Fig 1: rERK2-LOC expression faithfully reports localization of ERK2. (A) Non-transfected (top row) and transfected NIH-3T3 cells overexpressing either eGFP-rERK2 (middle row) or rERK2-LOC (bottom row) were serum starved for 24 h, and then were left untreated or were treated with U0126 or DMSO for 1 h. Next, they were stimulated with serum or FGF4 for 15 min, or were left unstimulated (baseline). All cells were fixed and non-transfected cells were processed for immunofluorescence using the anti-ERK1/2 antibody (top row) and all cells were imaged by confocal microscopy. Shown are representative images of ERK2 localization under the different treatments. Scale bars: 20 μm. (B) Quantitative comparison of the nucleo-cytoplasmic concentration index (CI) of ERK2 between endogenous ERK1/2 (blue bars), overexpressed eGFP-rERK2 (red bars) and rERK2-LOC (green bars). CI values were normalized between 0 and 1 (CI¯ values), where 0 and 1 are respectively the minimal and maximal CI values obtained. Bsln: baseline, S: serum, F: FGF4, U0: U0126, V: vehicle (DMSO). (C) Non-transfected (top row) and transfected NIH-3T3 cells overexpressing rERK2-LOC (bottom row) were serum starved for 24 h and then stimulated with serum or FGF4 for 1 or 2 h, or left untreated (baseline). All cells were fixed and non-transfected cells were processed for immunofluorescence using the anti-ERK1/2 antibody (top row) and all cells were imaged by confocal microscopy. Shown are representative images of ERK2 localization under the different treatments. Scale bar: 20 μm. (D) Quantitative comparison of nucleo-cytoplasmic concentration index (CI) of ERK2 at the indicated time-points between endogenous ERK1/2 (blue bars) and overexpressed rERK2-LOC (green bars). CI values were normalized between 0 (minimum obtained) and 1 (maximum obtained) (CI¯ values). Bsln: baseline; 1S and 1F: 1 h serum and 1 h FGF4; 2S and 2F: 2 h serum and 2 h FGF4. (E) Monitoring of the subcellular distribution of rERK2-LOC in (24h) serum-starved NIH-3T3 cells by time-lapse confocal microscopy every 2 min for 10 min (baseline) and after FGF4 stimulation (100 ng/mL) for 30 min. (F) Nuclear and cytoplasmic intensities of each rERK2-LOC transfected cell were measured with Volocity software for each time-point to calculate the concentration index values (CI). Vertical error bars represent the average ± SEM. Two-way ANOVA test, accepting p ≤ 0.05 as significant, was performed to compare CI¯ values differences between endogenous ERK1/2, eGFP-rERK2 and rERK2-LOC for a same treatment. One-way ANOVA test, accepting p ≤ 0.05 as significant, was performed to compare CI¯ values among all the treatments (Tables 2 and 3). At least two independent experiments were performed. The number of cells per condition (n) from fixed cells is indicated in Tables 2 and 3 for statistical analysis; at least 80 cells were measured for time-lapse microscopy.
