PBX1-dependent myeloma cell proliferation. (A) mRNA expression of PBX1 in 4 MM cell lines. (B) Immunohistochemical (IHC) analysis of trephine bone marrow biopsy samples from 11 patients with MM detects no (neg), medium (1), or high (2) PBX1 expression at the clonal or subclonal level (percentage of PBX1⁺ cells). (C) Time course flow cytometry–based analysis of MM.1S (left panel) and U226 (right panel) myeloma cell viability in vitro, upon lentiviral transduction with scrbl and anti-PBX1 shRNAs (P11, P31). Data collected from 3 biological replicates represent the fraction of GFP⁺ live cells at the time points shown, after normalization against day 3 (d3). Statistical analysis was performed using 2-way analysis of variance (ANOVA) with a post hoc multiple-comparisons test. Error bars represent standard error of the mean (n = 3). Knockdown of PBX1 in MM.1S cells using an in vivo plasmacytoma xenograft mouse model; tumor size (D) and tumor weight (E) at the termination day 32. Statistical analysis was performed using the Kruskal-Wallis test with Dunn’s post hoc multiple-comparisons test. (F) Relative fraction of transduced cells detected at the start (day 0 [D0], Live/GFP⁺ cells) and termination (D32, Live/HLA⁺GFP⁺ cells) day. (G) RNA-seq analysis of PBX1-depleted MM.1S and U266 cells 3 days after lentiviral transduction. Heat maps indicate differentially expressed genes shared between P11- and P31-depleted cells for each cell line. (H) Gene set enrichment analysis (GSEA) of upregulated (UP) or downregulated (DOWN) genes in MM.1S and U266 myeloma cells illustrates significantly enriched molecular pathways in each cell line. Enrichment plots for the prominent cell cycle regulation pathway (E2F targets), which was identified as a top hit, are also presented. (I) Flow cytometric cell cycle analysis of MM.1S and U266 cells 6 days after PBX1 knockdown. Data represent the summary of 3 biological experiments. Analysis was done using parametric 1-way ANOVA with a post hoc multiple-comparisons test. *P < .05; **P < .01; ***P < .001; ****P < .0001.