MEF cells lacking BNIP3 show no differences in background cell death and only a slight reduction in oxidative stress-induced cell death.

Mean cell death of BNIP3 wild-type (WT) and knockout (KO) Murine Embryonic Fibroblast (MEF) cells treated with hydrogen peroxide over a range of concentrations (10–1000 microM) for 24 hours. Total cell death was measured by flow cytometry with trypan blue staining. Bars represent standard error of three independent experiments.</p

Cellularity of wild-type and BNIP3 knockout E18.5 brains.

Cellularity of wild-type and BNIP3 knockout E18.5 brains.</p

Loss of BNIP3 increased proliferation in cultured primary astrocytes.

(A) Equivalent quantities of wild-type (WT) and BNIP3 knockout (KO) astrocytes isolated from neonatal brains of mice were seeded in 6-well plates, and plates were analyzed using the Cytation V instrument. The cell count shown is an average of three independent wells. (B) Equivalent numbers of wild type (WT) and BNIP3 knockout (KO) astrocytes were plated in triplicate in 96-well plates and allowed to replicate for 1 to 6 days. Relative cell density was measured by quantifying the amount of MTT reagent metabolized to purple formazan in each well by colorimetric assay. The error bars show standard error of the mean. Student’s t-test was used to calculate statistical significance. (ns: not significant, *: p<0.05, **: p<0.01).</p

Stable deoxycycline inducible expression of BNIP3.

HEK293 cells were engineered to stably co-express the Tet Repressor protein (TR) and tetracycline-inducible BNIP3 (TO-BNIP3) or beta-galactosidase (TO-β-gal) as described in the Materials & Methods. Multiple clones were established for each cell line. Addition of the tetracycline analog, doxycycline (dox), for 24 hours relieves TR repression and induces BNIP3 expression as detected by immunofluorescence (A), Western blot (B) and RT-PCR. In (A), nuclei are visualized by Hoescht dye (blue) and BNIP3 is seen in the cytoplasm (green fluorescence).</p

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.</p

MEF cells lacking BNIP3 show higher levels of MAPK activation compared to MEF cells expressing BNIP3.

(A) Total lysates from wild-type and BNIP3 knockout MEFs were western blotted for phospho-MAPK. The blot was stripped and reprobed with total MAPK and GAPDH as loading controls. (B) The protein levels of p-MAPK and total MAPK were measured using ImageJ software. The ratio of p-MAPK/total MAPK was plotted to compare the activation of MAPK. The results are an average of three replicates. The error bars show standard error of the mean. Tukey’s post hoc test was used to calculate statistical significance (*p<0.05). Results shown are representative of three independent experiments Total lysates from wild-type and BNIP3 knockout MEFs were western blotted for Cyclin D1. The blot was stripped and reprobed with GAPDH as loading control. The protein levels were quantified using ImageJ software. The ratio of Cyclin D1 to GAPDH was calculated and normalized to the highest level.</p

BNIP3 induction in HEK 293 cells reduces cell growth.

HEK293 cells were engineered to stably co-express the Tet Repressor protein (TR) and tetracycline-inducible BNIP3 (TO-BNIP3) or beta-galactosidase (TO-β-gal) as described in the Materials & Methods. Cells were untreated or treated with doxycycline (1 microg/mL) to induce gene expression. (A) Reactive oxygen species (ROS) production was increased upon BNIP3 (but not β-gal) induction. ROS were detected using the dye CM-H2DCFDA, which is oxidized to green fluorescent DCF (dichlorofluorescein) by hydrogen peroxide. (B) Autophagy was also increased upon BNIP3 (but not beta-gal) induction, as determined by GFP-LC3 localization. TO-cells were transiently transfected with GFP-LC3. 24 hours after transfection, cells were untreated or treated with doxycycline for an additional 24 hours. (C) Cell death was not induced upon BNIP3 or β-gal induction, as determined by the membrane permeability assay. (D) Cell growth was severely restricted upon BNIP3 (but not beta-gal) induction in HEK293 cells. For each cell type, 1.5x105 cells were seeded in one well of a 6-well culture dish, with or without doxycycline (gene induction). Three days later, the number of adherent cells in each well was determined using a Beckman Coulter Counter. The fold-increase represents the ratio of cells on day three compared to the number of cells originally seeded. Results represent the average of three independent experiments; error bars represent standard deviation. *p < .01.</p

HEK293 cells over-expressing nuclear BNIP3 have lower proliferation than cells transfected with empty control vector.

(A) HEK293 cells were transfected with either an empty control vector or nuclear BNIP3 expression vector. Total lysates from transfected cells were western blotted for BNIP3. The blot was stripped and reprobed with GAPDH as loading control. The results shown are representative of three independent experiments (three replicates each). (B) Proliferation of transfected cells was measured using the Click iT EdU cell proliferation assay. The results are an average of three independent experiments (three replicates each). The error bars show standard error of mean. Student’s t-test was used to calculate statistical significance (*p<0.0005). Results shown are representative of three independent experiments.</p

Morphology and cellularity changes in wild type and BNIP3 knockout brains.

Whole brains from 8-week old wild type and BNIP3-/- mice were fixed by cardiac perfusion and overnight immersion in paraformaldehyde (n = 3 pairs of littermates from different heterozygous crosses). Brains were embedded in paraffin, sectioned and stained with hematoxylin and eosin as described in Materials & Methods. Images were captured at 10x and 40x magnification and cell counting was performed using Image Pro Plus 5.0 software. These representative images depict the morphology of the cerebellum, hippocampus, cortex and inferior colliculus, with cell counts for the 40x images indicating increased cellularity in BNIP3-/- brains. E18.5 embryos from B) wild type and C) BNIP3-/- mice were fixed as described in the Material and Methods section. The paraffin embedded brains were sliced and placed on slides and stained for DNA with DAPI and antibodies against Ki67 and leaved caspase 3. This images represent three independent experiments.</p

Additional file 1 of Lessons learnt in the first year of an Australian pediatric cardio oncology clinic

Additional file 1. Supplementary data