Figure 5.

Doxycycline-induced CCRL2 overexpression increases the resistance of MDS-L and TF-1 cells to azacitidine. (A) Doxycycline-induced CCRL2 overexpression in MDS-L cells transduced with pLV-Puro-TRE-CCRL2- and pLV-Hygro-CMV>tTS/rtTA-expressing lentiviruses and selected with puromycin and hygromycin treatment was confirmed by western blot and flow cytometry. (B) CCRL2 overexpression by treatment of MDS-L cells with 1 mg/mL doxycycline led to a less prominent suppression of clonogenicity by 0.5 mM azacitidine (P=0.019) and increase of the azacitidine half maximal inhibitory concentration (IC₅₀) (P=0.003). (C) CCRL2 overexpression by treatment of MDS-L cells with 1 mg/mL doxycycline led to a small decrease of the percentage of apoptotic cells under treatment with 0.5 mM (P=0.010) and 1 mM azacitidine (P=0.001). (D) Doxycycline-induced CCRL2 overexpression in TF-1 cells with CRISPR-Cas9 CCRL2 deletion, transduced with pLV-Puro-TRE-CCRL2-and pLV-Hygro-CMV>tTS/rtTA-expressing lentiviruses and selected with puromycin and hygromycin treatment, was confirmed by western blot and flow cytometry. (E) CCRL2 overexpression by treatment of TF-1 cells with 1 mg/mL doxycycline led to a higher clonogenic capacity (P<0.001) in cells treated with dimethylsulfoxide and a less prominent suppression of clonogenicity by 0.5 mM (P=0.001) and 1 mM azacitidine (P=0.002). This led to a less prominent decrease of the relative number of colonies in cells treated with 1 mM azacitidine (P=0.044) and an increased azacitidine IC₅₀ (P=0.011). Doxy: doxycycline; Aza: azacitidine; DMSO: dimethylsulfoxide.