Fig 2: Equimolar co-expression of eGFP-rERK2 and mMEK1 restores cytoplasmic localization of eGFP-rERK2. (A) Schematic representation of all genetically encoded molecular constructs used in this study. The corresponding amino acid sequence of 2A (yellow box) encodes a T2A peptide isolated from plasmid Myr-TdTomato-2A-H2B-eGFP (#1). Amino acids (GSG) and (GGAP) improve cleavage efficiency. The red slash symbol at the peptide C-terminal end indicates the 2A peptide cleavage site. (B) Fluorescence confocal imaging of NIH-3T3 cells after transfection with different plasmids and serum starvation for 24 h: top, transfection with eGFP-rERK2 (#2) and mCherry-mMEK1 (#3) plasmids; bottom, transfection with mCherry-mMEK1-2A-eGFP-rERK2 (#5). Representative images are shown of rERK2 protein distribution (green: a, e), mMEK1 distribution (red: b, f) and the merged image (c, g). Corresponding scatter plots of green and red intensities of each pixel on the whole images are shown (d, h). Co-localized pixels are visualized in yellow. Scale bar: 20 μm. (C) Fluorescence confocal imaging of NIH-3T3 cells transfected with mCherry-mMEK1 (middle, red) and labeled with an anti-ERK2 antibody (left, green) after 24 h of serum starvation. Co-localization of rERK2 and mCherry-mMEK1 is shown in the merged images (right, yellow). White arrows point to nuclei of transfected cells. Scale bar: 20 μm. (D) Western blot analysis of NIH-3T3 cells transfected with rERK2-LOC at the indicated time-points. Cell lysates were analyzed by immunoblotting with the indicated antibodies (left of each blot). The percentage of uncleaved polypeptide (full-length mMEK1-2A-GFP-rERK2, red triangle) was quantified by densitometry. Quantitative comparison of the levels of overexpressed rERK2-LOC and endogenous ERK2 (green triangles, middle panel) is indicated below the blot as I rERK2-LOC / I rERK2. (E) After 24 h of serum starvation, NIH-3T3 cells transfected with rERK2-LOC were left untreated or were pretreated for 1 h with U0126 or DMSO, and then stimulated with serum or FGF4 for 15 min. Corresponding cell lysates were immunoblotted with the indicated antibodies (left of each blot). Relative phosphorylation levels of rERK2-LOC (green triangles) and endogenous ERK2 (blue triangles) were measured by densitometry. The ratios of phosphorylated protein to total proteins (I pYT-rERK2-LOC / I rERK2-LOC and I pYT-ERK2 / I ERK2) are indicated below the top blot. (F) rERK2-LOC—transfected NIH-3T3 cells were serum starved for 24 h and then left untreated or incubated or not with U0126 for 1 h before stimulation with serum or FGF4 for 15 min. Cells lysates were immunoprecipitated with anti-eGFP (top panel) or anti-ERK2 antibodies (middle panel), and ERK1/2 kinase activity was assayed in vitro. The phosphorylated form of MBP (pMBP) was detected by immunoblotting. Unconjugated beads and beads conjugated with anti-HA antibodies were used as a control in the assays. Lysate inputs for immunoprecipitation were probed with anti-β-actin antibody as a loading control. At least two independent experiments and 15 cells were measured from fixed cells. Biochemical data are representative of at least two independent experiments.
Fig 3: Overexpression of eGFP-rERK2 induces nuclear accumulation of eGFP-rERK2. (A) NIH3T3 cells were serum-starved for 24 h (a) and then stimulated with 10% serum (b) or 100 ng/mL FGF4 (f). In other conditions, cells were pretreated with 20 μM U0126 (c, e, g) or vehicle DMSO (d, h) for 30 min before stimulation with 10% serum (c, d) or FGF4 (g, h). Cells were fixed, processed for double immunofluorescence with antibodies against total ERK1/2 and activated di-phosphorylated YT-ERK1/2, and then imaged by confocal microscopy. A maximum-intensity projection of a 5-μm thick z-stack (step size: 0.3 μm) for each overlapping image is shown. (B) NIH-3T3 cells were transiently transfected with increasing amounts of eGFP-rERK2 plasmid as indicated on the top left of each image, serum-starved for 24 h, fixed, and then imaged by confocal microscopy. The total amount of DNA was kept at 500 ng/mL of medium in all conditions. Higher magnification images of representative eGFP-rERK2 localization are shown in white squares (bottom right). Scale bars: 50 μm. (C) Relationship between the concentration of eGFP-rERK2 plasmid and the concentration index (CI). Higher CI values reflect greater accumulation of eGFP-ERK2 in the nucleus. Average CI was determined by examination of at least 30 randomly selected cells for each of the transfected conditions from two independent experiments. Average CI value for endogenous ERK1/2 in serum-starved NIH-3T3 is also shown (green dotted line). (D) NIH-3T3 cells transfected with 25, 125 or 500 ng/mL of eGFP-rERK2 were observed under severe imaging conditions to visualize cells that express very low level of eGFP-rERK2 protein (upper panel, white squares). Higher magnification images of these cells exhibiting mainly cytoplasmic localization of eGFP-rERK2 are also shown (bottom panel, white arrows). Scale bars: 50 μm.
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