Figure 3. Ectopic expression of human RMEL3 protects NIH3T3 murine fibroblasts from serum withdrawal-induced growth arrest/ apoptosis.

(A) Schematic representation of RMEL3 locus (above), its transcript (center), and the region cloned into expression vectors (below). (B) Efficiency of exogenous RMEL3 induction in cells transduced with pLVX-RMEL3 or pLVX-TP (as control) after treatment with doxycycline (1 μM, 24 h), analyzed by RT-qPCR. ND (not-detected). Relative expression was calculated according to ΔCT using TBP (Tatabox binding protein) as endogenous control. (C-E) Functional assays using NIH3T3 cells stably transduced with pLVX-RMEL3 or pLVX-TP (as control) in the presence of doxycycline (1 μM). (C) Proliferation rates. During the time course of the assay, cells were maintained under serum starvation (in medium supplemented with 0.5% FBS). After the indicated time points, cells were stained with crystal violet and cell density was quantified according to the absorbance in an ELISA microplate reader. ***p < 0.0005 (D) Apoptosis rate. Flow cytometry-based detection of annexin-V- and propidium iodide (PI)-stained cells. Dot plot from one assay representative of three replicates, and below, a summary graphics of three independent replicates. Cells were cultured in FBS-free medium for 48 h, and afterward, culture medium was supplemented with 0.5% FBS and cells were cultured for additional 48 h, when they were assayed. *p < 0.05; **p < 0.005. (E) Clonogenic ability. Cells were seeded in 60-mm-diameter plates and allowed to grow for 9 days, when they were fixed with paraformaldehyde and stained with crystal violet to reveal the colonies. *p < 0.05; **p < 0.005. Error bars represent SEM of 3 independent experiments for B-E. Asterisks indicate statistically significant differences between groups based on unpaired parametric Student’s t test