BATF3 and JUNB are essential AP-1 factors in ATLL cells. (A) The indicated cell lines were infected with a lentivirus expressing sgBATF3_1 and sgIRF4_1 together with GFP. Shown is the fraction of GFP-positive cells over time relative to the GFP-positive fraction on day 3. (B) Immunoblot analysis of JUNB protein in sgJUNB-transduced ST1. (C) Toxicity assay using sgJUNB_1 was done as in Figure 2A. (D) Immunoblot analysis of JUNB, c-JUN, and JUND proteins in ATLL and MCL cell lines. (E) Immunoblot analysis of JUNB, c-JUN, and JUND proteins in ST1 ATLL cells transduced with indicated cDNA-expressing vectors. (F) ST1 cells were transduced with retroviruses expressing an sgRNA-resistant JUNB, JUN, JUND together with puromycin-resistance gene or with an empty vector expressing puromycin-resistance gene. After puromycin selection of transduced cells, cells were subsequently transduced with lentiviruses coexpressing GFP and either sgJUNB_1 or sgJUNB_2. sgAAVS1 and sgRPL6 were used as a negative and positive control sgRNA, respectively. The GFP-positive cell fraction was monitored as in Figure 2A. (G) Coimmunoprecipitation assay was performed by using anti-BATF3 antibody or anti-JUNB antibody. Immunoprecipitates were analyzed by immunoblot with anti-HBZ, anti-BATF3, anti-JUNB, or anti-IRF4 antibodies. Error bars represent the SEM of replicates (A,C,F).