Fig 2: Expression of CYTH4 in patients with AML. (A) Expression of CYTH4 in tumor and adjacent normal tissues across all TCGA tumors, analyzed by TIMER 2.0. (B) Comparison of CYTH4 expression between AML BM CD34+ cells and normal BM CD34+ cells, analyzed using the GSE30029 dataset. (C) Expression of CYTH4 in different AML FAB subtypes, analyzed using UALCAN. *P<0.05, **P<0.01 and ***P<0.001. TCGA, The Cancer Genome Atlas; ACC, adrenocortical cancer; BLCA, bladder cancer; BRCA, breast cancer; CESC, cervical cancer; CHOL, bile duct cancer; COAD, colon cancer; DLBC, large B-cell lymphoma; ESCA, esophageal cancer; GBM, glioblastoma; HNSC, head and neck cancer; KICH, kidney chromophobe; KIRC, kidney clear cell carcinoma; KIRP, kidney papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, lower grade glioma; LIHC, liver cancer; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian cancer; PAAD, pancreatic cancer; PCPG, pheochromocytoma & paraganglioma; PRAD, prostate cancer; READ, rectal cancer; SARC, sarcoma; SKCM, melanoma; STAD, stomach cancer; TGCT, testicular cancer; THCA, thyroid cancer; THYM, thymoma; UCEC, endometrioid cancer; UCS, uterine carcinosarcoma; UVM, ocular melanomas; BM, bone marrow; FAB, French-American-British classification system; CYTH4, cytohesin-4; TIMER 2.0, Tumor Immune Estimation Resource version 2.0; UALCAN, University of Alabama at Birmingham CANcer data analysis Portal.

Fig 3: Functional enrichment analysis of CYTH4-associated genes. (A) GO analysis result. (B) KEGG pathway enrichment analysis. (C) GSEA enrichment plots. (D) Genes interacting with CYTH4 in STRING. (E) CIBERSORTx result showing the different fraction of immune cells between the low- and the high-CYTH4 groups. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, Gene Set Enrichment Analysis; CYTH4, cytohesin-4.

Fig 4: Survival analysis based on the expression level of CYTH4. Comparison of (A) OS and (B) EFS between the high- and the low-CYTH4 group in TCGA dataset. Comparison of OS between the high- and the low-CYTH4 group in (C) GSE10358 and (D) GSE14468 datasets. The upper plots in A-D show the ROC curves of the effect of CYTH4 expression on survival. The bottom plots in A-D show the Kaplan-Meier survival curves. Comparison of OS between the high- and the low-CYTH4 group in (E) patients treated with chemotherapy alone or (F) treated with chemotherapy plus transplantation. Comparison of OS between chemotherapy and transplantation in patients (G) with low CYTH4 expression or (H) with high CYTH4 expression. Two-sided P<0.05 was considered to indicate a statistically significant difference. OS, overall survival; EFS, event-free survival; TCGA, The Cancer Genome Atlas; ROC, Receiver Operating Characteristic curve; CYTH4, cytohesin-4.

Fig 5: In vitro validation of the function of CYTH4. (A) Western blot showing reduced CYTH4 protein expression level after shRNA-mediated CYTH4 knockdown. (B) CCK-8 assay displaying the inhibition of cell growth in CYTH4-silenced cells compared with control cells. The number of living cells and dead cells on day 0 is displayed. (C) Flow cytometry analysis of cell cycle distribution in CYTH4-silenced and control cells. (D) Colony formation assays showing growth inhibition in CYTH4-silenced cells compared with control cells. (E) Flow cytometry showing increased apoptosis in CYTH4-silenced cells compared with control cells. Bars in (C), (D) and (E) show the mean ± SD from three independent biological replicates. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. CCK8, Cell Counting Kit-8; CYTH4, cytohesin-4; shRNA, short hairpin RNA.