Additional file 1: Figure S1. of Rheb1 promotes tumor progression through mTORC1 in MLL-AF9-initiated murine acute myeloid leukemia

The correlation of Rheb1 expression level with AML prognosis. (A) Kaplan-Meier event-free survival curves of AML patients, with low (≤50 % quantile, n = 426) and high (>50 % quantile, n = 3) expression based on Verhaak et al.’s dataset. (B) Kaplan-Meier overall survival curves of AML patients, w/o Rheb1 deletion based on the cBioPortal database. Figure S2. The genotype of established AML mouse model. (A) Western blotting of Rheb1 protein level in WT and AML cells (n = 3). (B) Flow cytometric analysis of AML cells. (C) Rheb1 mRNA expression of AML cells. (D) PCR genotype of AML mice. (E) Histopathological analysis of the spleens in AML mice. (F) Gating strategy for L-GMPs in the spleen. (G) L-GMP frequency in the spleen (n = 4). (H) The percentages of AML subpopulations in the spleen (n = 4). The data are the mean numbers ± SEM. Figure S3. The function and related signaling pathway of AML cells. (A) GFP+ cells homing to the BM (left panel) and spleen (right panel), n = 5. (B) The differentially expressed genes of AML cells. (C) Enrichment of M phase mitotic cell cycle-related genes in GFP+ cells. (D and E) The cell cycle status of K+G+ (D) and K−G+ cells (E) (n = 4). (F and G) The mRNA levels of the indicated genes in GFP+ (F) and K+G− cells (G). The data are the mean numbers ± SEM. (H and I) The normalized MFI of p-S6 (H) and p-4E-BP1 (I) of K+G+ and K−G+ cells in the BM (n = 4). The data are the mean numbers ± SEM. Figure S4. The signaling pathway in human and murine AML. (A and B) mTOR (A) and 4E-BP1 (B) mRNA expression of human AML cells in the HemaExplorer database. (C and D) KEGG analysis of the upregulated (C) and downregulated (D) pathways in Rheb1 Δ/Δ vs Rheb1 fl/fl GFP+ cells. Table S1. Primer sequence list. (PDF 30124 kb)