I: p-p9rsk I: p9rsk I: C I: p-p9rsk I: p9rsk 5 (ka) 5 5 (min) Ang II ( nm) p-p9rsk (Ser8) p9rsk p-p9rsk (Ser8) p9rsk (h) Mannitol 5 mm -Glucose 5 mm p9rsk (Ser8) (arbitrary units) p-p9rsk (Ser8) (arbitrary units) 4 7 6 5 4 5 5 (min) Mannitol 5 mm Ang II ( nm) (h) -Glucose 5 mm Online figure I. p9rsk activation in cardiomyocytes (A-) Cardiomyocytes were stimulated with Ang II ( nm) (A and ) or high glucose or mannitol (C and ) for the indicated times. p-p9rsk, p9rsk and tubulin were detected by Western blotting of total cell lysates with each specific antibody. ( and ) Intensities of p-p9rsk protein bands were quantified using a Fujifilm image-analysis program (Image Gauge 4.) and were calculated relatively to the intensity of the tubulin band at each time point. Results were expressed as fold increase of p-p9rsk in AngII-treated cells compared to untreated cells. Shown is mean ± S.. (n = ). p <., p <.5 compared to untreated cells.
p9rsk kinase activity (in vitro kinase assay, arbitrary units) Ad-LacZ vehicle Ad-LacZ Ang II Ad-N-p9RSK vehicle Ad-N-p9RSK Ang II I: p9rsk p9rsk I: 5 vehicle Ang II vehicle Ang II Ad-LacZ Ad-N-p9RSK I: p-p9rsk p-p9rsk (Ser8) I: p9rsk p9rsk I: 5 Ang II 6 6 (min) vehicle FMK-MEA ( µm) Online figure II. Ad-N-p9RSK and FMK-MEA, a specific p9rsk inhibitor, blocked Ang IImediated p9rsk activation in cardiomyocytes (A) Cardiomyocytes were stimulated with Ang II ( nm), and p9rsk kinase activity was detected using an in vitro kinase assay as described in methods. The p9rsk kinase activity (upper) was normalized to its expression level, which was evaluated by Western blotting of p9rsk (lower). () Cardiomyocytes were pretreated with vehicle or FMK-MEA for hrs and stimulated with Ang II ( nm) for the indicated times. p-p9rsk, p9rsk and tubulin were detected by Western blotting of total cell lysates with each specific antibody. The blots are representative of data obtained from three separate experiments.
Relative ICER/ mrna (arbitrary units) N.S Ang II ( nm) (min) Ad-LacZ Ad-N-p9RSK I : ICER I : p9rsk I : 5 7 5 ICER p9rsk ICER expression (abitrary units) Ad-WTp9RSK Ad- LacZ Ad-WTp9RSK Ad- LacZ Online figure III. Ad-N-p9RSK did not inhibit Ang II-mediated ICER mrna expression in cardiomyocytes (A), but Ad-WT-p9RSK enhanced ICER protein levels (). (A) Cardiomyocytes were transduced with Ad-LacZ or Ad-N-p9RSK for 4 hrs. Cells were then stimulated with Ang II ( nm) for min, and ICER mrna level was detected by qrt-pcr as described in methods. () Cardiomyocytes were transduced with Ad-LacZ or Ad-WT-p9RSK for 4hrs. ICER, p9rsk and tubulin were detected by Western blotting using the total cell lysates with each specific antibody (left). Intensities of ICER protein bands were quantified using a Fujifilm image-analysis program (Image Gauge 4.) and were calculated relatively to the intensity of the tubulin band at each time point. Results were expressed as fold increase of ICER in Ad-WT-p9RSK-transduced cells compared to the Ad-LacZ-transduced cells. Shown is mean ± S.. (n = ). p <.5.
