Additional file 1 of C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription
Creators: name: Du, Mengbao
name: Wang, Mowang
name: Liu, Meng
name: Fu, Shan
name: Lin, Yu
name: Huo, Yankun
name: Yu, Jian
name: Yu, Xiaohong
name: Wang, Chong
name: Xiao, Haowen
name: Wang, Limengmeng
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology

description: Additional file 1: Supplemental Materials and Methods. Supplementary Table 1. Patient characteristics. Supplementary Table 2. Human cDNA Primers for quantitative PCR. Supplementary Table 3. Mouse cDNA Primers for quantitative PCR. Supplementary Table 4. ChIP-qPCR primer sequences for putative C/EBPα binding sites in the promoter region of DDIT3 gene. Supplementary Fig. 1 (A) The RNA-seq data from Beat AML project of AML patients was analyzed using cBioPortal database, and a significant negative linear correlation was observed between CEBPA and DDIT3 expression in these cases (Pearson: -0.13, P = 5.136e-3). (B) The microarray data from GEO (GSE38987) (https://www.ncbi.nlm.nih.gov/gds) of AML patients was analyzed, and a negative linear correlation trend was observed in these cases. Supplementary Fig. 2 (A) Flow cytometric analysis of the apoptosis rates of AML cell lines (Kasumi-1, K562, HEL, NB4, KG-1, MOLM-13, MV4-11, THP-1, HL-60) after the treatment of tunicamycin (100 ng/ml, 48 hours). (B) Apoptosis rates of cells treated with tunicamycin (100 ng/ml) for 48 hours. The results of Kasumi-1, K562, and HEL were shown for better survival rate after treatment, which might be due to the activation of the adaptive UPR. (C) The sensitivity of AML cells to tunicamycin cytotoxicity was positively relevant to the expression level of DDIT3 in AML cell lines. Supplementary Fig. 3 Flow cytometric analysis revealed that compared with the negative control group, THP-1 cells with CEBPA knockdown displayed a significantly increased apoptosis rate after the treatment with tunicamycin (100 ng/ml, 48 hours). Supplementary Fig. 4. Flow cytometric analysis revealed that compared with the negative control group, 32Dcl3-shCebpa cells displayed a significantly increased apoptosis rate during granulocytic differentiation induced by G-CSF (100 ng/ml, 48 hours). Supplementary Fig. 5. The C/EBPα p42/p30 ratio was calculated based on the relative optical density. The Pearson's correlation test was applied to determine the correlation of the C/EBPα p42/p30 ratio and DDIT3. A negative linear correlation trend was observed between the C/EBPα p42/p30 ratio and DDIT3 at the protein level. Supplementary Fig. 6. (A) Flow cytometric analysis revealed that compared with the negative control group, NB4 cells with the induction of C/EBPα-p42 displayed a significantly reduced apoptosis rate after the treatment with tunicamycin (100 ng/ml, 48 hours). (B) Flow cytometric analysis revealed that compared with the negative control group, THP-1 cells with the induction of C/EBPα-p30 displayed a significantly increased apoptosis rate after the treatment with tunicamycin (100 ng/ml, 48 hours). Supplementary Fig. 7. (A) Other four fragments in the promoter region of DDIT3 were deleted (schematic diagram). (B) Deletions of other four fragments in the promoter region of DDIT3 including Chr12:57521027-57521326, Chr12:57521327-57521626, Chr12:57521627-57522036 and Chr12:57522037-57522236 did not affect the function of both C/EBPα-p42 and C/EBPα-p30. (C) Schematic diagram of C/EBPα regulating DDIT3 and outcome of UPR. In CEBPA wild type AML cells, C/EBPα-p42 is the dominant isoform and competitively inhibits the function of C/EBPα-p30 to keep a low basal DDIT3 level. When faced with ER stress, cells tend to initiate adaptive UPR, leading to cell survival. As for low p42/p30 ratio AML cells, overexpressed C/EBPα-p30 isoform strongly increases the basal DDIT3 level. When faced with