Fig 2: Increased Rac1 activity in APP mice and its reduction by expression of Rac1-N17. Western blots of active Rac1 assay (A) and summary graphs (B) showing increased level of active form of Rac1 in ventral hippocampus in APP compared to WT mice (WT: n = 8 independent experiments from 4 animals for each group, p = 0.0127, two-tailed paired t-test). AAV virus constructs of control EYFP and Rac1-N17 (C), western blots (D,F), and summary graph (E,G) showing decreased level of active Rac1 (n = 5 independent experiments from 3 animals for each group, p = 0.0335, two-tailed paired t-test) and phosphorylated active PAK1/2/3 (P-Pak) (n = 6 independent experiments from 3 animals for each group, p < 0.001, two-tailed paired t-test) in APP mice injected with Rac1-N17 virus (APP + Rac1-N17) compared to those injected with EYFP virus. Endogenous Rac1 refers to Rac1 protein expressed by the endogenous Rac1 gene, while exogenous Rac1 refers to EGFP-Rac1 fusion protein expressed by EYFP-Rac1-N17 viral infections. *P < 0.05, **P < 0.01, and P < 0.001.

Fig 3: Inhibition of Rac1 affects osteoblasts. (A) The expression of osterix to drive Cre recombinase without deletion of Rac1 (Osx+/+) in newborn calvarial osteoblasts suppresses their differentiation in vitro compared to littermate controls not carrying any gene (CT+/+). Newborn calvarial osteoblasts were isolated and cultured for 2-3 weeks in the mineralizing condition in the presence of vitamin C, β-glycerophosphate, and dexamethasone added fresh with each medium change (3 times/week). At the end, the wells are stained with von Kossa, the media evaluated for alkaline phosphatase, and mRNA from the cells evaluated for osteocalcin mRNA expression adjusted to HPRT. The mice were not exposed to doxycycline at any time pre- or postnatally. n = 19/16, 20/20, and 17/20. (B) Deletion of Rac1 using osterix-driven Cre recombinase (Osx Rac1) leads to diminished mineralization, alkaline phosphatase and osteocalcin/HPRT compared to littermate controls (CT Rac1fl/fl). The mice were not exposed to doxycycline at any time pre- or postnatally. n = 12/19, 32/32, and 3/5. (C) Using a chemical inhibitor of Rac1 (EHT1864) added with each medium change at a final concentration of 5 μM suppressed differentiation of newborn calvarial osteoblasts as evidenced by suppressed nodule formation in cultures stained with von Kossa, suppressed alkaline phosphatase in the conditioned media and lower levels of osteocalcin/HPRT mRNA expression compared to control cells treated with phosphate-buffered saline (PBS) containing dimethylsulfoxide (DMSO) at the same concentration as the inhibitor. n = 10/15, 19/19, 12/12. The values were compared between every two groups in all subpanels using t-tests.

Fig 4: Mechanism by which pleckstrin-2 supports proliferation, decreases apoptosis, and enhances enucleation in ineffective erythropoiesis.a The normal balance of differentiation and proliferation enables effective erythropoiesis in WT mouse bone marrow. In β-thalassemic bone marrows, ineffective erythropoiesis results from an imbalance, with relatively more proliferating erythroblasts that differentiate and enucleate to form mature RBCs. When ROS are increased in β-thalassemic erythroblasts, elevated Epo enables multiple downstream mechanisms. Plek2 serves as a mechanism downstream of Epo by which more β-thalassemic erythroblasts evade apoptosis and increase erythroblast enucleation (green line). In thal plek2+/− bone marrow, more erythroblasts undergo apoptosis (red X) and fewer enucleate (green line), resulting in worsening anemia compared with β-thalassemic mice. b When membrane-associated pleckstrin-2 is increased, it’s binding to cofilin and consequent prevention of cofilin translocation to the mitochondria results in decreased apoptosis despite elevation of ROS in β-thalassemic erythroblasts. In addition, interaction with and activation of Rac1 results in enhanced enucleation in high-pleckstrin-2 states. Thus, when membrane-associated pleckstrin-2 is decreased in the setting of increased erythroblast ROS, relatively more cofilin translocation to the mitochondria leads to apoptosis and decreased Rac1 activation results in relatively decreased enucleation, leading to worsening ineffective erythropoiesis in β-thalassemia. WT = wild type; thal = β-thalassemia; plek2 = pleckstrin-2; ROS = reactive oxygen species.

Fig 5: WNT4 knockdown decreased cell proliferation and RAC1 protein but not pJNK in early pTECs. Thymoma-derived pTECs and thymic carcinoma 1889c cell line were either transfected using pU6WNT4-shRNA or stimulated with either 200 ng/ml recombinant WNT4 or with the CM 25% (v:v) (Wnt4 Sup). Proteins of RAC1, pJNK, IKKα/β, and pAKT were extracted from the cells and analyzed by western blot after transfection of 48 h or after four days of stimulation. (A): Cell proliferation analysis in AB and B3 thymoma derived pTECs (n = 6) after transfection for 48 h with pU6-WNT4-shRNA. Cell proliferation was measured using MTT assay; (B): Protein analysis of RAC1, pJNK, IKKα/β, pAKT, in pTECs derived from 2 cases of AB thymomas; (C): Protein analysis of RAC1, pJNK, IKKα/β, pAKT, in pTECs derived from two cases of B3 thymomas; (D): Protein analysis of RAC1, pJNK, IKKα/β, pAKT in thymic carcinoma cell line, 1889c. GAPDH is used as loading control for cytosolic protein fractions.