I: ICER ICER I: 5 N-p9RSK-Tg WT-p9RSK -Tg ouble- Tg Sham MI sham Sham ERK5-CKO I: ICER ICER I: 5 Online figure IV. N-p9RSK-Tg, WT-p9RSK-Tg, ouble-tg, and ERK5-CKO mice showed no difference of ICER expression in sham operated mice. Expression of ICER and tubulin in heart samples collected from sham and MI in, sham in N-p9RSK-Tg, WT-p9RSK-Tg and ouble-tg mice (A), and heart samples from sham in and ERK5-CKO ().
C!STZ i.p. (mg/kg/ W) (%) Survival rate 8 6 4 Coronary ligation N-p9RSK 4 6 8 4 (days) (n = 5) (n = 7) p <.5 Random S (mg/dl) 4 M + MI - + + ody weight (g) Np9RSK-Tg Np9RSK-Tg 4 6 8 M + MI - + + LV/TL (mg/mm) Np9RSK-Tg 7 5.5.5. M + MI - + + Lung/TL (mg/mm) 5 5 M + MI + + E LVEd (mg/min) 6 5 4 M + MI + + 5 4 M + MI + + LVESd (mg/min) FS (%) 7 6 5 4 M + MI + + EF (%) 5 5 M + MI + + Np9RSK-Tg Np9RSK-Tg Np9RSK-Tg Np9RSK-Tg Np9RSK-Tg F G TUNEL API TUNEL-positive cells (%).6.4. M + MI - + + TUNEL/ API/αactinin Np9RSK-Tg Sham M + MI N-p9RSK-Tg Online figure V. Inhibition of p9rsk activation prevented the exacerbation of LV dysfunction after MI in diabetic mice. (A) Kaplan-Meier survival analysis in diabetic (n=7) and N-p9RSK-Tg (n=5) after MI. Overall survival was significantly higher in N-p9RSK-Tg compared to mice. p <.5 compared to group. () Random blood sugar (S) and body weight at one week after STZ injection for coronary ligation (M + MI) or vehicle-treated sham operation groups in or N-p9RSK-Tg mice. (mean ± S.., n =9-) (C) LV weight/tl (mean ± S.., n =9-) and () lung weight/tl in M + MI or vehicle-treated sham operation groups in or N-p9RSK-Tg mice one week after surgery. (p <., mean ± S.., n=9-). (E) Echocardiographic data obtained from M + MI, or vehicle treated sham operation groups in or N-p9RSK-Tg mice one week after surgery. LVEd, left ventricular end-diastolic dimension; LVESd, left ventricular end-systolic dimension; TL, Tibial length. (p<.5, p <., mean ± S.., n=9-).(f) Cardiomyocytes apoptosis in the remote area was increased in M + MI group, which was inhibited in N-p9RSK-Tg mice. TUNELpositive cardiomyocytes were counted in the remote area as described in Fig.6. Representative pictures of TUNEL (top), API (middle), and merged of α-actinin with TUNEL and API staining (bottom) of the remote area from mice subjected to sham or M+MI, and N-p9RSK-Tg mice subjected to M + MI operation. 4X objective lens. Scale bars: 4 µm (G) A bar graph showing TUNEL-positive cells (%) in and N-p9RSK-Tg. ( p <.5, mean ± S.., n =).
