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. 2023 Mar 15;13(1):37. doi: 10.1038/s41408-023-00809-7

Fig. 6. CYLD knockdown promotes cell growth and NF-κB activation.

Fig. 6

A Immunoblot analysis of CYLD in HBL1, LY10 and Mino transduced with lentiCRISPR-Cas9 (±sgCYLD) using an antibody raised against a C‐terminal epitope which detects full-length CYLD and a C‐terminal fragment of CYLD (CYLD‐Ct). Cells were treated with 50 nM BTK inhibitor Ibrutinib or 500 nM PKC inhibitor Sotrastaurin for 48 h as indicated. β-tubulin was used as loading control. B HBL1, LY10 and Mino transduced with lentiCRISPR-Cas9 without gRNA (empty vector; EV) or with sgCYLD were treated for 3 days with indicated concentrations of Ibrutinib or Sotrastaurin. The number of viable cells, as determined by 7-AAD staining, was normalized to the untreated condition. The mean ± SD of four independent experiments performed in triplicate is shown. *P < 0.05; **P < 0.01 using 2-way ANOVA with Sidak’s multiple comparisons test. C Immunoblot analysis of (phosphorylated) IkBα in HBL1, LY10 and Mino transduced with lentiCRISPR-Cas9 (±sgCYLD) treated for 48 h with 50 nM Ibrutinib or 500 nM Sotrastaurin as indicated. Cells were incubated with 5 µM proteasome inhibitor MG132 for 3 h before harvesting. β-tubulin was used as loading control. D Immunoblot analysis of (phosphorylated) STAT3 in LY10, HBL1 and Mino transduced with lentiCRISPR-Cas9 (±sgCYLD) treated 48 h with 50 nM Ibrutinib or 500 nM Sotrastaurin as indicated. β-actin was used as loading control.