Journal: bioRxiv
Article Title: Uncovering cancer dependencies in peptide-interacting protein pockets
doi: 10.64898/2026.03.13.711608
Figure Lengend Snippet: ( A ) Scheme showing the domains in TLE1-4 proteins, where the WD40 domain binds two types of motifs - EH1 and WRPW. ( B ) Superimposed AlphaFold3 structural models of a WRPW and an EH1 peptide bound to TLE1. ( C ) Sequences and a heatmap of the dropout ranks of WRPW peptides (top) with the TLE1-4 CRISPR knockout effects on proliferation (DepMap scores, bottom). ( D ) Pulldown of RIPPLY2 and HES1 WRPW peptides from HCT116 and NCIH358 cell lysates shows binding to all four TLE1-4 proteins. A representative example of two biological replicates is shown. ( E ) The effect of single and multiplex CRISPR knockout of TLE1/3/4 in NCIH358 cells measured by the relative proliferation rates of different subpopulations co-expressing specific gRNAs and fluorescent reporters. The proliferation rates were calculated from the change in subpopulation ratios over two cell doublings. Mean±standard deviation from three biological replicates. ( F ) The impact of TLE1-T2A-mCherry overexpression on the subpopulation proliferation rate of cells expressing RIPPLY2 and HES1 WRPW GFP fusions in a flow-cytometry-based competitive growth experiment. Plot shows changes in GFP positive cell subpopulation in 5 doublings. ( G ) The impact of RIPPLY2 and HES1 WRPW peptide overexpression in NCIH358 cells investigated by RNAseq. ( H ) Numbers of unique and overlapping upregulated genes in NCIH358 cells expressing RIPPLY2 or HES1 WRPW peptides. Data is from RNAseq presented in ‘G’. ( I ) Differential effect of the WRPW peptides observed with RNAseq on ATF4 targets, genes associated with negative regulation of Wnt pathway, and genes involved in response to IL-1. ( J ) Western blot shows differential regulation of the ATF4 response and TLE3 in NCIH358 cells expressing different WRPW peptides. A representative example of two biological replicates is shown.
Article Snippet: The following primary antibodies were used: ab13970 chicken polyclonal to GFP at 1:5000, p21 (12D1) Rabbit mAb (#2947, Cell Signaling Technologies) at 1:1000, p53 (1C12) Mouse mAb (#2524, Cell Signaling Technology) at 1:500, Hcfc1 Antibody (Amino-terminal Antigen) (#69690, Cell Signaling Technology) at 1:1000, TLE1 (F-4) (sc-137098, Santa Cruz Biotechnologies) at 1:500, TLE2 (D-10) (sc-374226, Santa Cruz Biotechnologies) at 1:500, TLE3 (D-10) (sc-514798, Santa Cruz Biotechnologies) at 1:500, TLE4 (E-10) (sc-365406, Santa Cruz Biotechnologies) at 1:500, TLE1/2/3/4 (#4681, Cell Signaling Technologies) at 1:500, ATF4 (PA5-27576, Thermo Fisher Scientific) at 1:2000, DDIT3 (R-20) (sc-793, Santa Cruz Biotechnologies) at 1:500, Vinculin (#13901, Cell Signaling Technology) at 1:1000, DHPS (A-10) (sc-365077, Santa Cruz Biotechnologies) at 1:500, ACOX3 (17360-1-AP, Proteintech) at 1:500, cyclin D1 (sc-20044, Santa Cruz Biotechnologies) at 1:500, cyclin D2 (D52F9) (#3741, Cell Signaling Technology) at 1:1000, cyclin D3 (DCS22) (#2936, Cell Signaling Technology) at 1:1000, eIF5A (D8L8Q) (#20765, Cell Signaling Technology) at 1:1000, hypusine (ABS1064-I, EMD Millipore) at 1:1000.
Techniques: CRISPR, Knock-Out, Binding Assay, Multiplex Assay, Expressing, Standard Deviation, Over Expression, Flow Cytometry, RNA sequencing, Western Blot
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