N-p9RSK-Tg Sham M + MI CHIP Ub E ligase assay 5 5 5 Quantification area Relative levels of ubiquitinated GST-ICER expressions.9.7.879.4.8.4.6.7.54 Online figure VI. Quantification of CHIP ubiquitin E ligase activity. The activities of CHIP Ub E ligase were assessed by quantifying ubiquitinated GST-ICER fusion proteins. riefly, protein was extracted by modififed RIPA buffer, and then immunoprecipitated with anti-chip antibody. CHIP proteins were immunopresipitated by protein A and G agarose mixture. After that, GST-ICER protein as added to each sample. CHIP Ub E ligase activity was detected using Ubiquitin-Protein Conjugation kit. Science Lab Image gauge software (version 4; Fuji Photo Film, Tokyo, Japan) was used for the analysis. Ubiquitinated GST-ICER expressions were calculated in equal area described in white broken squires. Relative levels were indicated as based on means of sham mice. 6
IP: ERK5 C IP ERK5 I: CHIP 7 CHIP I: CHIP 7 CHIP I: ERK5 ERK5 I: ERK5 ERK5 I: Myc 7 I: HA 5 I: Flag I: VP6 5 5 I: Myc-CHIP + - + + + + + + + Myc-CHIP HA- CA-MEK5α Flag-p9RSK VP6-ERK5 Fr (57-87) I: Myc I: HA I: Flag 7 5 Myc-CHIP + - + + + + + + + CA-MEK5α - + + + + + + + + Np9RSK WTp9RSK CHIP HA- CA-MEK5α Flag-N-p9RSK or WT-p9RSK CA-MEK5α - + + + + + + + + ERK5-CHIP binding (arbitraty units).5.5 WTp9RSK Fr VP6-ERK5 (57-87) ERK5-CHIP binding (arbitraty units)...8.6.4. Myc-CHIP + - + + + + + + + CA-MEK5α - + + + + + + + + Myc-CHIP + - + + + + + + + CA-MEK5α - + + + + + + + + Np9RSK WTp9RSK WTp9RSK VP6-ERK5 Fr (57-87) Online figure VII. p9rsk kinase activation inhibits ERK5-CHIP association. (A) oth wild type p9rsk (WT-p9RSK) and ERK5-Fr (aa57-87) disrupted ERK5-CHIP association. After plasmids were transfected as indicated, cell lysates will be immunoprecipitated with anti-erk5 and then immunoblotted with anti-chip. Protein expression was determined by immunoblotting with each specific antibody. () Relative ERK5-CHIP binding was quantified as described in Fig. (mean±s.., n=, (p<., p<.5 compared to both Myc-CHIP and CA-MEK5α transfected cells (the third black bar from left). Results are expressed as fold decrease in ERK5-CHIP binding in WT-p9RSK (grey bars) and ERK5-Fr (aa58-87) (white bars) transfected cells compared to the control cells (the third black bar from left). (C) WT-p9RSK but not N-p9RSK fully disrupted ERK5-CHIP interaction. ERK5-CHIP binding and each protein expression were detected as described in (A). () Relative ERK5-CHIP binding was quantified as described in Fig.. Results are expressed as fold decrease in ERK5-CHIP binding in cells over-expressing WT-p9RSK. p<., mean±s.., n=.
I: p-p9rsk p-p9rsk (Ser8) I: p9rsk p9rsk I: p-erk5 p-erk5 (T8/Y) I: ERK5 ERK5 I: p-erk / I: 7 5 p-erk / vehicle HO µm Ang II nm Online figure VIII. Ang II increased only p9rsk but not ERK5 kinase activity in cardiomyocyte. Cardiomyocytes were stimulated with Ang II ( nm) or HO ( µm) for 5 min. p- p9rsk, p9rsk, p-erk5, ERK5, p-erk/, and tubulin were detected by Western blotting using the total cell lysates with each specific antibody. Representative immunoblots from duplicate experiments are shown.
A ouble-tg (FV) WT (FV) MI M+MI WT (c57l) CKO (c57l) MI MI MI 5 Infarction size (%) 4 ou M +M bl ei Tg (F V )M W I T (c 57 L C )M KO I (c 57 L )M I (F V ) W T W T (F V )M I Online figure IX. Evaluation of infarction size at one week after MI surgery. (A) Representative pictures of LV tissue sections by Masson s trichrome staining. Infarct areas were described in black triangle between broken lines. 4X objective lens. Scale bars: µm. () Comparision of MI sizes. The sizes were not significantly changed in each groups. ata are shown as means ± S.. (n=). 9