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. 2023 Dec 18;13(1):188. doi: 10.1038/s41408-023-00962-z

Fig. 2. Effect of S100A9 silencing using S100A9-siRNA on cell viability, proliferation and apoptosis in human AML cell lines.

Fig. 2

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.