Genetic inhibition of PI3K catalytic subunit p110δ, but not p110α, normalizes GOF Shp2-induced hypersensitivity to GM-CSF. (A) and (B) Representative spleens from polyI:polyC-treated Shp2^D61Y/+;Mx1Cre− mice (D61Y;Cre−, negative controls), Shp2^D61Y/+;Mx1Cre+ mice (D61Y;Cre+, positive controls), and Shp2^D61Y/+;Pik3ca^flox/flox;Mx1Cre+ (D61Y;Cre+;p110α^Fl/Fl) or Shp2^D61Y/+;Pik3cd^D910A/D910A;Mx1Cre+ (D61Y;Cre+;p110δ^D910A/D910A) mice. (C) Quantification of spleen weight:body weight (n = 6 to 9 mice per group). *P = .02 for D61Y;Cre− vs D61Y;Cre+; **P = .04 for D61Y;Cre+;p110δ^D910A/D910A vs D61Y;Cre+, statistics by unpaired, 2-tailed student’s t test. (D) Bone marrow LDMNCs from 6 to 8 mice per genotype were plated in methylcellulose, and colony-forming unit-GM assays were carried out in duplicate in 3 to 4 independent experiments. Data are represented as percentage maximal colony formation, calculated by dividing the number of colonies at each GM-CSF concentration by the average number of colonies at GM-CSF 10 ng/mL. *P = .0005 for D61Y;Cre+;p110δ^D910A/D910A vs D61Y;Cre+. (E) Bone marrow LDMNCs from 6 to 8 mice per genotype were subjected to [³H]-thymidine incorporation assays in replicates of 6 in 3 to 4 independent experiments. ^P < .0001 for D61Y;Cre+;p110δ^D910A/D910A vs D61Y;Cre+. For colony-forming unit-GM and [³H]-thymidine incorporation assays, data were analyzed using mixed-effects models with random intercept, using GM-CSF concentration as a categorical variable. (F) Immunoblot demonstrating Erk and Akt hyperphosphorylation in D61Y;Cre+ mice compared with D61Y;Cre− mice, without normalization on successful knockout of p110α protein expression in 2 independent D61Y;Cre+;p110α^Fl/Fl mice. (G) Immunoblot demonstrating normalization of Erk and Akt hyperphosphorylation in 2 independent D61Y;Cre+;p110δ^D910A/D910A mice compared with 2 independent D61Y;Cre+ mice. (H) Representative flow cytometry analysis demonstrating increased megakaryocyte erythroid progenitor and decreased granulocyte macrophage progenitor in D61Y;Cre+ mice compared with D61Y;Cre− mice, as described previously.¹⁶  (I) Quantification of phenotypically defined bone marrow myeloid precursor distribution. *P = .02, with the D61Y;Cre+;p110δ^D910A/D910A group being significantly closer to D61Y;Cre− than the D61Y;Cre+;p110α^Fl/Fl group to D61Y;Cre−. Progenitor distribution between genotypes was analyzed by comparing Euclidian distance between mean vectors to quantify the similarity among group mean levels, with the P value determined using the bootstrapping resampling method.