Figure 5

Invalidation of AKR1C2 and 3 reduces preneoplastic transformation. Senescent NHEKs were subjected to AKR1C2 (si_1C2) and AKR1C3 (si_1C3) silencing by siRNAs or to non-target siRNAs (si_Ctrl) for 4 days, or were not transfected (NT). (A) AKR1C2 and 3 mRNA levels were analyzed by RT-qPCR and normalized to 18S levels (Student t-test; ***p ≤ 0.001). (B) Protein extracts from an equal number of si_1C2, si_1C3 or si_Crtl NHEKs were analyzed by immunoblotting with anti-AKR1C2 or anti-AKR1C3 antibodies. Quantification of immunoblots was performed using Image J software, results are presented as the ratio AKR1C isoform/GAPDH. (C) The emergence frequency was determined by plating cells at low density and monitoring the culture for the appearance of emergent clones. Once emergent clones have appeared (after 7days), the culture dishes were stained by Crystal Violet and emergent clones were counted under microscopic observation. Countings were performed in triplicate (Student t-test; *p < 0.05; **p < 0.01, by comparing the conditions si_Ctrl with si_1C2 or si_1C3). (D) Emergent clone as exemplified by phase-contrast microphotograph. (E) Emergent or young cells were subjected to AKR1C2 (si_1C2) and AKR1C3 (si_1C3) silencing by siRNAs or to non-target siRNAs (si_Ctrl) during 4 days, or were not transfected (NT). Cells were counted and cumulative doubling numbers were calculated. The barplots represent the mean +/− s.d. of the counts of three independent culture dishes (Student t-test; *p < 0.05, of the comparisons of the conditions si_Ctrl with si_1C2 or si_1C3). This experiment is representative of 3 independent ones.