Figure 6.
Dopamine regulates SCF/ERK signaling and Lck expression. (A) Representative line graph showing pERK in unstimulated Drd-DKO and control (Ctrl) LSK cells. (B) Normalized mean fluorescent intensity (nMFI) of ERK pathway protein phosphorylation in steady-state WT (n = 4-8) or Drd-DKO (n = 4-8) LSK cells. The P value was calculated using the Student t test. (C) ERK pathway phosphorylation in LSK cells from lethally irradiated mice at 14 days after transplantation with WT (n = 8) or Drd-DKO (n = 6) donor LSK cells. The P value was calculated using the Student t test. (D) ERK pathway phosphorylation in WT (n = 5-8) or Drd-DKO (n = 5-8) LSK cells with or without SCF treatment. The P values were calculated using analysis of variance (ANOVA). (E) Representative FACS line graphs showing flow cytometric analysis of c-Kit phosphorylation in Drd-DKO LSK cells and their controls. Normalized MFI of pKit in WT (n = 5) and Drd-DKO (n = 5) LSK cells with or without SCF treatment. The P values were calculated using ANOVA. nMFI of pERK (F) and pKit (G) in vehicle-treated (n = 5), SCF-stimulated vehicle-treated (n = 5), or SCF-stimulated 7-OH-DPAT–treated (n = 5) LSK cells. (H) Fragments per kilobase of transcript per million mapped reads (Fpkm) for transcripts encoding SFKs in WT and Drd-DKO LSK cells (n = 3 each). The P value was calculated using the Student t test. (I) Representative flow cytometric line graph of Lck protein in WT and Drd-DKO LSK cells (left panel). nMFI of total Lck under steady-state in WT (n = 4) and Drd-DKO (n = 4) LSK cells (right panel). (J) nMFI of total Lck in vehicle control–treated (n = 4) or 7-OH-DPAT–treated (n = 4) LSK cells. (K) Representative FACS line graph showing flow cytometric analysis of c-Kit phosphorylation in Lck inhibitor A-770041–treated LSK cells and their controls (left panel). nMFI of pKit in SCF-stimulated vehicle-treated (n = 4) or SCF-stimulated A-770041–treated (n = 4) LSK cells (right panel). ERK pathway protein phosphorylation in vehicle control (n = 3-4) or Lck inhibitor A-770041 (n = 3-4) treated LSK cells (L) and after SCF stimulation together with vehicle (n = 3-4) or A-770041 (n = 3-4) (M) The P value was calculated using the Student t test. (N) GFP+ cells (green) from Vav1-Cre Rosa26-mTmG donors and EdU incorporation (red) in sections from vehicle- or A-770041–treated long bone at 3 days after lethal irradiation and transplantation with Lin− cells. (O) Flow cytometric quantification of GFP+ cells and donor-derived CD45+ cells in BM (vehicle = 4; A-770041 = 4; upper panels). Quantification of the number and percentages of EdU+ cells in GFP+ donor cells by imaging (vehicle = 4; A-770041 = 4; lower panels). The P value was calculated using the Student t test.

Dopamine regulates SCF/ERK signaling and Lck expression. (A) Representative line graph showing pERK in unstimulated Drd-DKO and control (Ctrl) LSK cells. (B) Normalized mean fluorescent intensity (nMFI) of ERK pathway protein phosphorylation in steady-state WT (n = 4-8) or Drd-DKO (n = 4-8) LSK cells. The P value was calculated using the Student t test. (C) ERK pathway phosphorylation in LSK cells from lethally irradiated mice at 14 days after transplantation with WT (n = 8) or Drd-DKO (n = 6) donor LSK cells. The P value was calculated using the Student t test. (D) ERK pathway phosphorylation in WT (n = 5-8) or Drd-DKO (n = 5-8) LSK cells with or without SCF treatment. The P values were calculated using analysis of variance (ANOVA). (E) Representative FACS line graphs showing flow cytometric analysis of c-Kit phosphorylation in Drd-DKO LSK cells and their controls. Normalized MFI of pKit in WT (n = 5) and Drd-DKO (n = 5) LSK cells with or without SCF treatment. The P values were calculated using ANOVA. nMFI of pERK (F) and pKit (G) in vehicle-treated (n = 5), SCF-stimulated vehicle-treated (n = 5), or SCF-stimulated 7-OH-DPAT–treated (n = 5) LSK cells. (H) Fragments per kilobase of transcript per million mapped reads (Fpkm) for transcripts encoding SFKs in WT and Drd-DKO LSK cells (n = 3 each). The P value was calculated using the Student t test. (I) Representative flow cytometric line graph of Lck protein in WT and Drd-DKO LSK cells (left panel). nMFI of total Lck under steady-state in WT (n = 4) and Drd-DKO (n = 4) LSK cells (right panel). (J) nMFI of total Lck in vehicle control–treated (n = 4) or 7-OH-DPAT–treated (n = 4) LSK cells. (K) Representative FACS line graph showing flow cytometric analysis of c-Kit phosphorylation in Lck inhibitor A-770041–treated LSK cells and their controls (left panel). nMFI of pKit in SCF-stimulated vehicle-treated (n = 4) or SCF-stimulated A-770041–treated (n = 4) LSK cells (right panel). ERK pathway protein phosphorylation in vehicle control (n = 3-4) or Lck inhibitor A-770041 (n = 3-4) treated LSK cells (L) and after SCF stimulation together with vehicle (n = 3-4) or A-770041 (n = 3-4) (M) The P value was calculated using the Student t test. (N) GFP+ cells (green) from Vav1-Cre Rosa26-mTmG donors and EdU incorporation (red) in sections from vehicle- or A-770041–treated long bone at 3 days after lethal irradiation and transplantation with Lin− cells. (O) Flow cytometric quantification of GFP+ cells and donor-derived CD45+ cells in BM (vehicle = 4; A-770041 = 4; upper panels). Quantification of the number and percentages of EdU+ cells in GFP+ donor cells by imaging (vehicle = 4; A-770041 = 4; lower panels). The P value was calculated using the Student t test.

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