ShRNA-mediated silencing of SQSTM1 phenocopies miRNA overexpression. (A) Replating assay of mouse lin⁻ BM progenitors infected with 2 different hairpin vectors against SQSTM1, pSM2C-SH2024 and pSM2C-SH-2219, or empty vector control virus. Cells were plated in triplicate at densities of 1 × 10⁴ cells per dish in 1 mL methylcellulose medium containing IL-6 (10 ng/mL), IL-3 (supernatant 1/1000), SCF (10 ng/mL), CSF2 (10 ng/mL). Cells were grown for a week, isolated and replated in a secondary CFU-assay (10⁴ cells per plate). Colonies from the second plating were counted after 7 days of growth. Micrographs show size of CFUs on day 7 after plating. Black bar indicates 100 μm, red bars indicate 200 μm. Significance was calculated by comparing the samples with EV control and the different shRNAs with the Mann-Whitney test (asymoptotic significance [2-tailed]). *P < .05. (B) Mouse lineage negative progenitor cells isolated from the BM of C57BL/6 mice were infected with pSM2C-sh-2024 and pSM2C-sh-2219, empty vector control virus or with GFP containing control virus. Cells were selected in PURO 1.5 (μg/mL) containing expansion medium for 2 days. Recipient C57BL/6 mice were lethally irradiated (8.5 Gy) and reconstituted with 45% GFP positive cells mixed with shRNA or EV control infected cells. Six weeks after transplantation, mice were killed and BM cells were isolated. Lin⁻ cells were stained for flow cytometry analysis. The fold induction of the percentage of GFP⁻ Lin⁻; Sca-I⁺; c-Kit⁺ cells in the BM of pSM2C-EV (n = 4), sh-2024 (n = 5) and sh-2219 (n = 4) relative to the input are shown. Significance was calculated by comparing the samples of mice transplanted with EV control and the different shRNAs with the Mann-Whitney test (asymptotic significance [2-tailed]). *P < .05.