FIGURE 1 from Genome-wide CRISPR Screen Reveals RAB10 as a Synthetic Lethal Gene in Colorectal and Pancreatic Cancers Carrying SMAD4 Loss

SMAD4-inducible restoration restores the TGFβ-induced growth inhibition in colorectal and pancreatic cancer cells. A, Protein level of SMAD4 was assessed by Western blot analysis in SMAD4-reexpressing HT29 and SW620 cells. RKO, MDST8 and MIAPaCa-2 were used as positive control. B, Luciferase reporter gene assay of TGFβ-induced SMAD promoter. SMAD4 functionality was assessed using a reporter assay in the reexpressing cell lines HT29 and SW620. Luciferase activity was normalized to control pCM3586 transfected cells, in absence of any treatment [t test performed on n = 3 (SW620); or n = 4 (HT29) independent experiments; P = 0.0000027 (HT29) P = 0.008 (SW620)]. Control plasmid pCM3586 leads to cells expressing Cas9 under doxycycline treatment. C, SMAD4 expression level was assessed in isolated clones from the HT29-pSMAD4 and SW620-pSMAD4 population (POP). D, SMAD4-reexpressing HT29-pSMAD4 and SW620-pSMAD4 clones and populations were seeded in 2D, in flat-bottom 96-well plates and cultured for 10 days with or without doxycycline. Proliferation was assessed using the CellTiter-Glo assay. Proliferation was normalized to untreated transfected cells (DOX−). n = 3 independent experiments. E and F, SMAD4-reexpressing HT29-pSMAD4 and SW620-pSMAD4 clones and populations were seeded in 96-well 3D plates and cultured for 10 days with or without doxycycline. Sphere diameters were measured. The diameter of the doxycycline-treated spheres reexpressing SMAD4 are represented with bar (percentage with untreated spheres taken as reference; percentage mean with SD, n = 3 independent experiments; P = 0.00025 for HT29 C4 vs. C9; P = 0.000027 for SW620 C1 vs. C14).