, proliferation and apoptosis in human AML cell lines.KG-1a and MOLM-13 cells were exposed to 20 nM S100A9-siRNA and Lipofectamine 2000. A mock (only lipofectamine) and scramble condition (negative control) were included as controls. A Cell viability was detected by CellTiter-Glo at 72 h (n = 4). B Cell proliferation was investigated using BrdU staining at 72 h (n = 4). C Apoptosis was measured using an AnnexinV 7-AAD staining and flow cytometry at 72 h (n = 3). D RNA sequencing was performed on S100A9-siRNA treated KG-1a and MOLM13 cells at 72 h. The bubble plot shows the top 20 differentially regulated (activated/suppressed) pathways in the S100A9-siRNA group compared with mock (n = 3). E GSEA of the HEME_METABOLISM, MTORC1_SIGNALING and UNFOLD_PROTEIN_RESPONSE gene signature in KG-1a and MOLM-13 cells after treatment with 20 nM S100A9-siRNA for 72 h. GSEA of differentially expressed genes was determined by querying the MSigDB. False discovery rate (FDR) and normalized enrichment scores (NES) are indicated (n = 3). F–H KG-1a and MOLM-13 cells were cultured with 20 nM S100A9-siRNA for 72 h. Whole-cell lysates were subjected to Western blot analysis using anti-p-mTOR, mTOR, p-P70S6K, P70S6K, p-S6K, S6K, p-4E-BP1, 4EBP1, GRP78, p-eIF2a, eIF2a, ATF4, p21, p27, S100A9, puromycin, SLC7A11, SLC7A5 and anti-β-Actin antibodies (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, One-way ANOVA, Error bars indicate SD.

Fig 3: ALYREF directly targeted JunD which promote the transcription of SLC7A5 mRNA.A Venn Diagram showing the intersection of RIP-BisSeq, RNA-seq and JASPAR database. JunD is identified as a potential target. B, C Expression of JunD following ALYREF knockdown was detected by RT-qPCR and western blotting. D RIP assay was used to validate the direct binding between ALYREF and JunD. E The mRNA decay rate of JunD in shNC and shALYREF PDAC cells treated with Actinomycin D. F The dot blot assay was used to assess the change of mRNA m5C level after NSUN2 knockdown in PDAC cells. G MeRIP-qPCR assay was conducted using m5C-specific antibody to measure the m5C levels. H RIP-qPCR assay was performed using anti-ALYREF antibody to determine the binging efficiency between ALYREF and JunD mRNA. I, J Expression of SLC7A5 following JunD knockdown was detected by RT-qPCR and western blotting. K Schematic illustration showing the position of ChIP- qPCR primers. L, M ChIP assay was performed to test whether JunD could bind to the promoter of SLC7A5. Dual-luciferase assays verified that whether JunD could promote the transcription of SLC7A5 mRNA. Data are presented the mean ± SD of 3 independent experiments, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: The correlation among ALYREF, JunD and SLC7A5 and the clinical significance of ALYREF-JunD-SLC7A5 axis.A The expression levels of ALYREF, JunD and SLC7A5 in TMA were measured by multiplex immunohistochemical and two groups of representative multiplexes immunohistochemical images are shown (scale bar, 50 μm). B The correlation of among the expression levels of ALYREF, JunD and SLC7A5 in TMA samples was analyzed by Pearson correlation coefficient (n = 139). C Overall survival analysis based on TMA cohort showed that patients with high co-expression of ALYREF, JunD and SLC7A5 have poor prognosis. D The graphic illustration of ALYREF modulating tumor progression and immune escape in PDAC.

Fig 5: Knockdown of SLC3A2 decreases intracellular polyamine and branched-chain amino acid concentration.A Gene dependency effects (CRISPR, Chronos) of SLC3A2 with either SLC7A5 or SLC7A11 in NB cell lines (n = 37) from DepMap (https://depmap.org/portal/). B CLB-GAR cells were treated with either SLC3A2-targeting or control siRNAs. (B) Intracellular polyamine and (C) branched-chain amino acids (BCAA) concentrations were determined relative to controls (n = 3, mean ± s.e.m.). Intracellular polyamine (D) and BCAA (E) concentrations were determined at 0, 1 and 6 h of NB1 cell treatment with ALKAL2 (1 µg/ml) and lorlatinib (0 or 30 nM) (n = 3, mean ± s.e.m.). Intracellular polyamine (F) and BCAA (G) concentrations were determined at 0, 6 and 24 h of CLB-BAR cell treatment with lorlatinib (0 or 30 nM) (n = 3, mean ± s.e.m.). H, I Intracellular polyamine (H) and BCAA (I) concentrations were determined at 72 h after transection of HEK293T cells with either vector control, MARCH11-WT, or MARCH11-Y371F (n = 3, mean ± s.e.m.). J, K CLB-BAR cells were transfected with either control siRNA or MARCH11-targeting pool siRNAs for 24 h and lorlatinib (30 nM) added for additional 24 h. Intracellular polyamine (J) and BCAA (K) concentrations were determined (n = 3, mean ± s.e.m.). Results were analyzed by Student t test (unpaired, two-tailed). p-values are indicated (****P < 0.0001, ***0.0001 < P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05, ns P ≥ 0.05); exact P values are shown in Supplementary Table 1.