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. 2020 Feb 24;9:e50041. doi: 10.7554/eLife.50041

Figure 1. Locating the p40MET fragment by immunofluorescence and subcellular fractionation.

(a) Schematic representation of MET receptor cleavage by caspase during apoptosis, with besides representation of the GFP-p40MET fragment and of the GFP-p40MET D1374N fragment which still possesses the C-terminal tail. (b, c, d) MCF10A (b–c) or IHH (d) cells were transiently transfected with a vector expressing GFP, GFP-p40MET, GFP-p40MET D1374N, or flag-p40MET. After 24 hr transfection for MCF10A and 48 hr for IHH, the cells were fixed and labeled with an appropriate antibody: anti-flag when transfected with the vector expressing flag-p40MET, anti-cytochrome C or anti-cleaved caspase 3 antibody to evaluate apoptosis. The percentage of cytochrome C release or of cleaved-caspase-3-positive cells was determined with respect to the number of GFP- or flag-positive cells. At least 150 cells per well (n = 6;± S.D.) (b), 200 cells per well (n = 6;± S.D.) (c) and 60 cells per well (n = 4;± S.D.) (d) were counted. (e) MCF10A epithelial cells were transfected with a vector expressing GFP, GFP-p40MET, or GFP-p40MET D1374N. Twenty-four hours after transfection, the nuclei were stained with Hoechst (blue staining) and immunofluorescence staining was performed with anti-FACL4 to label the MAMs (red staining). Cells were observed by fluorescence confocal microscopy. Weighted colocalization coefficients were determined by means of Manders coefficients for green staining (of GFP, GFP-p40MET, or GFP-p40MET D1374N) and FACL4 staining (red) on the basis of the fluorescence confocal microscopy images (n = 30;±S.D.). (f) MCF10A cells were starved overnight and treated for 4 hr with 1 µM staurosporine (STS). After treatment, the cells were fractionated into ER, MAM and mitochondrial fractions. Proteins from whole-cell lysates (50 µg) and from the different fractions (20 µg) were analyzed by western blotting with antibodies against the MET kinase domain, the reticular protein calnexin, the MAM protein FACL4, and the inner mitochondrial membrane protein MCU. The positions of prestained molecular weight markers are indicated. Arrows indicate the positions of p40MET, calnexin, FACL4 and MCU; scale bar = 10 µm, ns, nonsignificant; *, p<0.05; **, p<0.01; ***, p<0.001 as determined by Student’s t test.

Figure 1—source data 1. Source data of Figure 1b–c–d and Figure 1—figure supplement 1d reporting counting of GFP, active caspase 3, and cytochrome C release positive cells, calculation of the percentage, mean and SD, diagram conception and statistical analyses; source data of Figure 1e reporting the coefficient of fluorescence colocalisation, calculation of the mean, SD and statistical analyses.

Figure 1.

Figure 1—figure supplement 1. Validation of the vectors expressing GFP-p40MET and GFP-p40MET D1374N.

Figure 1—figure supplement 1.

(a) HEK 293 cells were transfected with a vector expressing GFP, GFP-p40MET, GFP-p40MET D1374N or Flag-p40MET. Twenty-four hours after transfection, the cells were lysed. The protein mixture was resolved by 4–12% SDS-PAGE and analyzed by western blotting with antibodies against the MET kinase domain, GFP, and GAPDH. (b–c) Representative pictures of transfected cells immuno-labeled with a cytochrome-c (b) or cleaved-caspase 3 antibody (c) are shown. White arrowheads indicate transfected cells positive for cytochrome-c release or cleaved caspase 3; scale bars = 10 µm (b) and 50 µm (c). (d) MCF10A epithelial cells were transiently transfected with a vector expressing GFP, GFP-p40MET or GFP-p40MET D1374N and treated with zVAD (20 µM). Twenty-four hours after transfection, the cells were fixed and labeled with anti-cytochrome C antibody. The percentage of cells displaying cytochrome C release was determined with respect to the number of GFP-positive cells. At least 60 cells were counted per well (n = 3; mean ± S.D; **p<0.01 as determined by Student’s t test).
Figure 1—figure supplement 2. p40MET fragment generation in IHH cells.

Figure 1—figure supplement 2.

(a) IHH hepatocyte cells were cultured for 24 hr on 6 well plates coated with collagen. Cells were then starved overnight in a serum-free medium and treated 7 hr with staurosporine (STS) or ABT 737 at the indicated concentration before cell lysis. As a positive control, 16HBE epithelial pulmonary cells were starved overnight in a serum-free medium and treated 7 hr with staurosporine (STS) at the indicated concentration before cells lysis. (b), IHH cells were cultured for 24 hr on 6 well plates coated with collagen. Then cells were starved overnight in a serum-free medium and treated 7 hr with 1 µM ABT 737 with or without the pan-caspase inhibitor zVAD-FMK or Q-VD (20 µM). (c) IHH cells were transfected with a control siRNA or a pool of three MET-targeting siRNAs for 48 hr on 12 well plates coated with collagen. Cells were then treated 7 hr with ABT 737 (1µM) before cells lysis. The same amount of proteins was resolved by SDS-PAGE and analyzed by immunoblotting with antibodies against the MET kinase domain, PARP, to assess apoptosis induction, and GAPDH, to assess loading.
Figure 1—figure supplement 3. Partial overlap between GFP-p40MET and mitotracker signals acquired by immunofluorescence.

Figure 1—figure supplement 3.

MCF10A epithelial cells were transfected with a vector expressing GFP, GFP-p40MET, or GFP-p40MET D1374N. Twenty-four hours after transfection the mitochondria were stained with MitoTracker (red), fixed, and observed by fluorescence confocal microscopy.