Fig. 4: CALCRL controls cell cycle and DNA repair pathways through E2F1.
From: Adrenomedullin-CALCRL axis controls relapse-initiating drug tolerant acute myeloid leukemia cells
a Volcano plot of most differentially expressed (278 upregulated and 623 downregulated, FDR < 0.05 and p-value < 0.05) genes identified in transcriptomes of MOLM-14 shCAL#1 and #2 vs shCTR. The FDR values based on −log10 were plotted against the log2 ratio of gene expression level for all genes. b, c Genomatix analysis of cell cycle (b) or DNA integrity (c) pathways negatively affected by CALCRL depletion. d Western blot results showing expression of RAD51, CHK1, BCL2, and β-ACTIN proteins in MOLM-14 and OCI-AML3 four days after transduction with indicated shRNA. e Representative FACS graphs of cell cycle upon shCAL. f Cell cycle distribution (n = 3 independent experiments, two-way ANOVA). g Genomatix analysis of co-cited transcription factors negatively affected by CALCRL depletion. h E2F1 activity is followed by flow cytometry through the level of mKate2 fluorescence intensity (n = 5 independent experiments, unpaired t-test). i Western blot results showing expression of Cyclin A, B1, D1, E, RB, and p-RB. j Western blot results showing expression of E2F1, RAD51, CHK1, BCL2, and β-ACTIN proteins in MOLM-14 and OCI-AML3 four days after transduction with indicated shRNA. k Graph shows cell proliferation of MOLM-14 or OCI-AML3. Three days after transduction, cells were plated at 0.3 M cells/ml (D0) and cell proliferation was followed using trypan blue exclusion (n = 6 independent experiments, two-way ANOVA). l Representative FACS graphs of cell cycle upon shE2F1. m Cell cycle distribution (n = 5 independent experiments for MOLM-14 and n = 6 independent experiments for OCI-AML3, two-way ANOVA). n Graph shows the percentage of Annexin-V + or 7-AAD + cells 4 days after cell transduction (n = 6 independent experiments for MOLM-14 and n = 7 independent experiments for OCI-AML3, unpaired t-test). o Correlation between clonogenic capacities of primary AML samples (n = 11) and CALCRL protein expression assessed by western blot analysis (Supplementary Fig. 1m). Linear regression was performed to determine R2 and p-value. p Colonies in methylcellulose were counted 1 week after transfection of primary AML samples with siCTR or siCAL (unpaired t-test). q Western blot results showing expression of RAD51, BCL2, CALCRL, and β-ACTIN proteins in primary AML samples 7 days after transfection with indicated siRNA. Data are mean ± s.e.m.
