AP-1 maintains KIT+ erythroid precursors and opposes differentiation. (A) Intracellular flow cytometry for total JUN and phospho(Ser73)-JUN in cells isolated from spleens at up to 72 h post-PHZ. N = 3. (B) Experimental layout for testing JUN function in stress precursors. (C) Western blotting of JUN and actin, beta protein in G1E proerythroblasts infected with Jun-targeting shRNA (shJun1 and shJun2) or shControl. (D) Flow cytometry analysis of the percentage of shRNA-infected (GFP+) cells expressing the KIT surface receptor. (E) Flow cytometry analysis of the percentage of live (DAPI−) cells expressing the KIT cell surface receptor. SR11302 treatment 20 µM for 24 h (see Methods). (F) Flow cytometry analysis of shRNA-infected cells after 3 days culture, stained with the erythroid-specific cell surface marker LY76 (also known as TER-119). (G) Quantitative RT-PCR of Jun mRNA expression in LIN− cells isolated from spleens of PHZ-treated mice and cultured for 3 days, followed by stimulation with BMP4 (50 ng/mL). Statistical significance was determined by two-tailed unpaired Student's t-test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.
