Journal: Molecular Medicine

Article Title: Activation of dopamine receptor D1 promotes osteogenic differentiation and reduces glucocorticoid-induced bone loss by upregulating the ERK1/2 signaling pathway

doi: 10.1186/s10020-022-00453-0

Figure Lengend Snippet: Dex inhibited the differentiation of osteoblasts and reduced D1R expression in vitro. A CCK-8 analysis of Dex-induced MC3T3-E1 cells. B Representative images showing ALP staining. Scale bar: 200 μm. C Quantitative analysis of ALP staining. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the Dex (−) group. D Representative images showing ARS staining. Scale bar: 200 μm. E Quantitative analysis of ARS staining. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the Dex (−) group. F Representative images of western blots probed with antibodies against the osteogenesis-specific proteins ALP, OSX, and Runx2 and the dopamine receptors D1–D5. G – N Quantification of the protein levels. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the Dex (−) group

Article Snippet: The cells were stained with primary antibodies against D1R (Abcam, ab20066, 1:1000) at 4 °C overnight, washed and incubated with secondary Alexa Fluor 555 antibodies (ab150078, Abcam 1:1000) and Molecular Probes Alexa Fluor 488 phalloidin (Cell Signaling Technology, Danvers, USA) for 1 h in the dark.

Techniques: Expressing, In Vitro, CCK-8 Assay, Staining, Western Blot

Journal: Molecular Medicine

Article Title: Activation of dopamine receptor D1 promotes osteogenic differentiation and reduces glucocorticoid-induced bone loss by upregulating the ERK1/2 signaling pathway

doi: 10.1186/s10020-022-00453-0

Figure Lengend Snippet: Dex inhibited the differentiation of osteoblasts and reduced D1R expression in vitro. A Representative images of western blots probed with antibodies against ALP, OSX, Runx2, and D1R. B – E Quantification of ALP, OSX, Runx2, and D1R protein levels. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the Dex (−) group at the same time point. F Representative images showing immunofluorescence staining of D1R. Scale bar: 50 μm

Article Snippet: The cells were stained with primary antibodies against D1R (Abcam, ab20066, 1:1000) at 4 °C overnight, washed and incubated with secondary Alexa Fluor 555 antibodies (ab150078, Abcam 1:1000) and Molecular Probes Alexa Fluor 488 phalloidin (Cell Signaling Technology, Danvers, USA) for 1 h in the dark.

Techniques: Expressing, In Vitro, Western Blot, Immunofluorescence, Staining

Journal: Molecular Medicine

Article Title: Activation of dopamine receptor D1 promotes osteogenic differentiation and reduces glucocorticoid-induced bone loss by upregulating the ERK1/2 signaling pathway

doi: 10.1186/s10020-022-00453-0

Figure Lengend Snippet: Activation of D1R alleviated Dex-induced inhibition of osteoblast differentiation in vitro. A Representative images showing ALP and ARS staining. Scale bar: 200 μm. B and C Quantitative analysis of ALP and ARS staining. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the control group. D Representative images of western blots probed with antibodies against ALP, OSX and Runx2. E – G Quantification of ALP, OSX and Runx2 protein levels. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the control group. H Representative images of western blots probed with antibodies against ALP, OSX and Runx2 in the overexpression experiment. I – K Quantification of ALP, OSX and Runx2 protein levels. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the control group

Article Snippet: The cells were stained with primary antibodies against D1R (Abcam, ab20066, 1:1000) at 4 °C overnight, washed and incubated with secondary Alexa Fluor 555 antibodies (ab150078, Abcam 1:1000) and Molecular Probes Alexa Fluor 488 phalloidin (Cell Signaling Technology, Danvers, USA) for 1 h in the dark.

