chrm1 tango  (Addgene inc)

Journal: Science advances

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

doi: 10.1126/sciadv.abn4007

Figure Lengend Snippet: Fig. 3. DOPA antagonizes CHRM1. (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 pro- teins 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 M l-DOPA and 6.25 M 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 M l-DOPA and 6.25 M 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 M l-DOPA and 6.25 M carbidopa. n = 3. (G) CHRM1 overexpression (OE) in WM2664 and RPMI-7951 human melanoma (DOPA nonresponders) in the presence or absence of 25 M l-DOPA and 6.25 M 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: CHRM1 overexpression vector was cloned from a codon-optimized plasmid available on Addgene (plasmid no. 66248).

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: Fig. 5. Schematic overview of CHRM1 signaling in melanoma. Proposed mech- anism of oncogenic CHRM1 signaling in melanoma. Red text denotes inhibitors of this pathway used in this manuscript.

Article Snippet: CHRM1 overexpression vector was cloned from a codon-optimized plasmid available on Addgene (plasmid no. 66248).

Techniques: