Journal: iScience

Article Title: Understanding cancer drug resistance with Sleeping Beauty functional genomic screens: Application to MAPK inhibition in cutaneous melanoma

doi: 10.1016/j.isci.2023.107805

Figure Lengend Snippet: Performance of candidates (A) Schematic of potential mechanisms by which candidates drive drug resistance. (B) Representative western blot with indicated antibodies in a panel of melanoma cell lines engineered to overexpress the screen candidates, empty vector (EM), VAV1, PDGFRB, or CSF1R. (C) Dose-response of modified cell lines against ENCOR and COBI. Top row shows ENCOR data and bottom row shows COBI data. Each data point represents three replicate measurements. Viability is shown after three days of drug treatment for A375 and 451Lu and four days for SKMEL28 and PDX10. The error bars denote the 95% confidence interval. (D) Alternate visualization of data in (C) showing viability of indicated cell lines relative to VEH for 21 nM of ENCOR or COBI. Asterix indicates two-tailed Welch’s t-test p value of <0.05. The error bars denote the 95% confidence interval. (E) Viability of indicated cell lines relative to day zero after three days for A375 and 451Lu and four days for SKMEL28 and PDX10. Conditions included either treatment with VEH or ENCOR. Asterix indicates two-tailed Welch’s t-test p value of <0.05. The error bars denote the 95% confidence interval. (F) Representative western blot for indicated antibodies in A375 modified with enforced expression of empty vector (EM), CSF1R L301S, Y969 (CSF1R∗), and PDGFRB N666K (PDGFRB∗). Cells were treated either VEH or 300nm of ENCOR for 6 h. Right panel shows quantification of three technical replicate blots. Asterix indicates two-tailed Welch’s t-test p value of <0.05. The error bars denote the 95% confidence interval. (G) Dose-response curves of A375 modified with EM, CSF1R∗, and PDGFRB∗ against ENCOR. Right panel is alternate visualization that displays data from dose-response curves for 21 nM of ENCOR. Asterix indicates two-tailed Welch’s t-test p value of <0.05. The error bars denote the 95% confidence interval.

Article Snippet: The antibodies we used were alpha-tubulin (12G10 UIOWA hybridoma bank), ARAF (CST 4432), VAV1 (Sigma AB-174), PDGFRB (CST 28E1), CSF1R (CST D3O9X), ERK1/2 (CST 9102), p-ERK1/2 (CST 9101), CAV1 (CST 3267S), and AXL (CST C89E7).

Techniques: Western Blot, Plasmid Preparation, Modification, Two Tailed Test, Expressing

Journal: iScience

Article Title: Understanding cancer drug resistance with Sleeping Beauty functional genomic screens: Application to MAPK inhibition in cutaneous melanoma

doi: 10.1016/j.isci.2023.107805

Figure Lengend Snippet: VAV1 drives phenotype switching by engaging a RAC1-like transcriptional program (A) Volcano plot of differentially expressed genes induced by enforced expression of VAV1 (left) or RAC1 P29S (right) in A375. Solid triangles are genes that belong to the Tsoi Un-differentiated gene set. Top right of each volcano plot shows GSEA enrichment of the Tsoi-Undifferentiated gene set in the entire list of genes. (B) GSEA normalized enrichment scores of MSigDB Hallmark pathways in genes differentially expressed by enforced expression of VAV1 or RAC1 P29S in A375. (C) Venn diagram showing the overlap of genes differentially expressed by the enforced expression of VAV1 or RAC1 P29S in A375. Only genes with an adjusted p value of <0.01 and abs(log 2 FC) > 2 are included. (D) Enrichment of gene set upregulated by enforced expression of VAV1 in genes differentially expressed upon saracatinib treatment. (E) Violin plots of scaled expression values of genes in Tsoi-Undifferentiated gene set in A375 with empty vector and treated with vehicle (E + VEH), A375 with enforced VAV1 expression and treated with vehicle (VAV1+VEH), and A375 with enforced expression of VAV1 treated with saracatinib (VAV1+SRCi). (F) Top five similar LINCs L1000 chemical perturbation consensus signatures identified by Enrichr for genes overexpressed with VAV1 and RAC1 P29S and genes in the Tsoi-Undifferentiated and Verfaillie-TEADs gene sets. Overexpression is defined as adjusted p value of <0.01 and log 2 FC > 2. OE stands for overexpression. (G) Violin plot of normalized expression of indicated genes in de-differentiated and differentiated melanomas in the TCGA SKCM dataset (n = 472, de-differentiated = 85). P-value was computed using Welch’s t-test. Significance was determined using a p value cutoff of 0.05. The edges of the boxes denote the 1st and 3rd quartiles, and the line denotes the 2nd quartile. (H) log 2 FC and adjusted p value of indicated genes elicited by overexpression of VAV1 or RAC1 P29S .

Article Snippet: The antibodies we used were alpha-tubulin (12G10 UIOWA hybridoma bank), ARAF (CST 4432), VAV1 (Sigma AB-174), PDGFRB (CST 28E1), CSF1R (CST D3O9X), ERK1/2 (CST 9102), p-ERK1/2 (CST 9101), CAV1 (CST 3267S), and AXL (CST C89E7).

Techniques: Expressing, Plasmid Preparation, Over Expression

Journal: iScience

Article Title: Understanding cancer drug resistance with Sleeping Beauty functional genomic screens: Application to MAPK inhibition in cutaneous melanoma

doi: 10.1016/j.isci.2023.107805

Figure Lengend Snippet:

Article Snippet: The antibodies we used were alpha-tubulin (12G10 UIOWA hybridoma bank), ARAF (CST 4432), VAV1 (Sigma AB-174), PDGFRB (CST 28E1), CSF1R (CST D3O9X), ERK1/2 (CST 9102), p-ERK1/2 (CST 9101), CAV1 (CST 3267S), and AXL (CST C89E7).

Techniques: Recombinant, Transfection, RNA Extraction, Plasmid Preparation, Software

Journal: iScience

Article Title: Understanding cancer drug resistance with Sleeping Beauty functional genomic screens: Application to MAPK inhibition in cutaneous melanoma

doi: 10.1016/j.isci.2023.107805

Figure Lengend Snippet:

Article Snippet: The antibodies we used were alpha-tubulin (12G10 UIOWA hybridoma bank), ARAF (CST 4432), VAV1 (Sigma AB-174), PDGFRB (CST 28E1), CSF1R (CST D3O9X), ERK1/2 (CST 9102), p-ERK1/2 (CST 9101), CAV1 (CST 3267S), and AXL (CST C89E7).

Techniques: Recombinant, Transfection, RNA Extraction, Plasmid Preparation, Software