Identification of SQSTM1 as a target for AAAGUGC seed-containing miRNAs. The myeloid cell line 32D was infected with miR-93 or empty vector (EV) control viruses. The EV control cells were grown in RPMI media containing ¹³C-labeled Lys and Arg (heavy) and 32D–miR-93 cells in medium containing regular Lys and Arg (light). Cells were counted and miR-93 containing cells were either mixed with EV control cells in a 1:1 ratio for proteomics analysis or subjected for mRNA isolation. (A) Samples were prepared and analyzed by quantitative proteomics. The ratios of protein expression in steady state (ss) conditions and at 4 days of CSF3 treatment are plotted. The correlation of the average log (L/H) values (n = 2 independent measurements) of a biologic duplicate (rep1 = x-axis and rep2 = y-axis) of all the identified proteins are indicated by the black dots. Single identifications from one experiment are indicated by the green dots. SQSTM1 is indicated by the red arrow and dot. (B) The average SQSTM1 protein levels (n = 2 independent measurements) of a biologic duplicate and the average mRNA abundances relative to GAPDH and EV control cells in steady state (ss) conditions and at 4 days of CSF3 treatment are shown (n = 3). Error bars represent SD (mRNA). Significance was calculated by comparing the EV control with the steady state and CSF3 condition with the Mann-Whitney test (asymptotic significance [2-tailed]). *P < .05. (C) Luciferase reporter plasmids containing the full-length 3′UTR sequence of Sqstm1 with a wt or mutated miRNA binding site were generated and transfected with the Firefly control vector into HEK293 cells. Two days after transfection, cells were lysed and assayed for Luciferase activity. Luciferase values were normalized against Firefly activity. Normalized values for wt 3′UTR were put to 100%. The Luciferase activity values of the mutant 3′UTR relative to the wt is shown. Error bars represent SD of 3 experiments. (D) The Luciferase reporter plasmids containing the full-length 3′UTR sequence of Sqstm1 were cotransfected with indicated MSCV-miRs. The Luciferase activity values of the miRNA expressing cells relative to the Empty vector control are shown. Error bars represent SD of 3 experiments. (E) The Luciferase reporter plasmids containing the mutant 3′UTR sequence of Sqstm1 were cotransfected with indicated MSCV-miRs. (F) 32D-CSF3R cells were switched from IL-3 to CSF3-containing medium on t = 0 days (steady state, SS). Total RNA was isolated at indicated time points. Sqstm1 levels were measured by qPCR in triplicate. Sqstm1 expression relative to Gapdh and to SS condition is shown. (G) Western blot analysis of normal karyotype AML samples exhibiting low and high miR-106 with monoclonal anti-SQSTM1 antibodies. Actin was stained for loading control.