Fig. 5

The increased ALDH activity correlated with chemoresistance and ALDH1A3 silencing partially reverted the resistance. a The flow cytometry analysis by Aldefluor Assay revealed 9.5-fold increased ALDH activity in chemoresistant cells when compared to chemonaïve counterparts. b The chemoresistant cells switched the expression of ALDH isoforms as demonstrated by qPCR. The expression in parental HT-29/EGFP cells was set as a reference, and GAPDH and HPRT1 served as an internal control. The data are expressed as means of quadruplicates ± SD. Mann-Whitney U test was used for statistical analysis. c The Western blot analysis confirmed increased expression of ALDH1A3 also on the protein level. The longer cultivation of chemoresistant cells in the medium with 5-FU, the higher amount of protein. 1. HT-29/EGFP; 2. HT-29/EGFP/FUR low passage; 3. HT-29/EGFP/FUR middle passage; 4. HT-29/EGFP/FUR high passage. β-actin was used as an internal loading control. d The Western blot analysis after RNA silencing proved successful ALDH1A3 inhibition as shown 48 and 72 h post nucleofection. 1. HT-29/EGFP/FUR no treatment at 48 h; 2. FUR/ctrl siRNA at 48 h; 3. FUR/ALDH1A3 siRNA at 48 h; 4. HT-29/EGFP/FUR no treatment at 72 h; 5. FUR/ctrl siRNA at 72 h; 6. FUR/ALDH1A3 siRNA at 72 h. β-actin was used as an internal loading control. e The molecular silencing of ALDH1A3 by specific siRNA did not affect the proliferation rate of chemoresistant cells (referred to as FUR/ALDH1A3 siRNA) as detected by the kinetic imaging system. The control siRNA was used as a negative control (FUR/ctrl siRNA). f The molecular ALDH1A3 silencing did not either affect the migration potential of chemoresistant cells as determined by the wound healing assay. FUR/ALDH1A3 siRNA-cells filled in 29% of wound in comparison to cells silenced with control siRNA (FUR/ctrl siRNA) which filled in 26% of the wound. g-h The silencing of ALDH1A3 partially reversed chemoresistance of HT-29/EGFP/FUR cells as demonstrated by flow cytometry. The increased sensitivity to chemotherapeutics added 48 h post nucleofection (5-FU, CisPt - cisplatin, PTX - paclitaxel, Dox - doxorubicin, CPX - cyclophosphamide) was induced by the molecular inhibition of ALDH1A3. The apoptosis was subsequently determined 48 h later by the Annexin V assay. g The data demonstrating apoptosis are expressed as means of quadruplicates ± SD. Mann-Whitney U test was used for statistical analysis. h Density plots demonstrating cell death. The gates were defined based on the cells nucleofected by control siRNA without chemotherapeutics. The cells positive for Annexin V are apoptotic, positivity for DAPI stands for necrotic ones