Fig. 5

Loss of MYB expression decreases viability, colony formation, and anchorage-independent growth of LS174T cells. a qRT-PCR and western Blot analyses of MYB expression in LS174T cells stably transduced with lentiviral vectors for Dox-inducible shRNA expression. Cells were treated with 1 μg ml⁻¹ Dox for 96 h or were left untreated. Left: MYB transcript levels are depicted as expression relative to GAPDH (rel. expr.). Shown are the mean and SEM; n = 3. Right: Immunodetection of MYB. α-TUBULIN (TUBULIN) was used to control for equal loading. MW molecular weight. b The MTT assay with LS174T cells stably transduced with lentiviral vectors for Dox-inducible shRNA expression. Cells were treated with 1 μg ml⁻¹ Dox for 96 h or were left untreated. Values of untreated cells were set to 1. Shown are the mean and SEM; n = 3. c Western Blot analyses of PARP1 and CASPASE3 cleavage in LS174T cells stably transduced with lentiviral vectors for Dox-inducible shRNA expression. Cells were treated with 1 μg ml⁻¹ Dox for 96 h or were left untreated. Immunodetection of α-TUBULIN (TUBULIN) was used to control for equal loading. MW molecular weight. d Cell cycle analysis of LS174T cells stably transduced with lentiviral vectors for Dox-inducible shRNA expression. Cells were treated with 1 μg ml⁻¹ Dox for 72 h or were left untreated. Cells were stained with propidium iodide (PI). Shown are the mean and SEM; n = 3. e The 2D colony formation assay of LS174T cells stably transduced with lentiviral vectors for Dox-inducible shRNA expression. Cells were treated with 1 μg ml⁻¹ Dox for 12 days or were left untreated. Left: quantification of colony numbers after 12 days. Colonies were counted using ImageJ. Shown are the mean and SEM; n = 3. Right: representative image of a six-well plate with colonies stained with crystal violet after 12 days of incubation. f Anchorage-independent growth of LS174T cells stably transduced with lentiviral vectors for Dox-inducible shRNA expression. Cells were treated with 1 μg ml⁻¹ Dox for 12 days or left untreated. Left: quantification of colony numbers using ImageJ. Shown are the mean and SEM; n = 3. Center: colony size. Diameters of at least 50 colonies from each condition were measured using ImageJ. Shown are the mean and SEM; n = 3 independent experiments. Right: representative images of colonies. Scale bar: 50 µm. a–f shNON non-silencing control shRNA, shMYB1, shMYB2 MYB-specific shRNAs. Statistical significance was calculated using a two-tailed unpaired Students’ t test. ***p-value < 0.001; **p-value < 0.01; *p-value < 0.05; ns: not significant