Although we could confirm inhibition of the CCR4-NOTcore complex, it is unclear whether NIP-2 could also inhibit the full complex with all its subunits. Several of these components have been described to be able to bind RNA directly and targeting of NOT9 alone could therefore not be sufficient. The full CCR4-NOT complex can be reconstituted from purified proteins, but using this approach any adapter proteins binding to NOT9 would be absent. Since these can compete with NIP-2 we wanted to evaluate inhibition in a more biologically relevant context. It was previously shown that cell lysates can be used in combination with model RNAs to evaluate deadenylation. To verify this, we incubated the A20 RNA in HeLa cell lysate and indeed observed degradation after electrophoretic separation of the products (Fig. , supplemental Fig. ). Both NIP-WT-Ac and NIP-2-Ac were able to inhibit deadenylation of A20 while NIP-MUT-Ac did not. Interestingly, NIP-2-Ac seemed to be more potent in this assay than NIP-WT-Ac although higher concentrations were required in comparison to the experiments using CCR4-NOTcore. Hydrocarbon stapling has been known to enhance the cell permeability of α-helical peptides by two factors. First, through restricting the backbone in an α-helical conformation forcing the peptide to be amphipathic. Second, the hydrophobic nature of the linker further improves the overall amphipathic character of the helical peptide. Nonetheless, the permeability of stapled peptides is highly dependent on the position of the staple and the overall charge. To determine the cell permeability, we utilized the recently reported NanoClick assay which requires only minimal modification of the peptide with an azide and is particularly suitable for stapled peptides. When cells are treated with such peptides, and only if the peptide can reach the cytosol, the azide will react with a DBCO-modified Halotag-NanoLuc fusion protein. If this reaction occurs it inhibits the generation of a BRET signal that is generated by an azide-modified dye added after peptide treatment providing a readout for cell permeability. The cellular uptake was analyzed by incubating HeLa cells with a dilution series of the peptides starting at 10 μM for 18 hours and the output was recorded as the BRET ratio and normalized as BRET ratio inhibition. The cellular uptake was further compared with the known cell-penetrating peptide (CPP) octaarginine (R8-N3) as a positive control (Fig. , supplemental Fig. ). NIP-2-N3 had an improved EC50 value (2.44 ± 0.91 μM) in comparison to NIP-WT-N3 (EC50 > 10 μM) demonstrating that hydrocarbon stapling significantly promoted cell permeability. However, in comparison to R8-N3 (EC50 = 0.31 ± 0.03 μM), NIP-2-N3 has an almost 7-fold lower uptake. In comparison to previously reported EC50 values for stapled peptides (in the sub-micromolar range), NIP-2-N3 does not perform very well which is likely due to its limited amphipathic character.