Techniques: Activation Assay, Inhibition, In Vitro, Staining, Western Blot, Over Expression

Journal: Molecular Medicine

Article Title: Activation of dopamine receptor D1 promotes osteogenic differentiation and reduces glucocorticoid-induced bone loss by upregulating the ERK1/2 signaling pathway

doi: 10.1186/s10020-022-00453-0

Figure Lengend Snippet: The ERK1/2 pathway was involved in the protective effect of D1R against Dex-induced osteoblasts in vitro. A Representative images of western blots probed with antibodies against the MAPK signaling pathway proteins p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, and p38 after 30 min’ Dex treatment. B – D The ratios of p-ERK1/2/ERK1/2, p-JNK/JNK, and p-p38/p38. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the control group. E Representative images of western blots probed with antibodies against the MAPK signaling pathway proteins p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, and p38 in the overexpression experiment. F – H The ratios of p-ERK1/2/ERK1/2, p-JNK/JNK, and p-p38/p38. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the control group

Article Snippet: The cells were stained with primary antibodies against D1R (Abcam, ab20066, 1:1000) at 4 °C overnight, washed and incubated with secondary Alexa Fluor 555 antibodies (ab150078, Abcam 1:1000) and Molecular Probes Alexa Fluor 488 phalloidin (Cell Signaling Technology, Danvers, USA) for 1 h in the dark.

Techniques: In Vitro, Western Blot, Over Expression

Journal: Molecular Medicine

Article Title: Activation of dopamine receptor D1 promotes osteogenic differentiation and reduces glucocorticoid-induced bone loss by upregulating the ERK1/2 signaling pathway

doi: 10.1186/s10020-022-00453-0

Figure Lengend Snippet: The ERK1/2 pathway was involved in the protective effect of D1R against Dex-mediated inhibition of osteoblast differentiation. A Representative images showing ALP and ARS staining. Scale bar: 200 μm. B and C Quantitative analysis of ALP and ARS staining. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the control group. D Representative images of western blots probed with antibodies against ALP, OSX and Runx2. E – G Quantification of ALP, OSX and Runx2 protein levels. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the control group. H Representative images of western blots probed with antibodies against ALP, OSX and Runx2. I – K Quantification of ALP, OSX and Runx2 protein levels in the overexpression experiment. n = 3 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the control group

Article Snippet: The cells were stained with primary antibodies against D1R (Abcam, ab20066, 1:1000) at 4 °C overnight, washed and incubated with secondary Alexa Fluor 555 antibodies (ab150078, Abcam 1:1000) and Molecular Probes Alexa Fluor 488 phalloidin (Cell Signaling Technology, Danvers, USA) for 1 h in the dark.

Techniques: Inhibition, Staining, Western Blot, Over Expression

Journal: Molecular Medicine

Article Title: Activation of dopamine receptor D1 promotes osteogenic differentiation and reduces glucocorticoid-induced bone loss by upregulating the ERK1/2 signaling pathway

doi: 10.1186/s10020-022-00453-0

Figure Lengend Snippet: Activation of D1R alleviated Dex-induced osteoporosis in vivo. A Representative 3D reconstructions of μCT images and B representative paraffinized sections stained with H&E. C BMD within the region of interest (ROI) was calculated by μCT. D BV/TV E BS/BV, F BS/TV, G Tb. Th, H Tb. N. and I Conn.Dn. J and K BV/TV and BS within the ROI were analyzed by H&E staining. n = 5 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the vehicle group, # p < 0.05, ## p < 0.01, vs. the control group

Article Snippet: The cells were stained with primary antibodies against D1R (Abcam, ab20066, 1:1000) at 4 °C overnight, washed and incubated with secondary Alexa Fluor 555 antibodies (ab150078, Abcam 1:1000) and Molecular Probes Alexa Fluor 488 phalloidin (Cell Signaling Technology, Danvers, USA) for 1 h in the dark.

Techniques: Activation Assay, In Vivo, Staining

Journal: Molecular Medicine

Article Title: Activation of dopamine receptor D1 promotes osteogenic differentiation and reduces glucocorticoid-induced bone loss by upregulating the ERK1/2 signaling pathway

doi: 10.1186/s10020-022-00453-0

Figure Lengend Snippet: Activation of D1R promoted bone formation in vivo. A Representative images of toluidine blue staining. B and C Representative images showing IHC staining of Runx2 and ALP. D Quantitative analysis of the area of newly formed bone calculated by toluidine blue staining. E and F Quantitative analysis of the number of positive cells stained with Runx2 and ALP were analyzed by IHC staining, n = 5 per group. NS: Not statistically significant, * p < 0.05, ** p < 0.01, vs. the vehicle group, # p < 0.05, ## p < 0.01, vs. the control group

Article Snippet: The cells were stained with primary antibodies against D1R (Abcam, ab20066, 1:1000) at 4 °C overnight, washed and incubated with secondary Alexa Fluor 555 antibodies (ab150078, Abcam 1:1000) and Molecular Probes Alexa Fluor 488 phalloidin (Cell Signaling Technology, Danvers, USA) for 1 h in the dark.

