shRNA-mediated PBGD and ALAS2 silencing reduces porphyrin accumulation in UROS-deficient erythroid TF1 cells. WT and CRISPR/Cas9–engineered UROS-deficient (UROS^E4i and UROS^E10i) TF1 cells were transduced with 4 different lentiviral vectors coexpressing EGFP and shRNA-targeted to DsRed (control), ALAS2 (#1-4), or PBGD (#1-4) genes. Cells were transduced at a multiplicity of infection of 5, enabling a high transduction efficiency (>90%) as determined by flow cytometry analysis of EGFP expression (supplemental Figure 2). (A) Real-time semi-quantitative reverse transcription–PCR analysis of ALAS1, ALAS2, and PBGD mRNA expression. The relative gene expression was normalized with the ribosomal protein P0 gene and expressed as a ratio vs untransduced control cells (NT) for each condition. (B) Western blot analysis of ALAS2, PBGD, and GAPDH proteins in TF1-UROS^E4i and TF1-UROS^E10i cells. The signal density was determined by using ImageJ software (National Institutes of Health) to perform semi-quantitative analysis of protein expression. Semi-quantitative results are expressed as a ratio vs GAPDH expression. (C) Analysis of porphyrin accumulation in TF1-UROS^WT and TF1-UROS^E4i or TF1-UROS^E10i cells. Total porphyrins were extracted from cell lysates and quantified by spectrofluorometry using commercially available calibrators. (D) Relationship between porphyrin accumulation and ALAS2 or PBGD expression. The regression curves fitted to the data were determined by using Prism software (GraphPad Software). All the results are mean ± SD of 4 different experiments. *P < .05 vs NT. ND, not detectable; NT, nontransduced.