Fig. 3

Effect of DNMT3B knockdown on MM cell biology. (A) Visual representation of the inducible lentiviral vectors containing a shRNA cassette against DNMT3B (shDNMT3B). (B) Validation of DNMT3B knockdown (KD) in the AMO-1 (upper panel) and XG-2 (lower panel) cells on mRNA level. DNMT3B levels were determined after 3 days of doxycycline treatment using qPCR. ABL was used as reference gene. The relative expression levels in stimulated (+ D) compared to unstimulated (-D) cells are shown (n = 3). (C) Validation of DNMT3B KD in AMO-1 (upper panel) and XG-2 (lower panel) cells on protein level. DNMT3B levels were determined by western blot 5 days post-doxycycline treatment. Tubulin was used as loading control. Left: one experiment representative of at least three is shown, right: quantification of DNMT3B levels relative to tubulin as measured by Image Studio and normalized to unstimulated (-D) cells. D-F) Effect of DNMT3B KD on apoptosis (D), proliferation (E) and clonogenic outgrowth (F). (D) Cells were stimulated for 5 days with or without doxycycline and apoptosis was measured by an AnnexinV/7’AAD staining followed by flow cytometric analysis. The % apoptotic cells are the sum of AnnexinV (+) and AnnexinV (+)/7’AAD (+) cells. (E) Cells were treated for 3 days with doxycycline after which the effect on bromo-deoxyuridine (BrdU) incorporation (left panel) and cell cycle progression (right panel) was determined using BrdU and PI-stainings respectively. (F) Transduced AMO-1 and XG-2 cells were treated for 5 or 3 days respectively with doxycyline and were then plated to perform a colony forming assay. The number of colonies were determined after 14 days using the EVOS M7000 Imaging System (left) and counted with ImageJ software (right). The mean ± SD of at least three independent experiments is shown. *p ≤ 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to unstimulated (-D) cells