MiR-34a and miR-152 expression inversely correlate with ATG4B and ATG4D transcript levels in CD34⁺ CML cells, and ATG4B is a target of miR-34a. (A) Quantitative real-time PCR analysis of transcript levels of miR-34a, miR-152, ATG4B, and ATG4D in CD34⁺ NBM isolated from healthy individuals (n = 5) and CD34⁺ cells from CML patients (n = 8). Each data point represents the average of a triplicate quantitative measurement of each transcript normalized to β2M or RNU48. (B) Comparison of transcript levels of the same miRNAs and ATG genes in K562 cells. Each data point represents 4 quantitative measurements of each transcript normalized to β2M or RNU48. Cross bars represent the mean ± SEM of data for each group. (C) Quantitative real-time PCR validation of increased miR-34a and miR-152 expression levels 48 hours after transient transfection of miRNA mimics into K562 cells. (D) Western blot analysis of protein expression of ATG4B and ATG4D in miR-34a and miR-152 transfected K562 cells and scramble controls. Protein expression of ATG4B and ATG4D relative to GAPDH was compared. (E) Schematic representation (top) of ATG4B 3′ UTR and ATG4D 3′ UTR sequences containing potential miR-34a and miR-152 binding sites. The positions of mutations generated are marked in red. Luciferase assay (bottom) of the reporter vector containing ATG4B or ATG4D binding sites and their corresponding mutations 48 hours after transient cotransfection into HEK-293T cells with miRNA mimics. (F) K562 cells were transfected with miR-34a mimic and scramble control and CFC assays were performed in the presence of 0.5 µM IM or DMSO (control). Values represent the mean ± SEM of measurements from 3 independent experiments.