Figure 4.
GNG5 upregulation mediates the CC-122 resistance rendered by KCTD5 inactivation. (A) Cell fitness assessment of SU-DHL-4 cells with knockout of KCTD5 in the presence or absence of CC-122, compared with control cells by a flow cytometry–based CRISPR competition assay, as described in Figure 3A. Data are shown as the mean ± standard deviation (SD); 2 biological replicates. (B) Heat map showing the CC-122–induced enrichment of DLBCL cells with loss of CRBN or KCTD5 in the CRISPR competition assay. Enrichment score is calculated as the FC of the RFP+/GFP+ ratios of cells treated with 50 µM CC-122 vs their DMSO controls on day 10. (C) Immunoblot analysis of SU-DHL-4-Cas9 cells transduced with lentiviral vectors expressing the indicated sgRNAs. Cells were treated with either DMSO or 10 µM CC-122 for 24 hours. (D) A volcano plot of differentially abundant proteins in response to CC-122 treatment relative to DMSO control. SU-DHL-4-Cas9 cells transduced with lentiviral vectors expressing sgNT-1 or sgKCTD5-7 were subjected to TMT proteomics analysis. The x-axis indicates the log2-FC of each protein in cells expressing sgKCTD5-7 vs cells expressing sgNT-1. P values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method to arrive at an adjusted P value (Padj, also known as an FDR). The y-axis shows the log10 (FDR) values indicating statistical significance, such that proteins lying above the dotted red line are statistically significant findings with Padj < .01. (E) Immunoblot analysis of SU-DHL-4-Cas9 cells transduced with lentiviral vectors expressing the indicated sgRNAs. SU-DHL-4-Cas9 cells were first transduced with a lentiviral vector expressing sgNT-1, sgGNG5-7, or sgGNG5-8, and then selected by 400 µg/mL hygromycin for 10 days. The selected cells were then transduced with a lentiviral vector expressing sgNT-1 or sgKCTD5-7 followed by selection with puromycin (2 µg/mL) for 3 days. (F) Cell fitness assessment of SU-DHL-4 cells with knockout of KCTD5, GNG5, or both in the presence or absence of CC-122, compared with control cells, by flow cytometry–based CRISPR competition assay. SU-DHL-4-Cas9 cells stably expressing sgNT-1, sgGNG5-7, or sgGNG5-8 were transduced with a lentiviral vector coexpressing GFP and sgNT-1 or with a lentiviral vector coexpressing RFP and sgNT-1 or sgKCTD5-7. Three days after infection, RFP+ and GFP+ cells were mixed at a 1:1 ratio and treated with DMSO or CC-122 at the indicated concentrations. The change in the RFP+/GFP+ ratio was then monitored by flow cytometry at day 7. RFP+/GFP+ ratios of cells after transduction with the indicated sgRNA were first normalized to their RFP+/GFP+ ratios on day 0 and then to that of DMSO controls at day 7. (A,F) Data were analyzed by 2-tailed unpaired Student t test. *P < .05; **P < .01; NS, P > .05.

GNG5 upregulation mediates the CC-122 resistance rendered by KCTD5 inactivation. (A) Cell fitness assessment of SU-DHL-4 cells with knockout of KCTD5 in the presence or absence of CC-122, compared with control cells by a flow cytometry–based CRISPR competition assay, as described in Figure 3A. Data are shown as the mean ± standard deviation (SD); 2 biological replicates. (B) Heat map showing the CC-122–induced enrichment of DLBCL cells with loss of CRBN or KCTD5 in the CRISPR competition assay. Enrichment score is calculated as the FC of the RFP+/GFP+ ratios of cells treated with 50 µM CC-122 vs their DMSO controls on day 10. (C) Immunoblot analysis of SU-DHL-4-Cas9 cells transduced with lentiviral vectors expressing the indicated sgRNAs. Cells were treated with either DMSO or 10 µM CC-122 for 24 hours. (D) A volcano plot of differentially abundant proteins in response to CC-122 treatment relative to DMSO control. SU-DHL-4-Cas9 cells transduced with lentiviral vectors expressing sgNT-1 or sgKCTD5-7 were subjected to TMT proteomics analysis. The x-axis indicates the log2-FC of each protein in cells expressing sgKCTD5-7 vs cells expressing sgNT-1. P values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method to arrive at an adjusted P value (Padj, also known as an FDR). The y-axis shows the log10 (FDR) values indicating statistical significance, such that proteins lying above the dotted red line are statistically significant findings with Padj < .01. (E) Immunoblot analysis of SU-DHL-4-Cas9 cells transduced with lentiviral vectors expressing the indicated sgRNAs. SU-DHL-4-Cas9 cells were first transduced with a lentiviral vector expressing sgNT-1, sgGNG5-7, or sgGNG5-8, and then selected by 400 µg/mL hygromycin for 10 days. The selected cells were then transduced with a lentiviral vector expressing sgNT-1 or sgKCTD5-7 followed by selection with puromycin (2 µg/mL) for 3 days. (F) Cell fitness assessment of SU-DHL-4 cells with knockout of KCTD5, GNG5, or both in the presence or absence of CC-122, compared with control cells, by flow cytometry–based CRISPR competition assay. SU-DHL-4-Cas9 cells stably expressing sgNT-1, sgGNG5-7, or sgGNG5-8 were transduced with a lentiviral vector coexpressing GFP and sgNT-1 or with a lentiviral vector coexpressing RFP and sgNT-1 or sgKCTD5-7. Three days after infection, RFP+ and GFP+ cells were mixed at a 1:1 ratio and treated with DMSO or CC-122 at the indicated concentrations. The change in the RFP+/GFP+ ratio was then monitored by flow cytometry at day 7. RFP+/GFP+ ratios of cells after transduction with the indicated sgRNA were first normalized to their RFP+/GFP+ ratios on day 0 and then to that of DMSO controls at day 7. (A,F) Data were analyzed by 2-tailed unpaired Student t test. *P < .05; **P < .01; NS, P > .05.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal