Panel A shows a combination of microscopic images and line graphs. The images depict the morphology of transformed cells at the 6th passage (MC 6) stained with May-Grünwald-Giemsa (MGG). The line graphs display the colony numbers at different replating stages (MC 2, MC 4, MC 6) for various constructs: Vector, Retinoic Acid Receptor Alpha (RARA), Retinoic Acid Receptor Beta (RARB), POZ/RARB, and Promyelocytic Leukemia Protein fused with Retinoic Acid Receptor Alpha (PML fused with RARA). The x-axis represents the replating stages, and the y-axis represents the colony number. The graphs show that RARA and PML fused with RARA significantly increase colony numbers compared to the Vector, RARB, and POZ/RARB. Panel B consists of Western blot images showing the expression of RARA, PML fused with RARA, RARB, and POZ-RARB in Lin minus progenitors at the 2nd passage (MC 2). The left blot shows bands for RARA and PML fused with RARA, while the right blot shows bands for FLAG and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Panel C contains two bar graphs representing the relative luciferase activity in 293T cells transfected with RARA, PML fused with RARA, and their I396E mutants, with and without All-trans Retinoic Acid (ATRA) treatment. The x-axis lists the constructs, and the y-axis shows the relative luciferase activity. The graphs indicate that ATRA treatment significantly increases luciferase activity in cells with RARA and PML fused with RARA compared to the Vector and I396E mutants. Panel D includes images, a line graph, and additional images. The images show the morphology of transformed cells at the 4th passage (MC 4) stained with MGG. The line graph displays colony numbers at different replating stages (MC 2, MC 3, MC 6) for various constructs: Vector, RARA, RARA I396E, PML fused with RARA, and PML fused with RARA I396E. The x-axis represents the replating stages, and the y-axis represents the colony number. The graphs show that RARA and PML fused with RARA significantly increase colony numbers compared to the Vector and their I396E mutants. The additional images show the representative morphology at the 3rd plating (MC 3). Panel E consists of Western blot images showing the expression of RARA, PML fused with RARA, and their I396E mutants in Lin minus progenitors at the 2nd passage (MC 2). The blots show bands for RARA and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Transcriptional repression is critical for RARA and PML::RARA-driven progenitor immortalization. (A) Primary murine Lin− hematopoietic progenitors were retrovirally transduced with RARA, RARB, or their fusions and cultured in methylcellulose with cytokines. MGG staining at the sixth passage (MC VI, upper panel) illustrates the morphology of transformed cells; colony numbers were quantified at the indicated replating (middle panel), and representative colony morphology at the third plating (MC III, lower panel) is shown. Scale bars, 10 μm (upper panel) and 100 μm (lower panel). N = 3. (B) Western blot analysis of RARA, PML::RARA (left), RARB, and POZ-RARB (right) expression in Lin− progenitors at the second passage (MC II). The asterisk indicates an unidentified posttranslational modification of RARA. N = 2. (C) RARE/DR5 luciferase reporter assay in 293T cells transfected with RARA, PML::RARA, their I396E mutants that are deficient for NCoR binding. Cells were treated for 12 h with ATRA (1 μM) or left untreated (Unt). Change fold of ATRA treatment over vector without treatment was indicated on the top of each bar in the ATRA panel. N = 2. (D) MGG staining at the fourth passage (upper panel), colony counts (middle panel) at the indicated replating, and representative morphology at the third plating (lower panel) of primary mouse Lin− hematopoietic progenitors transformed with RARA fusions or their I398E mutants. N = 3. Scale bar, 10 mm (upper panel) and Scale bar, 100 mm (lower panel). (E) Western blot of RARA, PML::RARA, and their I396E mutants from Lin− progenitors at the second passage. N = 2. (A and D) Cells were replated every 5–7 days; MC II, III, IV, and VI denote the second, third, fourth, and sixth methylcellulose replating. (A, C, and D) Data are presented as the mean ± SD of three technical triplicates from one representative experiment out of at least two biologically independent experiments. Statistical significance was assessed by two-way ANOVA (A and D) or two-tailed unpaired t test (C), ***P < 0.001. Source data are available for this figure: SourceData F1.