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

Fig. 4. Ectopic expression of non-cleavable CYLD inhibits cell growth and NF-κB pathway activity.

Fig. 4

A Flow cytometric analysis of LY10, RIVA and Mino cells transduced with an empty vector (EV) or a CYLD (wildtype or R324A mutant) containing bicistronic vector co-expressed with YFP. The percentage of YFP positive cells was followed in time and plotted as the percentage of YFP+ cells, normalized to the value at day 3 following retroviral transduction. The mean ± SD of at least three independent transductions is shown. *P < 0.05; **P < 0.01 using 1-way ANOVA with Tukey’s multiple comparisons test. B Immunoblot analysis of CYLD in LY10, RIVA and Mino 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 transduced with an empty vector (EV) or an expression vector for CYLD (WT or non-cleavable R324A mutant) and sorted for YFP expression. β-actin was used as loading control. C Flow cytometric analysis of LY1 and Z138 cells transduced with an empty vector (EV) or a CYLD (wildtype or R324A mutant) containing bicistronic vector co-expressed with YFP. The percentage of YFP positive cells was followed in time and plotted as the percentage of YFP+ cells, normalized to the value at day 3 or day 4 following retroviral transduction. The mean ± SD of three independent transductions is shown. P > 0.05; ns (non-significant) using 1-way ANOVA with Tukey’s multiple comparisons test. D Immunoblot analysis of CYLD in LY1 and Z138 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 transduced with an empty vector (EV) or an expression vector for CYLD (WT or non-cleavable R324A mutant) and sorted for YFP expression. β-tubulin was used as loading control. E Immunoblot analysis of (phosphorylated) IkBα in LY10 and Mino transduced with an empty vector (EV) or a CYLD (wildtype or R324A mutant) expressing vector. Three days after sorting cells were incubated with or without 5 µM proteasome inhibitor MG132 for 3 h before harvesting. β-tubulin was used as loading control. F Heatmap representing the RT-qPCR analysis of NF-κB target gene expression in LY10 and Mino transduced with an empty vector (EV) or an expression vector for CYLD (WT or non-cleavable R324A mutant). Cells were sorted for YFP expression and allowed to recover for 48 h before RNA isolation. RPLP0 was used as an input control and data are normalized to the EV control expression levels. The mean of three independent experiments performed in triplicate is shown. G Immunoblot analysis of (phosphorylated) STAT3 in LY10 transduced with an empty vector (EV) or an expression vector for CYLD (WT or non-cleavable R324A mutant). β-tubulin was used as loading control.