Figure 2.

Dynamic regulation of Lamin A during erythroid differentiation and impaired terminal erythroid differentiation following CRISPR-Cas9-mediated Lmna disruption. (A) Western blot of Lamin A protein levels following transition of fetal liver erythroid progenitors to differentiation culture. One million cells were loaded per lane. (B) Densitometry quantification of relative changes in Lamin A protein levels following transition of fetal liver erythroid progenitors to differentiation culture. Each data point represents the mean of 3 biological replicates with error bars denoting standard deviation. (C) Surveyor nuclease assay of disruption efficiency of the Lmna gene in fetal liver erythroid progenitor cells following delivery of Cas9 with indicated sgRNAs or Cas9 alone. Arrows denote cleavage products and lanes without indel quantification indicate less than 1% disruption efficiency. (D) Western blot assessing levels of Lamin A protein in fetal liver erythroid progenitor cells following delivery of empty vector, or Cas9 with and without a sgRNA targeting Lmna. (E) Proliferation of fetal liver erythroid progenitors in differentiation medium following delivery of empty vector, or Cas9 with and without Lmna sgRNA. Data points represent the mean of 3 biological replicates and error bars denote standard deviation. P-values comparing Cas9 with Lmna sgRNA to other conditions were calculated using Student’s t-test. (F) Hemoglobin content per reticulocyte generated from fetal liver erythroid progenitors following delivery of empty vector, or Cas9 with and without sgRNA targeting Lmna. Data represent the mean of 3 biological replicates and error bars denote standard deviation. P-value calculated using Student’s t-test.