Additional file 7 of NAT10/ac4C/JunB facilitates TNBC malignant progression and immunosuppression by driving glycolysis addiction
Creators: name: Li, Guozheng
name: Ma, Xin
name: Sui, Shiyao
name: Chen, Yihai
name: Li, Hui
name: Liu, Lei
name: Zhang, Xin
name: Zhang, Lei
name: Hao, Yi
name: Yang, Zihan
name: Yang, Shuai
name: He, Xu
name: Wang, Qin
name: Tao, Weiyang
name: Xu, Shouping
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Supplementary Material 7: Supplementary Fig. S1. Ac4C level in TNBC tissues and efficiency of targeting NAT10. (A) Heat map showed the expression of NAT10 in different molecular types. (B)The ac4C level in TNBC tissues was determined by dot blot. (C) The NAT10 expression in different breast cancer cell lines. (D-F) Western blot and qRT-PCR measured NAT10 expression by using siRNA and remodelin. (G) Dot-blot quantification of ac4C abundance in mRNA transcripts. Ac4C dot blot assay was performed with methylene blue (MB) as a loading control. The data are shown as the means ± SDs; ****P &lt; 0.0001. Supplementary Fig. S2. NAT10 facilitates the TNBC tumorigenesis.  (A-D) NAT10 overexpression promotes the TNBC cells invasion and migration. (E) NAT10 overexpression promotes the TNBC cells proliferation. The data are shown as the means ± SDs; ****P &lt; 0.0001. Supplementary Fig. S3. NAT10 facilitates the 4T1 cell development. (A) The loss of NAT10 inhibits the 4T1 proliferation. (B-E) The loss of NAT10 inhibits the 4T1 invasion and migration. The data are shown as the means ± SDs; ***P &lt; 0.001, ****P &lt; 0.0001. Supplementary Fig. S4. NAT10 function is required for immunosuppressive TME in TNBC. (A) NAT10 expression in BLIS and non-immunosuppressive subtype (Others), generated from TCGA database (B) Correlation of NAT10 expression with immune infiltration levels in breast cancer based on the TIMER2 analysis. (C) Kaplan-Meier survival curves comparing immune cell infiltration of breast cancer in TIMER2. (D) Heatmap of phenograph clusters of CD45+ cells. The relative expression levels of markers across cells were shown and sorted by cell type. The data are shown as the means ± SDs; *P &lt; 0.05. Supplementary Fig. S5. Targeting NAT10 increases cytokines that could activate T cell and macrophages. (A-B) Heatmap showing serum cytokine concentration of remodeling-treated mice bearing 4T1 tumor compared with control. (C-D) Mice tissues were used to detect the mRNA expression of cytokines. The data are shown as the means ± SDs; *P&lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001, ns, nonsignificant. Supplementary Fig. S6. The loss of NAT10 facilitates T cell infiltration.. (A-B) Quantification of CD4+ and CD8+ populations among CD3+ T cells after co-culture with remodelin-treated TNBC cells. The data are shown as the means ± SDs; *P&lt; 0.05, **P &lt; 0.01. Supplementary Fig. S7. The loss of NAT10 does not affect the macrophage activation. (A) THP-1 was induced and differentiated into M1- and M2-like macrophages. (B) CM from NAT10-Kd cells were used to culture M1- and M2-like macrophages. Supplementary Fig. S8. NAT10 contributed to LDHA upregulating by interacting with JunB mRNA. (A)The sequence on JunB mRNA containing ac4C modification. (B) RNA-pulldown detected the protein interacted with JunB mRNA. Cells were treated with 1mM puromycin at 37℃ for 30 min, followed by protein isolation and western blotting. (C-D) qRT-PCR and western blot measured expression efficiency upon the knockdown and overexpression of JunB. (E) LDHA expression upon remodelin treatment. The data are shown as the means ± SDs; **P&lt; 0.01, ****P &lt; 0.0001. Supplementary Fig. S9. NAT10 did not affect the expression of LDHA and lactate production in T cells and macrophages. (A) Quantification of ATP production in control and NAT10-Kd cells. (B-C) Quantification of NAD+ and NADH in control and NAT10-Kd cells. (D) Jurkat was treated with remodelin, and LDHA expression and lactate production were measured. (E) M1- and M2-like macrophages were treated with remodelin, and LDHA expression and lactate production were measured. The data are shown as the means ± SDs; **P &lt; 0.01, ***P &lt; 0.001,****P &lt; 0.0001, ns, nonsignificant. Supplementary Fig. S10. Remodelin promotes the expression of CTLA-4 on T cells. (A) CTLA-4 expression on CD8+ and CD4+ T cells was measured based on the CyTOF results. (B-C) The number of Treg and CTLA-4+ Treg were measured based on the CyTOF results. (D) The expression of CLTA-4 in four tumor tissues is based on the TCGA database. The data are shown as the means ± SDs; *P &lt; 0.05, ****P &lt; 0.0001, ns, nonsignificant. Supplementary Fig. S11. Molecular mechanism