Fig 1: Restoration of ER-mitochondrial proximity without BOK does not rescue apoptosis or stimulus-induced Ca2+ transfer(A) Time course of FRET fluorescence during early treatment with 2 µM thapsigargin (arrowhead) in WT and Bok-/- cells.(B) Time course of FRET fluorescence during induction of the linkage by 100 nM rapamycin (arrowhead).(C) Percent annexin-V-positive WT and Bok-/- cells treated with DMSO (DMSO), treated with 100 nM rapamycin for 10 min followed by fresh media containing 2 µM thapsigargin for 16 h (Rapa + TG) after mock transfection, or transiently transfected with the plasmid and treated with rapamycin and thapsigargin (Rapa +TG + linkers). Data are represented as mean ± SD. Significance was calculated using an ANOVA test (ns, not significant).(D) Fluorescence intensity is measured in defined regions of interest in WT and Bok-/- MEFs by the Ca2+ indicator dyes Mag-Fluo-4 for the ER (first column), Fluo-3 for the cytoplasm (second column), or Rhod-2 for the mitochondria (third column). Cells were untreated (top row) or treated with 100 nM rapamycin (bottom row) for 10 min before being treated with 2 µM thapsigargin (arrowhead). A representative curve from at least three independent experiments is plotted. Measurements were made on at least 5 fields with more than 8–10 cells per field (at least 50–60 cells total) for each condition within each experiment.See also Figure S5.
Fig 2: Bok-deficiency sensitizes against DNA damaging agents.a DNA damage in Etoposide-treated LKR10 LacZ and Bok-/- cells. Cells were treated with 6 µg/ml Etoposide for indicated times and the level of DNA damage (% DNA in tail) analyzed by alkaline comet assay. Percent tail in DNA (% DNA in tail as measured in comet assay) after exposure to Etoposide (left panel). Representative microscopic images of the comet assay (right panel). Data are means ± SEM of at least 25 cells per group analyzed across two slides. Statistical differences at the indicated times were determined by unpaired t-test with Welch correction. b Cell viability of LKR10 LacZ and Bok-/- cells treated with Etoposide (4 µg/ml) or Irinotecan (80 µM) for indicated times. Data are means ± SEM of three independent experiments. Significant differences from LacZ were revealed by two-way ANOVA, Bonferroni’s multiple comparison test (*: P < 0.05; **: P < 0.01; ***: P < 0.001).
Fig 3: Mitochondria-associated membrane (MAM) proteins are mis-localized in the absence of BOK by immunofluorescence(A–C) Localization in a single plane by confocal microscopy of MitoTracker (first column) with respect to (A) FACL-4, (B) IP3R1, and (C) IP3R2 (second column). The merged image is shown at low (third column) and high (fourth column) power. (D) Quantification of percent co-localization of the individual protein with MitoTracker in 20–30 images per condition from three independent experiments. Data are represented as mean ± SD. Significance was calculated using an ANOVA test (*p < 0.05).See also Figure S3.
Fig 4: The effect of Bok in murine NSCLC is dependent on functional p53.a Representative images of H&E staining of lungs of the indicated genotypes 13 weeks after infection with 5 × 106 PFU of AdenoCre. Lower panels are higher magnification of indicated areas. Scale bars represent 1 mm or 100 µm, respectively. b Quantification of the H&E staining expressed as the ratio between the area of the lesions over the total lung area. Data were analyzed by one-way ANOVA (p = 0.9654, F(2,20) = 0.0325). c Quantification of the H&E staining expressed as number of lesions per mm2. Data were analyzed by one-way ANOVA (p = 0.5803, F(2,20) = 0.5593). d, e Representative images, and quantification of Ki67 staining. Data are reported as number of positive cells normalized to lesion area. Data were analyzed by Kruskal–Wallis test (p = 0.1013, H(3) = 4.579). f Quantification of ?-H2AX staining. Data are reported as percentage of positive staining normalized to lesion area. Data were analyzed by Kruskal–Wallis test (p = 0.0783, H(3) = 5.094). For the quantifications reported in panel (b, c), three sections per animal, separated by 100 µm, were analyzed. For the quantification in panel (e) and (f), six lesion per slide/animal were evaluated. n = number of animals. Data in panel (b, c, e, and f) are presented as dot plot and report mean ± SEM.
Fig 5: Disruption of the BOK-IP3R interaction decreases MAM proteins with minimal impact on ER-mitochondrial apposition(A) Representative western blot analysis (n = at least 3) for MAM proteins in WT and Bok-/- MEFs as well as Bok-/- MEFs with stable expression of WT FLAG-BOK (+WT) or L34G FLAG-BOK (+L34G). Equal total protein was loaded for each fraction as described in Figure 4. Molecular weight markers are shown in kDa on the left.(B) Representative transmission EM images of Bok-/- MEFs stably transfected with WT (left) or L34G (right) FLAG-BOK. The bottom row shows bounding image of the middle row with mitochondria in red and ER in green as completed in ImageJ.(C) The top scatterplot shows the distance between the mitochondria and ER in WT (n = 125 sites), Bok-/- (n = 117 sites), Bok-/- + WT FLAG-BOK (n = 127 sites), and Bok-/- + L34G FLAG-BOK (n = 124 sites) MEFs from more than 30 EM images. The bottom scatterplot shows the length of each contact site for those that are less than 15 nm between the ER and mitochondria in WT (n = 27), Bok-/- (n = 25), Bok-/- + WT FLAG-BOK (n = 38), and Bok-/- + L34G FLAG-BOK (n = 32) MEFs. WT and Bok-/- MEF data are from Figure 2C. Data are represented as individual values and mean (bar). Significance was calculated using an ANOVA test (ns, not significant).See also Figure S6.
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