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. 2016 Jun 30;7(1):95–109. doi: 10.1016/j.stemcr.2016.05.015

Figure 4.

Figure 4

Loss-of-Function and Gain-of-Function Studies of the Dopamine D2 Receptor DRD2 in MIN6 Cells

The effects of DPD and dopamine (DA) in control (A, B, C, G) and Drd2-knockdown (D, E, F) or Drd2-overexpressing (H, I) MIN6 cells.

(A) The number of MIN6 cells per well after 4 days of chemical treatment (days 3–7). Dopamine treatment decreased the number of cells, whereas DPD or cAMP treatment increased the number of MIN6 cells (five independent experiments, each with three to six replicates).

(B) DPD increased proliferation of MIN6 cells revealed by EdU incorporation (48 hr) assay (four independent experiments, each with four replicates).

(C) Relative cAMP levels after 6 hr of chemical treatment on day 3. DPD increased intracellular cAMP levels, whereas dopamine decreased intracellular cAMP levels, and DPD reversed the dopamine-mediated decrease.

(D–F) Drd2 knockdown increased cAMP levels and phenocopied DPD effects. (D) Upper panel: a schematic of the lentiviral vector used for Drd2 shRNA-mRFP expression. Lower panel: fluorescence and transmission images of the Drd2-knockdown MIN6 cells (D2KD). (E) D2KD cells showed a partial decrease in Drd2 expression (two independent experiments, each with three replicates). (F) Responses of D2KD cells to dopamine chemicals. DPD increased cell number for 9 days culture, but not in D2KD cells. The effect of dopamine decreased in knockdown cells (four independent experiments, each with four replicates).

(G–I) Dopamine treatment of wild-type (G) or Drd2-overexpressing (I) MIN6 cells on days 3–7. Dopamine dose-dependently increased the number of apoptotic cells (G) (three independent experiments, each with four replicates). Upper panel in (H) shows a schematic of the FLAG-tagged Drd2-expressing vector, and lower panel shows RT-PCR analysis of the exogenously overexpressed Drd2 in MIN6 cells. MIN6 cells overexpressing Drd2 (D2-OE) showed increased sensitivity to dopamine. Up to 37% of the cells became apoptotic after treatment with 10 μM dopamine (I) (three independent experiments, each with three replicates). ∗∗p < 0.01, ∗p < 0.05 compared with control DMSO-treated cells. Data represent mean ± SD. Student's t test. Scale bar, 100 μm.