The BH3 only Bcl-2 family member BNIP3 regulates cellular proliferation
Fig 1
MEF cells lacking BNIP3 have increased proliferation compared with MEF cells expressing BNIP3.
A) Bnip3-null allele was created by replacing exons 2 and 3 with a neomycin resistance cassette. (B) Reverse transcription polymerase chain reaction (RT-PCR) was performed on total RNA isolated from MEFs using primers against neomycin-resistance gene. The PCR products were run on a 1% agarose gel at 80V for 1.5 hours and visualized under UV light with Chemidoc XRS+ instrument (Bio-Rad). A single band at ~500bp is observed (expected size is 492 bp). (C) Total lysates from MEFs were western blotted for BNIP3. Cells were incubated under hypoxia for 24 hours before lysing to over-express BNIP3. The blot was stripped and reprobed with GAPDH as loading control. (D) The Click iT EdU cell proliferation assay was performed and Edu positive cells were counted using a flow cytometer. The results show Edu positive cells as a percentage of total cells in the sample, and are an average of three replicates. The error bars show standard error of the mean. Tukey’s post hoc test was performed to measure statistical significance (***p<0.0001). (E) Cell counts were monitored using an RTCA instrument over 5 days. The results shown are representative of three independent experiments. The error bars show standard deviation. (F) Cells were incubated under normal conditions and counted with a Cytation V instrument over 6 days. The cell count shown is an average of three independent wells (4 measurements per well). The error bars show standard error of the mean. Tukey’s post hoc test was used to calculate statistical significance (* p<0.05, ** p<0.02, *** p<0.005). Results shown are representative of three independent experiments.