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. 2020 Jan 3;21(1):327. doi: 10.3390/ijms21010327

Figure 3.

Figure 3

DDI2-deficient MDA-MB-231 cells are more sensitive to carfilzomib-induced cell death. (A) MDA-MB-231 cells stably expressing a doxycycline-inducible DDI2 shRNA construct (inducible shDDI2 cells) were incubated with or without doxycycline for ≥72 h, then were treated with 200 nM CFZ or equal volume DMSO for 4 h and analyzed by western blot using the antibodies indicated. β-Actin was used as a loading control. Blots shown are representative of three independent experiments. (B) MDA-MB-231 inducible shDDI2 cells were treated with or without doxycycline for ≥72 h, then were treated with increasing doses of CFZ (6.25–800 nM) for 24 h and analyzed by a luminescent cell viability assay. Viability percentage was determined via comparison to viability of untreated cells (100%). Error bars denote standard deviation (n = 3). (C) MDA-MB-231 inducible shDDI2 cells were treated with or without doxycycline for ≥72 h, then were treated with DMSO or 10, 25, or 50 nM CFZ for 24 h and analyzed by western blot using the antibodies indicated. β-Actin was used as a loading control. Blots shown are representative of three independent experiments. (D) MDA-MB-231 control (expressing EGFP sgRNA) and DDI2−/− cells were treated with 200 nM CFZ or equal volume DMSO for 4 h and analyzed by western blots using the indicated antibodies. β-Actin was used as a loading control. Blots shown are representative of three independent experiments. (E) MDA-MB-231 control (expressing EGFP sgRNA) and DDI2−/− cells were treated with DMSO or increasing doses of CFZ (6.25–400 nM) for 24 h and analyzed by MTT assay. Viability percentage was determined via comparison to viability of DMSO-treated cells (100%). Error bars denote standard deviation (n = 3). (F) MDA-MB-231 control (expressing EGFP sgRNA) and DDI2−/− cells were treated with 10 nM CFZ or equal volume DMSO for 24 h, then the cell lysates were analyzed by western blot with the indicated antibodies. β-Actin was used as a loading control. Blots shown are representative of three independent experiments. (G) MDA-MB-231 DDI2−/− cells expressing a vector control or CRISPR-resistant Flag-WT-DDI2 were treated with 20 nM CFZ or DMSO for 24 h and the cell lysates were subjected to immunoblotting with indicated antibodies. GAPDH was used as a loading control. Blots shown are representative of three independent experiments. Significance (p value < 0.05) is denoted by asterisk.