Figure 2.

Validation of BIRC6 dependency in vitro and in vivo.A, Consequences of CRISPR-mediated BIRC6 knockout on cell viability. Five putatively dependent cells and six putatively nondependent cells [as defined by Chronos score (see Methods)], all of which constitutively express Cas9, were analyzed using an ATP-based assay 7 days after transducing an sgRNA against BIRC6 (three different sgRNA sequences were tested). Viability scores relative to the average viability of cells transduced with cutting control sgRNAs and the average viability of cells with knockout (KO) of common essential genes are shown. Values = means ± SD (n = 9). ****, P < 0.0001 (dependent vs. nondependent; for each guide). B, Consequences of CRISPR interference (CRISPRi)–mediated BIRC6 knockdown on long-term cell fitness. Clonogenic growth of the cells was evaluated 14 days after the transduction of an all-in-one CRISPRi construct targeting the indicated gene. Two sgRNA sequences against BIRC6 were tested. Presented are the representative images of cells with crystal violet staining (left) and the mean staining intensities per sample (n = 3, right). *, P < 0.05; ****, P < 0.0001 (sgCiCh2-2 vs. sgCiBIRC6). C and D, Cell cycle (C) and cell death (D) analysis following BIRC6 knockout. Cas9-expressing derivatives of indicated cells were transduced with a cutting control sgRNA (sgCh2-2) or an sgRNA targeting BIRC6 (sgBIRC6-1, sgBIRC6-4). Cells were harvested 4 (C) or 7 (D) days later, stained, and analyzed by flow cytometry. In C, the proportion of cells in the S-phase was reduced upon BIRC6 knockout in the three dependent models, but not in the three nondependent models. In D, the proportion of dead cells (late apoptosis + nonapoptotic death + early apoptosis) was increased following the knockout of BIRC6 in all of the three dependent cell lines, but only one of the three nondependent cell lines. ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (n = 3). E–G,In vivo validation of the BIRC6 dependency. In E, ZR751 breast cancer cells expressing a doxycycline (DOX)-inducible shRNA against BIRC6 (shBIRC6–2) were implanted into the mammary fat pads of NRG mice. Following tumor formation, some of these mice were treated with DOX, while others were left untreated. In F and G, KYSE450 esophagus cancer cells (F) and HCC95 lung cancer cells (G), both of which were engineered to express an sgRNA against BIRC6 in a tamoxifen (TAM)-inducible fashion, were implanted subcutaneously via intraperitoneal injection (IP) into the NSG (NOD-scid Il2rg^−/−) mice. Following tumor formation, some mice were injected with TAM, while others were treated with vehicle control. In both cases, the tumor growth is plotted to compare the two different groups of mice. Data are represented as means ± SEM [n = 8 (Keep w/o TAM group, G), 9 (Keep w/o TAM and TAM(-) groups, F; TAM hereafter group, G), 10 (Keep w/o DOX and DOX(-) groups, E; TAM hereafter and TAM (+) groups, F; TAM(-) and TAM(+) groups, G), 12 (DOX hereafter and DOX (+) groups, E)]. ns, P ≥ 0.05; ****, P < 0.0001 (for each of the last five time points for the tumor growth curves). All the experiments were performed twice except for A, which was conducted three times, and E–G, which were conducted once.