Supplementary Figure 1. Confocal immunofluorescence showing mitochondrial translocation of Drp1. Cardiomyocytes treated with H 2 O 2 were prestained

Size: px

Start display at page:

Download "Supplementary Figure 1. Confocal immunofluorescence showing mitochondrial translocation of Drp1. Cardiomyocytes treated with H 2 O 2 were prestained"

Sheryl May
5 years ago
Views:

1 Supplementary Figure 1. Confocal immunofluorescence showing mitochondrial translocation of Drp1. Cardiomyocytes treated with H 2 O 2 were prestained with MitoTracker (red), then were immunostained with anti-drp1 antibody (green). 1

2 Supplementary Figure 2. Verification of cardiac-specific Pink1 transgenic mice. (a) Schematic map showing the transgenic construct of Pink1. (b) qrt-pcr analyzes the expression of mature Pink1 in different tissues isolated from Pink1 transgenic mice (Tg) and wild-type mice (WT). (c) Detection of Pink1 levels in Pink1 transgenic mice. The expression of Pink1 was analyzed by qrt-pcr from wild type and different lines of mir-421 transgenic mice. (d) Mitochondrial ATP levels in the hearts of control and Pink1 transgenic mice. Wild type mice and Pink1 transgenic mice were subjected to I/R injury. Hearts were harvested and ATP levels measured as described in Methods. Mitochondrial ATP levels were significantly increased in the hearts of Pink1 transgenic mice compared with controls in response to I/R treatment. Data are shown as mean ± s.e.m. of six independent experiments. Analysis was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis. *p<0.05. (e) Mitochondrial fusion is increased in cardiomyocytes from the Pink1 transgenic mouse. Mitochondrial fusion was determined by electron microscopy in hearts from wild type mice and Pink1 transgenic mice. Data are shown as mean ± s.e.m. of six independent experiments. *p<0.05 vs WT in Student s t-test. 2

3 Supplementary Figure 3. Wild-type Pink1 but not kinase dead mutant Pink1 protects against H 2 O 2 -induced mitochondrial fragmentation and apoptosis. (a) Cardiomyocytes were treated with H 2 O 2. Parkin levels in mitochondria were analyzed by immunoblot. n=3. (b) Mice were subjected to Sham or I/R, Parkin levels in mitochondria were analyzed by immunoblot. n=5. (c and d) Cardiomyocytes were infected with adenovirus harboring Pink1, kinase dead Pink1 mutant (Pink1-KD; K219A/D362A/D384A) or -gal, and then exposed to H 2 O 2. The cells were stained with mitotracker-red and the cells with fragmented mitochondria were counted (c). TUNEL was employed to analyze apoptotic cells. TUNEL-positive cells were counted and calculated (d). Data are shown as mean ± s.e.m. of four independent experiments. Analysis was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis. *p<

4 Supplementary Figure 4. The protective effects of Pink1 on mitochondrial fragmentation and apoptosis depend on phosphorylating TRAP1. (a) Cardiomyocytes were treated with H 2 O 2 (upper panel) or mice were subjected to Sham or I/R (lower panel). In vivo phosphorylation of endogenous Miro was determined by immunoprecipitation with anti-miro antibody followed by immunoblotting using anti-phosphoserine and anti-miro antibodies. n=3. (b) Cardiomyocytes and mice were treated as described in (a). In vivo phosphorylation of endogenous Mfn2 was determined by immunoprecipitation with anti-mfn2 antibody followed by immunoblotting using anti-phosphoserine and anti-mfn2 antibodies. n=3. (c) Cardiomyocytes and mice were treated as described in (a). In vivo phosphorylation of endogenous TRAP1 was determined by immunoprecipitation with anti-trap1 antibody followed by immunoblotting using anti-phosphoserine and anti-trap1 antibodies. n=4. (d) Pink1 knockdown reduces TRAP1 phosphorylation. Cardiomyocytes were infected with adenoviral Pink1-siRNA or its scramble form (Pink1-sc). In vivo phosphorylation of endogenous TRAP1 was 4

5 determined by immunoprecipitation with anti-trap1 antibody followed by immunoblotting using anti-phosphoserine and anti-trap1 antibodies. n=3. (e) Cardiomyocytes were infected with adenovirus harboring Pink1, kinase dead Pink1 mutant (Pink1-KD; K219A/D362A/D384A) or -gal, and then exposed to H 2 O 2. In vivo phosphorylation of endogenous TRAP1 was determined by immunoprecipitation with anti-trap1 antibody followed by immunoblotting using anti-phosphoserine and anti-trap1 antibodies. n=4. (f) Cardiomyocytes were infected with adenoviral TRAP1-siRNA or its scramble form (TRAP1-sc). The levels of TRAP1 and actin in the cell lysates were analyzed by immunoblotting with anti-trap1 and anti-actin antibodies. n=3. (g and h) TRAP1 depletion abolishes the effects of Pink1 on mitochondrial fragmentation and apoptosis in response to H 2 O 2. Cardiomyocytes were infected with adenovirus harboring Pink1 and TRAP1-siRNA or TRAP1-sc, and then exposed to H 2 O 2. The cells were stained with mitotracker-red and the cells with fragmented mitochondria were counted (g). TUNEL was employed to analyze apoptotic cells. TUNEL-positive cells were counted and calculated (h). Data are shown as mean ± s.e.m. of four independent experiments. Analysis was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis. *p<

