Figure 2.

The miR-106b∼25 cluster allows cells to bypass doxorubicin-induced senescence and to generate drug resistance. (a–d) MTMEC cells were stably infected with a lentiviral vector expressing the miR-106b∼25 cluster genomic sequence (intron 13 of MCM7). (a) MiR expression was determined by reverse transcription and real-time PCR using TaqMan probes and was normalized to U6 RNA. (b) Bypass of doxorubicin-induced senescence in cells overexpressing the miR-106b∼25 cluster. Cells were treated with 60 ng/ml doxorubicin for 24 h and stained for SA-β-galactosidase 3 weeks after. Percentage of senescent cells is indicated after monitoring at least six fields of view (typical variation approximately 10%). Bar represents 30 μm. (c) Cells overexpressing the miR-106b∼25 cluster are able to generate doxorubicin-resistant clones. Cells were treated for 24 h with 60 ng/ml doxorubicin and drug-resistant clones were stained with crystal violet after 3 weeks (left panel), counted (middle panel) and crystal violet was solubilized (right panel). (d) MiR-106b∼25 overexpressing cells were able to proliferate after doxorubicin challenge. Cells were treated with doxorubicin as above and 3 weeks later, trypsinized and seeded (10 000 cells per well in a 96-well plate). EdU was added to the medium and left for 18 h to allow its incorporation into newly synthesized DNA. Cells were monitored using both brightfield and fluorescence and the percentage of cells incorporating EdU determined after examination of at least three fields of view (left panels). EdU incorporation was determined in the well using a fluorimeter (right panel). (e–g) Downregulation of miR-106b∼25 cluster expression in drug-resistant MD60 cells restores drug sensitivity. MD60 cells were stably infected with a lentiviral ZIP construct to downregulate endogenous miR-106b∼25 cluster expressions and were treated with 240 ng/ml doxorubicin for 24 h and then were left drug free for 3 weeks to enable the generation of doxorubicin-resistant clones. (e) SA-β-galactosidase. Percentage of senescent cells is indicated after monitoring at least six fields of view (typical variation approximately 10%). Bar represents 30 μm. (f) Cultures were stained with crystal violet (left panel), the number of drug-resistant clones counted (middle panel) and crystal violet solubilized and the optical density determined (right panel). (g) EdU incorporation as in d. Numerical data represent the average±S.D. of at least three different experiments (*P<0.05). Pictorial data were repeated at least in triplicate and a representative picture is shown