Fig 1.
PDCD6 gene knock-out in DT-40 cells and ectopic ALG-2 expression.
A. Top: Strategy of PDCD6 gene knock-out. The genomic organization of the chicken PDCD6 gene; Nco1 restriction digestion of genomic DNA was used for Southern blot analysis, the sites within the PDCD6 gene are indicated; the probe used for hybridization is shown as a red box; the size of the Nco1 digested DNA fragment to which it hybridizes in the wt cells is indicated above the gene structure. Bottom: the genomic situation after homologous recombination and the length of the expected Nco1 fragments. The open arrows indicate the drug resistance genes and their transcriptional orientation. B. Southern blot of genomic DNA from all the cell lines used and generated during PDCD6 gene disruption hybridized with a chicken PDCD6 cDNA probe. C. Western blot of protein extracts from the same cell lines as in B using a specific ALG-2 antibody and ERK-1 as a loading control.
Fig 2.
ALG-2 knock-out leads to reduced cell viability after electroporation induced membrane damage.
A. Western blot analysis of cell extracts from DT-40 wt, an ALG-2 KO cell line 17-2-11, and two 17-2-11 clones, H8 and H10 transfected with an ALG-2 expressing vector. ERK-1 was used as a loading control. Numbers below represent the relative ALG-2 signal intensities normalized to the ERK1 signals. Quantification was performed using ImageJ software. B. DT-40 wt cells, an ALG-2 KO cell line 17-2-11 and a rescue clone H10 were exposed to electroporation in order to investigate the involvement of ALG-2 in cell survival after mechanical membrane damage. Manual cell counting was performed 24 hours post electroporation. Individual data from four independent experiments are shown. Means +/- SEM are indicated. The p-values (unpaired t-test) are indicated.
Fig 3.
ALG-2 overexpression leads to increased cell viability after digitonin treatment.
HeLa cells were transfected with four different constructs: EGFP (control), EGFP-ALG-2 (wt ALG-2), EGFP- ALG-2ΔGF (ALG-2 short isoform missing Gly121 and Phe122), EGFP-ALG-2EF-1, -3 (ALG-2 protein with mutated high affinity Ca2+-binding sites). 50 μM digitonin was used to induce membrane damage. Cell viability was assessed by imaging and quantifying EGFP expressing cells one hour (blue symbols) and three hours (red symbols) post-treatment. Data were normalized to the EGFP transfected control set as 100%. Individual data from three independent experiments are shown. Means +/- SEM are indicated. The p-values (unpaired t-test) are indicated.
Fig 4.
A peptide with the ALG-2 binding sequence of ALIX impairs the ALG-2 pro-survival function.
A. HeLa cells expressing EGFP or EGFP-ALG-2 were treated with 50 μM digitonin in the presence of 10 μM ALIX peptide with ALG-2 binding sequence, ALIX mutant peptide incapable of ALG-2 binding, unrelated peptide or vehicle, as indicated. Cell viability was assessed by quantifying EGFP expressing cells (blue symbols) or cells expressing EGFP-ALG-2 (red symbols) one hour post-treatment. Individual data from at least three independent experiments are shown. Means +/- SEM are indicated. Statistical analysis was performed using unpaired t-test with Welch’s correction. B. Cellular uptake of TMRA-labeled ALIX peptides (top panels: wt ALG-2 binding ALIX peptide, bottom panels: mutated version incapable of ALG-2 binding) with the corresponding brightfield images in the absence (left panel) or the presence (right panel) of digitonin.