6 Supplementary Figure 5. The expression pattern of mir-421. (a) MiRNAs expression levels upon treatment with H 2 O 2. Cardiomyocytes were untreated (control) or treated with H 2 O 2 for 24 hours. MiRNAs were analyzed by qrt-pcr. Data are shown as mean ± s.e.m. of four independent experiments. *p<0.05 vs control in Student s t-test. (b) The copy numbers of mir-421 in hearts from heart failure mouse were determined by qrt-pcr using standard curve method. Data are shown as mean ± s.e.m. of three independent experiments. (c) The copy numbers of mir-421 in hearts from heart failure human were determined by qrt-pcr using standard curve method. Data are shown as mean ± s.e.m. of three independent experiments. (d) The expression levels of mir-421 and mir-1 were analyzed by qrt-pcr. Data are shown as mean ± s.e.m. of three independent experiments. *p<0.05 vs mir-1 in Student s t-test. (e) Putative mir-421 binding site in the 3 UTR region of Pink1. 6

7 supplementary Figure 6. The effects of mir-421 on Drp1 expression and translation. (a) MiR-421 has no effect on the expression of Drp1. Cardiomyocytes were infected with adenoviral mir-421 or b-gal. The levels of Drp1 were analyzed by immunoblot. n=3. (b) MiR-421 has no effect on Drp1 translation. HEK293 cells were infected with adenoviral mir-421 or β-gal, then transfected with the luciferase constructs of the wild type Drp1-3 UTR. The luciferase activity was analyzed. 7

8 Supplementary Figure 7. Pink1 target protector attenuates the effects of mir-421 on mitochondrial fragmentation and apoptosis. (a) Knockdown of mir-421 attenuates mir-421 levels upon I/R. Adult male C57BL/6 mice (8 weeks old) were delivered in three consecutive days, intravenous injections of mir-421 antagomir (anta-421) or antagomir control (anta-nc) at doses of 30 mg/kg body weight. 3 days after injection the mice were subjected to I/R. Mir-421 levels were analyzed by qrt-pcr. Data are shown as mean ± s.e.m. of six independent experiments. Analysis was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis. *p<0.05 vs I/R alone. (b) Knockdown of mir-421 ameliorates cardiac function after I/R. Mice were treated as described in (a). Transthoracic echocardiographic analysis was performed. LVIDd, diastolic left ventricular internal diameters; FS, fractional shortening of left ventricular diameter. Data are shown as mean ± s.e.m. of six independent experiments. Analysis was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis. *p<0.05 vs I/R alone. (c and d) Pink1 target protector attenuates mir-421-induced mitochondrial fragmentation and apoptosis. Cardiomyocytes were infected with adenoviral mir-421 or b-gal, then transfected with the target protector (Pink1-TP mir-421 ) or the control (Pink1-TP control ). The cells with fragmented mitochondria were counted (c), TUNEL-positive cells were counted and calculated (d). Data are shown as mean ± s.e.m. of three independent experiments. Analysis was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis. *p<

9 Supplementary Figure 8. MiR-421/Pink1 bitransgenic mice exhibit reduced mitochondrial fragmentation, apoptosis and myocardial infarction. (a-c) WT, mir-421 transgenic mice and mir-421/pink1 bitransgenic mice were exposed to I/R. Mitochondrial fragmentation (a), apoptosis (b) and infarct sizes (c) were analyzed. Data are shown as mean ± s.e.m. of seven independent experiments. Analysis was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis. *p<

10 Supplementary Figure 9. E2F1 knockout mice present markedly preserved cardiac function after I/R. E2F1 knockout mice (E2F1 KO) or wild type mice (WT) were subjected to sham-operation or 45 min of ischemia followed by 1 week of reperfusion (I/R). Transthoracic echocardiographic analysis was performed. LVIDd, diastolic left ventricular internal diameters; LVIDs, systolic left ventricular internal diameters; FS, fractional shortening of left ventricular diameter. Data are shown as mean ± s.e.m. of seven independent experiments. Analysis was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis. *p<0.05 vs I/R +WT. 10

11 Supplementary Figure 10. Knockdown of Pink1 attenuates the effect of E2F1 knockdown on Pink1 expression, mitochondrial fragmentation and apoptosis upon H 2 O 2. Cardiomyocytes were infected with adenoviral E2F1-siRNA or E2F1-sc, Pink1-siRNA or Pink1-sc, and then exposed to H 2 O 2. (a) Pink1 expression was analyzed by immunoblot. n=4. (b) Mitochondrial fragmentation (upper panel) and apoptosis (low panel) were analyzed. Data are shown as mean ± s.e.m. of four independent experiments. Analysis was performed with one-way ANOVA followed by Tukey-Kramer post hoc analysis. *p<

12 Supplementary Figure 11. Uncropped images of blots presented in the main paper. Molecular weight markers are indicated in kda. 12

13 Supplementary Figure 12. Uncropped images of blots presented in the main paper. Molecular weight markers are indicated in kda. 13

c Ischemia (30 min) Reperfusion (8 w) Supplementary Figure bp 300 bp Ischemia (30 min) Reperfusion (4 h) Dox 20 mg/kg i.p.

c Ischemia (30 min) Reperfusion (8 w) Supplementary Figure bp 300 bp Ischemia (30 min) Reperfusion (4 h) Dox 20 mg/kg i.p. a Marker Ripk3 +/ 5 bp 3 bp b Ischemia (3 min) Reperfusion (4 h) d 2 mg/kg i.p. 1 w 5 w Sacrifice for IF size A subset for echocardiography and morphological analysis c Ischemia (3 min) Reperfusion (8