Journal: The Journal of Biological Chemistry
Article Title: The Yeast Homolog of Heme Oxygenase-1 Affords Cellular Antioxidant Protection via the Transcriptional Regulation of Known Antioxidant Genes *
doi: 10.1074/jbc.M110.187062
Figure Lengend Snippet: Heme oxygenase-1 translocates to the (peri)nuclear region in response to H2O2. A, confocal microscopy images of HA-tagged Hmx1p cells treated with 4 mm H2O2 for 6 h and then stained as described under “Experimental Procedures.” Arrows denote areas (white) that show staining with all three markers, DAPI (blue), anti-HA (green), and ER marker DiOC6(3) (red), indicating (peri)nuclear localization of Hmx1p. Scale bars 10 or 5 μm, as indicated. B, quantification of the experiments shown in A. The extent of (peri)nuclear localization of Hmx1-HAp (open bars) and HA-Hmx1p (closed bars) in H2O2-treated cells was compared by counting cells that displayed distinctive (peri)nuclear staning, as assessed by acquisition and analysis of z-stacks taken of >1,000 cells for each of two separate experiments. C, cells were treated as in A, followed by cell fractionation into cytosolic (C) and nuclear fraction (N), and Western blotting using antibodies directed against HA (Hmx1p), Pgk1 (cytosol), Nop2 (nucleus), Dpm1p (ER), and Kar2 (ER). The results shown are representative of three separate experiments. D, quantification of the data obtained in C. The extent of (peri)nuclear/ER-localized Hmx1-HAp (open bars) and HA-Hmx1p (closed bars) in cells treated with H2O2 (4 mm, 6 h) was assessed by determining the respective density ratios of Hmx1p to either Nop2, Dpm1, or Kar2 from three separate experiments using ImageJ, with the value for the respective ratio for Hmx1-HAp set at 1. *, p < 0.05 compared with corresponding Hmx1-HAp (Wilcoxon Rank Sum Test).
Article Snippet: Blots were incubated with either mouse monoclonal anti-HA (to localize Hmx1p) (Sigma, 1:5,000 dilution), rat anti-tubulin (loading control) (Abcam, 1:5,000), mouse anti-Pgk1 (cytosol marker) (Invitrogen, 1:5,000), mouse anti-Nop2 (nucleus marker) (Abcam, 1:5,000), mouse anti-Dpm1 (marker for ER and nuclear membrane-ER network) (Molecular Probes, 1:500) or rabbit anti-Kar2 antibody (ER marker) (Santa Cruz Biotechnology, 1:1,000), and bound antibody visualized by chemiluminescence (ECL, Amersham Biosciences) following incubation with sheep anti-mouse immunoglobulin-horseradish peroxidase conjugate (Amersham Biosciences, 1:5,000), sheep anti-rat immunoglobulin-horseradish peroxidase conjugate (Sigma, 1:5,000), or goat anti-rabbit immunoglobulin-horseradish peroxidase conjugate (Abcam, 1:5,000).
Techniques: Confocal Microscopy, Staining, Marker, Cell Fractionation, Western Blot
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![Receptor tyrosine kinase (RTK) signaling is restricted to the front of migrating leader cells. (a,b) bFGF-induced tyrosine phosphorylation was higher in the front of migrating cells (white arrows). Addition of the pan-RTK inhibitor Ponatinib blocked tyrosine kinase signaling in the front, but not in the back of leader cells. Follower cells did not respond to bFGF. HUVECs were fixed and stained with pY20 anti-phospho-tyrosine antibody (n = 107, 105, 115, 110 and 107 cells for SFM, follower cells, and Ponatinib 0 nM, 25 nM and 100 nM, respectively). SFM: serum-free medium. (c,e) Fluorescence ratio images of leader cells co-expressing YFP-Akt-PH (PIP 3 sensor) or YFP-C1AC1A (DAG sensor) and a <t>plasma</t> <t>membrane</t> marker <t>(CFP-mCD4).</t> PIP 3 (c) and DAG (e) were enriched in the front of migrating cells. (d,f) Front-to-back gradients of PIP 3 and DAG were present in leader, but not follower cells (24 leader and 42 follower cells in (d) , 28 and 62 cells in (f) ). (g) Ca 2+ pulses in migrating HUVECs were measured as relative increases in local PM targeted GCaMP6s fluorescence intensity. Higher activities were observed in the front (#1) compared to the middle (#2) or back (#3) of migrating cells. (h) Relative mean amplitudes of local Ca 2+ fluctuations measured over 3 minutes in the front of migrating cells in response to serum or serum plus Ponatinib (see also ). Amplitudes of Ca 2+ fluctuations were normalized to basal cytosolic levels (0.3 R.U. means the fluctuation is 30% of the average cytosolic [Ca 2+ ] level; n = 24 cells). (i,j) Migrating HUVECs expressing GCaMP6s-CAAX and the reference membrane marker mCD4 were used to measure Ca 2+ gradients in leader and follower cells (n = 83 leader and n = 86 follower cells). Bars denote mean ± SEM in Fig. 1b,h. Student t test was used for Fig. 1b,d,f,h,j. In Fig. 1d,f,j, p values were calculated by comparing the ratio of the sensor / PM intensity ratios in the front and back (both regions were 10% of cell length).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3390/pmc03953390/pmc03953390__nihms548211f1.jpg)
