Fig 1: Biological properties of ALDH1A1 knockouts in vitro. (A) Response to 5‐fluorouracil (5‐FU) and oxaliplatin (OxaliPt), analyzed by unpaired T‐test (n = 6), representative results of three independent experiments, mean ± SD. (B) Migration ability, scratch wound assay (n = 10), representative results of three independent experiments, mean ± SD, analyzed with paired Wilcoxon test. (C) Immunofluorescent staining of Actin filaments (Alexa Fluor 488 Phalloidin) and nuclei (DAPI). Axio Imager Z2 fluorescence microscope (Carl Zeiss), magnification 630×, Metafer 3.6 software (MetaSystems). (D) Detection of apoptosis, Annexin V staining, flow cytometry (n = 3). (E, F) Colony‐forming assay in soft agar. (E) Representative pictures of pts80 and HT‐29 colonies and their size, magnification 50×, ZEISS Axiovert microscope 1.0, scale bar: 100 μm. (F) Capability of pts80 and HT‐29 cells and their engineered counterparts to proliferate and form one cell‐derived colonies in soft agar, calculated as a ratio of growth colonies and seeded cells × 100% (n = 12 for HT‐29, n = 18 for pts80), each dot represents individual value, analyzed by Mann–Whitney test. (G–J) Colonosphere‐forming assay. (G, I) Representative images of single cell‐derived colonies, prepared from pts80 cells and HT‐29 cells, magnification 25×, Leica microscope Mateo/FL, scale bar: 400 μm. (H, J) Relative luminescence corresponding to viable cells proliferating in non‐adherent conditions as colonospheres. Assay was performed in triplicates, t‐test was used for statistical analysis, mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns—not significant P ≥ 0.05).