Knockdown of Ptbp1/PTBP1 causes a metabolic phenotype. (A) Overview of the pLEPIR lentiviral all-in-one vector for tetracycline-inducible expression of shRNAs. (B) Knockdown efficiency after shRNA treatment in murine MLL-ENL–transformed primary HSPCs and human MOLM13 cells. Shown are qPCR results obtained with RNA isolated from cells selected for the presence of LNGFR from shRNA-expressing and vector-only (nontargeting shRNA) populations. (C) Ptbp1/PTBP1 knockdown affected long-term fitness of the cells. The graph depicts the percentage of shRNA⁺ (knockdown) cells, as detected by coupled expression of a truncated LNGFR in FACS analysis. Cultures were induced at day 0. Further explanations are given in the text. A representative of 2 experiments is depicted. (D) Visual aspect of Ptbp1 knockdown. An equal number of selected knockdown (shRNA) and control (vec) cells was cultivated overnight demonstrating reduced medium acidification as a consequence of shRNA activity. This phenotype was observed in murine and human cells; only the murine sample is shown. (E) Glucose and lactate metabolism changed after knockdown of Ptbp1/PTBP1. An equal number of enriched LNGFR⁺ cells from cultures expressing shRNA or vector as controls were seeded, and glucose and lactate concentrations were determined in supernatant medium sampled at the indicated time points.