Journal: Science Advances

Article Title: Endogenous DOPA inhibits melanoma through suppression of CHRM1 signaling

doi: 10.1126/sciadv.abn4007

Figure Lengend Snippet: ( A ) Schematic diagram depicting melanin synthesis. Pharmacologic inhibitors used in this paper are shown in red. ( B ) LC-MS quantitation of DOPA content in LMCs and DMCs. Biologic, n = 3. ( C ) Dose-response curve of l -DOPA in representative LMC and DMC after 4 days of l -DOPA treatment. Technical, n = 5. ( D ) LMCs and DMCs treated with either 25 μM l -DOPA, 75 μM phenylthiourea (PTU), or a combination for 4 days. Image is representative of one biologic replicate of LMC and DMC; technical replicate, n = 5. ** P = 0.0033, *** P = 0.0009, **** P < 0.0001 analyzed via two-way analysis of variance (ANOVA). Control populations relative to themselves for both LMC and DMC. ( E ) Panel of melanoma cell lines treated with vehicle, 25 μM l -DOPA, or the combination 25 μM l -DOPA and 6.25 μM carbidopa. **** P < 0.0001, analyzed via two-way ANOVA. Technical, n = 5. ( F ) YUMM1.7 murine melanoma growth in syngeneic BL/6 mice treated with vehicle or l -DOPA methyl ester (300 mg/kg) and carbidopa (75 mg/kg). ** P = 0.0065. n = 5 for each group. ns, not significant.

Article Snippet: For l -DOPA and carbidopa experiments, l -DOPA methyl ester (300 mg/kg; Tocris, # 0455) and carbidopa (75 mg/kg; Cayman, # 16149) were injected intraperitoneally daily for 3 weeks, then 5 days on and 2 days off for the remainder of the experiment.

Techniques: Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Control

Journal: Science Advances

Article Title: Endogenous DOPA inhibits melanoma through suppression of CHRM1 signaling

doi: 10.1126/sciadv.abn4007

Figure Lengend Snippet: ( A ) DOPA-mediated GPCR activation or inhibition as determined by the PRESTO-Tango reporter assay. Data points are shaded on the basis of relative expression determined using RNA-seq in MCs (FPKM). ( B ) Log fold enrichment of CRISPR gRNAs selected for or against. Controls for protumorigenic proteins included CDK9 and PCNA. GPER1 served as an internal GPCR tumor suppressor control. High-confidence hits are targets with at least five guides that are selected for (>5-fold) or against (<0.1-fold), and where those five guides represent at least 50% of total guides for that gene. ( C ) siRNA-mediated CHRM1 depletion in A375 human melanoma in the presence of 25 μM l -DOPA and 6.25 μM carbidopa after 5 days of treatment. Technical, n = 8. ( D ) qPCR for CHRM1 mRNA in A375 after siRNA treatment confirming knockdown. Time point taken 24 hours after siRNA transfection. Technical, n = 3. ( E ) Effect of 25 μM l -DOPA and 6.25 μM carbidopa on proliferation of A375 cells in which CHRM1 was depleted using CRISPR-Cas9 versus control gRNA against green fluorescent protein (GFP). Cell number was determined at day 5. Technical, n = 8. ( F ) Low CHRM1 expression, determined via qPCR, correlates with lack of response to 25 μM l -DOPA and 6.25 μM carbidopa. n = 3. ( G ) CHRM1 overexpression (OE) in WM2664 and RPMI-7951 human melanoma (DOPA nonresponders) in the presence or absence of 25 μM l -DOPA and 6.25 μM carbidopa after 5 days of treatment. *** P = 0.0002, **** P < 0.0001 analyzed via two-way ANOVA. n = 5. ( H ) Western blot for CHRM1 in WM2664 and RPMI-7951 after transduction with either empty vector or CHRM1.

Article Snippet: For l -DOPA and carbidopa experiments, l -DOPA methyl ester (300 mg/kg; Tocris, # 0455) and carbidopa (75 mg/kg; Cayman, # 16149) were injected intraperitoneally daily for 3 weeks, then 5 days on and 2 days off for the remainder of the experiment.

Techniques: Activation Assay, Inhibition, Reporter Assay, Expressing, RNA Sequencing, CRISPR, Control, Knockdown, Transfection, Over Expression, Western Blot, Transduction, Plasmid Preparation

Journal: Science Advances

Article Title: Endogenous DOPA inhibits melanoma through suppression of CHRM1 signaling

doi: 10.1126/sciadv.abn4007

Figure Lengend Snippet: ( A ) Relative fluorescence intensity (RFU) of diacylglycerol (DAG) sensor in HEK293T upon addition of 5 nM ACh or combination of 100 μM DOPA and 5 nM ACh. Cells treated with combination of 100 μM DOPA and 5 nM ACh were pretreated with 100 μM DOPA 1 hour before plate reading. Drugs injected where arrows are pointed. n = 10. ( B ) FOXM1 mRNA level determined over time via qPCR in A375 human melanoma treated with 25 μM l -DOPA and 6.25 μM carbidopa. * P = 0.0142, ** P = 0.0054, **** P < 0.0001. n = 3. ( C ) Western blot for FOXM1 and c-Myc in lysates from A375 human melanoma cells treated with 25 μM l -DOPA and 6.25 μM carbidopa. ( D ) Western blot of FOXM1 and c-Myc at baseline in light and dark MCs. Biologic, n = 2 for LMC and DMC. ( E ) Proliferation in A375 cells following transduction with FOXM1C versus empty vector ± 25 μM l -DOPA and 6.25 μM carbidopa. **** P < 0.0001. n = 8. ( F ) Western blot confirming FOXM1C overexpression in A375 human melanoma.

Article Snippet: For l -DOPA and carbidopa experiments, l -DOPA methyl ester (300 mg/kg; Tocris, # 0455) and carbidopa (75 mg/kg; Cayman, # 16149) were injected intraperitoneally daily for 3 weeks, then 5 days on and 2 days off for the remainder of the experiment.

Techniques: Fluorescence, Injection, Western Blot, Transduction, Plasmid Preparation, Over Expression