A Novel CRISPR/Cas9-Based Cellular Model to Explore Adenylyl Cyclase And

MOL #111849 A Novel CRISPR/Cas9-Based Cellular Model to Explore Adenylyl Cyclase and Cyclic AMP Signaling Monica Soto-Velasquez, Michael P. Hayes, Aktan Alpsoy, Emily C. Dykhuizen, and Val J. Watts Molecular Pharmacology SUPPLEMENTAL FIGURES Supplemental Table 1. Sequence of primers used for RT-qPCR 1 MOL #111849 SUPPLEMENTAL FIGURES A. B. Supplemental Figure 1. Relative mRNA levels of AC isoforms in HEK293, HEK-ACΔ6, and HEK-ACΔ3/6 cell lines determined by RT-qPCR. (A). Agarose gel electrophoresis of RT-qPCR products from total RNA isolated from HEK293 cells. (B). Fold-change in relative gene expression between HEK293 cells and the knockout cell lines. Data represents the mean and SEM of three biological replicates independently run and conducted in duplicate. Statistical analysis was performed using unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared ΔCt values of knockout cell line to the ΔCt values of the HEK293 parental cell line for each target gene. 2 MOL #111849 A. B. HEK-ACΔ6 cell line C. HEK-ACΔ3/6 cell line Supplemental Figure 2. Genotypic characterization of HEK-ACΔ6 and HEK-ACΔ3/6 cell lines Schematic for the generation and characterization of the CRISPR/Cas9-based cell lines (A). Genomic sequencing and predicted protein sequence alignment for the ADCY3 and/or ADCY6 edited genes in the HEK-ACΔ6 (B) and the HEK-ACΔ3/6 (C) cell lines. 3 MOL #111849 The sgRNA target sequences are in bold, and the black triangles indicate the Cas9- mediated cleavage site upstream of the protospacer adjacent motif (PAM) sequence (box). The resulting indels for the ADCY3 and/or ADCY6 genes were determined by sequencing a PCR product that contained the nuclease cleavage site from the genomic DNA of the clonal cell lines. The black arrows show the mutation (deletion/insertion) introduced for each allele of the targeted genes, and the deleted/inserted base pairs are underlined. These indels caused a frame-shift on both ADCY3 and ADCY6 genes that introduced a premature STOP codon as illustrated by the amino acid sequence alignment between wild-type ADCY3 or ADCY6 genes and the predicted protein sequences of the mutated genes. Amino acid sequence for the wild-type genes are in bold and aligned regions with 100% identity are shaded in grey. 4 MOL #111849 Supplemental Figure 3. Ca2+ inhibition of AC5 and AC6 activity. HEK-ACΔ3/6 cells transiently transfected with Venus, AC5, or AC6 were treated for 1- hour with 10 μM forskolin in the absence (black) or presence (white) of the Ca2+ ionophore, A23187, and cAMP accumulation was measured. Data represents the mean and SEM of four to five independent experiments conducted in duplicate. Statistical analysis was performed using paired t-test. *P < 0.05, **P < 0.01 compared to the cAMP levels of the vehicle-treated cells. 5 MOL #111849 Supplemental Figure 4. Ca2+ stimulation of AC1 mutants expressed in HEK293 and HEK-ACΔ3/6 cells. HEK293 and HEK-ACΔ3/6 cells transiently transfected with Venus, AC1-WT, or the AC1 mutant constructs were stimulated with 3 μM A23187 for 1-hour, and cAMP accumulation was measured. Data represents the mean and SEM of three independent experiments conducted in duplicate. 6 .

Load more