Journal: Cancer Research

Article Title: AP-2α–Mediated Activation of E2F and EZH2 Drives Melanoma Metastasis

doi: 10.1158/0008-5472.can-21-0772

Figure Lengend Snippet: Figure 2. Loss of AP-2a represses E2F signaling. RNA-seq reveals a gene signature shared among four TFAP2A knockouts and an shRNA-mediated knockdown that is consistent with inhibition of E2F signaling. A, Heatmap illustrating all consistent and statistically significant (per Cuffdiff) differential expression; selected genes within the E2F pathways are highlighted. Differential expression with knockout of TFAP2A was calculated relative to the parental cell line and differential expression with shTFAP2A (denoted shRNA) was calculated relative to a nontargeting (shNT) control. B, qRT-PCRs showing loss of TFAP2A represses E2F1, E2F2, and E2F8 in four TFAP2A knockout clones (TFAP2AKO1–4 relative to the parental cell line, dashed line) and three human (A375, SKMEL28, and M21) and one mouse (TKLP) melanoma cell lines by shRNA (relative to a shNT control; dashed line). Data are represented as mean SEM. , P < 0.01; , P < 0.001; , P < 0.0001 (Student t test). C, Propidium iodide staining showing knockout of TFAP2A disrupts cell-cycle progression. See also Supplementary Fig. S2. D, 10X Genomics scRNA-seq of the A375 cell line shows a high degree of heterogeneity (left) and violin plots show E2F signaling, as epitomized by EZH2 and E2F1, is active in four of six clusters (right). Clustering and violin plots were made with the Seurat package for R.

Article Snippet: Reads from parental A375, A375 shNT, and A375 shTFAP2A scRNA-seq had the 28 base 10X Genomics UMIs/barcodes removed from the start of R1 and the first lowquality base removed from the start of R2 using the HEADCROP operation of Trimmomatic.

Techniques: RNA Sequencing, shRNA, Knockdown, Inhibition, Quantitative Proteomics, Knock-Out, Control, Clone Assay, Staining