ER stress, cells tend to initiate terminal UPR, leading to cell apoptosis. Supplementary Fig. 8. (A) The RNA-seq data from Beat AML project of AML patients was analyzed using cBioPortal database. A positive linear correlation was observed between CEBPA and BCL2 expression in these cases, and negative linear correlations were observed between CEBPA and BCL2L1 and MCL1. (B) The microarray data from GEO (GSE38987) (https://www.ncbi.nlm.nih.gov/gds) of AML patients was analyzed. A positive linear correlation was observed between CEBPA and BCL2 expression in these cases, and negative linear correlations were observed between CEBPA and BCL2L1 and MCL1. (C) The Pearson's correlation test was applied to determine the correlations between the protein levels of C/EBPα and BCL2, MCL1, and BCL-XL in AML cell lines. A positive linear correlation was observed between C/EBPα and BCL2 expression, and negative linear correlations were observed between C/EBPα and BCL-XL and MCL1. (D) The Pearson's correlation test was applied to determine the correlation between the C/EBPα p42/p30 ratio and BCL2, MCL1, and BCL-XL in AML cell lines. Supplementary Fig. 9. Flow cytometric analysis of the apoptosis rates of AML cell lines after the treatment of venetoclax (5 μM, 48 hours) and sorafenib (3 μM, 48 hours). Supplementary Fig. 10. (A) The histogram of the apoptosis rates of AML cell lines after the treatment of venetoclax (5 μM, 48 hours) and sorafenib (3 μM, 48 hours). The results of Kasumi-1 were not shown because of a better survival rate was detected after venetoclax treatment. (B)-(C) The sensitivity to venetoclax cytotoxicity was positively relevant to the mRNA (B) and protein (C) levels of BCL2 in AML cell lines. Supplementary Fig. 11. (A) Quantitative analysis of protein levels of BCL2, BCL-XL and MCL1 in THP-1 cells with CEBPA knockdown. (B) Quantitative analysis of protein levels of BCL2, BCL-XL and MCL1 in NB4 cells with induction of C/EBPα-p42 and C/EBPα-p30. (C) Quantitative analysis of protein levels of BCL2, BCL-XL and MCL1 in THP-1 cells with induction of C/EBPα-p30. Supplementary Fig. 12. (A) The mRNA levels of DDIT3 were increased in NB4 cells after the treatment of tunicamycin (100 ng/ml, 24 hours) and sorafenib (3 μM, 24 hours). (B) Quantitative analysis of protein levels of DDIT3 in NB4 cells after the treatment of tunicamycin (100 ng/ml, 24 hours) and sorafenib (3 μM, 24 hours). (C) Correlation analysis using the Pearson model revealed that the mRNA levels of CEBPA in AML cell lines tended to be negatively correlated with tunicamycin (100 ng/ml, 48 hours) and sorafenib (3 μM, 48 hours) induced apoptosis rates. Supplementary Fig. 13. (A) Quantitative analysis of protein levels of BCL2, BCL-XL and MCL1 in NB4 cells with combined treatment with tunicamycin and venetoclax and each drug alone. (B) Quantitative analysis of protein levels of BCL2, BCL-XL and MCL1 in NB4 cells with combined treatment with sorafenib and venetoclax and each drug alone. Supplementary Fig. 14. (A) The protein levels of p-ERK in NB4 cells were significantly downregulated by treatment with venetoclax+tunicamycin and venetoclax+sorafenib. (B) Quantitative analysis of protein levels of p-ERK and ERK after the treatment of DMSO, venetoclax, tunicamycin, venetoclax+tunicamycin, sorafenib and venetoclax+sorafenib. Supplementary Fig. 15. (A)-(B) Flow cytometric analysis revealed that combined treatment with tunicamycin (100 ng/ml) and venetoclax (5 μM) resulted in increased apoptosis/cell death of NB4 cells compared to either drug alone (A), and combined treatment with sorafenib (5 μM) and venetoclax (5 μM) also resulted in increased apoptosis/cell death compared to either drug alone (B). Supplementary Fig. 16. (A) BLI of leukemia growth on day 7 and the histogram. (B) BLI of leukemia growth on day 14 and the histogram.