Techniques: Activation Assay, In Vivo, Staining, Immunohistochemistry

Journal: PLoS ONE

Article Title: Dopamine Receptors in Human Adipocytes: Expression and Functions

doi: 10.1371/journal.pone.0025537

Figure Lengend Snippet: A , comparison of DAR expression in human pituitary (P), striatum (S) and sc adipose tissue (A), as determined by conventional RT-PCR. B , expression of selected DAR in the stromo-vascular cell (SVC) fraction and mature adipocytes, as determined by real-time PCR. Data are expressed as relative changes over SVC. Each value is a mean±SEM of 3 determinations; *, p<0.05. C , Immunoblot of selected DAR proteins in tissues and cells. Lanes: 1, pituitary; 2, proliferating primary preadipocytes; 3, proliferating LS14; 4, proliferating SW872; 5, differentiated primary adipocytes; 6, differentiated LS14; 7, differentiated SW872. Each lane was loaded with 40 µg proteins, except for the pituitary (30 µg proteins). β-actin (β-Act) was used as a loading control. Expression of D1R, D2R and D4R ( panel D ), and PRL vs PRLR ( panel E ) during adipogenesis in LS14 cells was determined by qPCR. Data are expressed as relative changes over day 0, and were calculated from the cycle threshold and efficiency measurements (Mean±SEM of 3 determinations).

Article Snippet: For DAR, validated antibodies – against D1R (Calbiochem, San Diego, CA: 324390; 1∶2,000), D2R (Santa Cruz, CA: SC-5303; 1∶250), and D4R (Calbiochem: 324405; 1∶1000) were used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Dopamine Receptors in Human Adipocytes: Expression and Functions

doi: 10.1371/journal.pone.0025537

Figure Lengend Snippet: Sc explants ( panel A ), isolated mature adipocytes ( Panel B ) or differentiated primary adipocytes ( panel C ) were incubated with 1 or 100 nM DA or with 10 nM of SKF38393, a D1R/D5R agonist, for 24 hr. Leptin concentration in CM was determined by ELISA. Each value is a mean±SEM of 5 determinations; *, p<0.05. D , differentiated primary adipocytes were incubated as above and adiponectin release was determined by ELISA. E , proliferating primary adipocytes were incubated with 1 or 100 nM DA or with 10 nM of SKF for 6 hr and IL-6 release was determined by ELISA.

Article Snippet: For DAR, validated antibodies – against D1R (Calbiochem, San Diego, CA: 324390; 1∶2,000), D2R (Santa Cruz, CA: SC-5303; 1∶250), and D4R (Calbiochem: 324405; 1∶1000) were used.

Techniques: Isolation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Dopamine Receptors in Human Adipocytes: Expression and Functions

doi: 10.1371/journal.pone.0025537

Figure Lengend Snippet: DA can reach the adipocytes from infiltrating lymphocyte/macrophages, sympathetic nerve endings or via the circulation in the form of DA-S. DA-S can become de-conjugated to bioactive DA by ARSA which is secreted from the lysosomes. DA binds to either D2R-like or D1R-like membrane receptors. Activation of D2R causes suppression of cAMP and inhibition of PRL gene expression and release. Activation of D1R-like receptors results in the inhibition of leptin release and stimulation of adiponectin and IL-6, an effect which may be mediated via the cGMP or MAPK signaling. Other catecholamines (i.e. NE and Epi) from the circulation or sympathetic neurons activate β-AR and modulate, by yet an unknown fashion, adipokine release. See text for additional explanations. ARSA, arylsulfatase A; β-AR, β-adrenergic receptors; D2R/D1R, type 1 or type 2 dopamine receptors; DA, dopamine; DA-S, dopamine sulfate; Epi, epinephrine; NE, norepinephrine; MAPK, mitogen-activated protein kinase; PRL, prolactin.

Article Snippet: For DAR, validated antibodies – against D1R (Calbiochem, San Diego, CA: 324390; 1∶2,000), D2R (Santa Cruz, CA: SC-5303; 1∶250), and D4R (Calbiochem: 324405; 1∶1000) were used.

Techniques: Activation Assay, Inhibition, Expressing