Fig 1.
Synthetic flavagline (FL3) binds PHB1 and PHB2 and increases PHB1 leves in H9c2 cells.
A. Whole-cell extracts of the H9c2 line (input) were either incubated with the beads Affi-Gel 10 conjugated with FL3 or blocked with ethanolamine (unconjugated Affi-Gel) [7,17]. The bound and eluted proteins (Affi-Gel-FL3) and output proteins (output Affi-Gel-FL3) were analyzed by western blotting using antibodies against PHB1 and PHB2 (n = 3). B and C. Representative western blot analyses and histogram based quantification of total PHB1 levels in the cell lysates by. FL3 alone increased PHB1 protein levels within 10h as compare to non-treated cells (NT). However PHB1 level was lower in the present of both FL3 and doxorubicin (n = 3). D and E. Representative western blot analyses and histogram based quantification of nuclear PHB1 levels by. Doxorubicin accumulates PHB1 in the nucleus but lowers in the cytoplasm that was reduced by preconditioning with FL3. * Indicates p<0.05 as compare to control, ** indicates p<0.05 as compare to the doxorubicin alone group.
Fig 2.
The flavagline FL3 induces translocation of PHB1 to mitochondria in cardiomyocytes.
A. H9c2 cells were incubated with FL3 (100 nM) and analyzed by confocal microscopy. The cells were co-labeled with the anti-PHB1 antibody (green staining), mitotracker (red staining), and (DAPI; blue staining). The latter two dyes stained mitochondria and the nucleus, respectively. Merged confocal images show that FL3 induced the translocation of PHB1 to mitochondria (white arrows show PHB1 and mitotracker co-localization). B. The histogram shows quantitative analyses of co-localization of PHB1 and Mito Tracker in each cell by confocal analyses (n = 6). C. Representative illustration of PHB1 levels in mitochondrial and nuclear fractions upon FL3 treatment. In the mitochondrial fraction PHB1 accumulation by FL3 occurred within 20 min. PHB1 was initially increased in nucleus and rapidly reduced within 20 min. D and E. The histogram shows quantitative analyses of mitochondrial and nuclear PHB1 levels upon treatment of H9c2 cells with FL3 (100 nM). * Indicates p<0.05 as compare to vehicle (n = 3).
Fig 3.
The mitochondrial STAT-3 phosphorylation is correlated with PHB1 translocation to mitochondria by FL3 in cardiomyocytes.
A and B. Representative western blot analyses and histogram based quantification of mitochondrial STAT3 activation by phosphorylation. STAT3 was phosphorylated by FL3 in mitochondrial fraction. C and D. Representative western blot analyses and histogram based quantification of nuclear STAT3 activation by phosphorylation. STAT3 activation was only detected in nucleus after FL3 treatment. E and F. The western blot and histogram show quantitative analyses of nuclear phosphorylated STAT3 levels upon treatment of H9c2 cells with control (DMSO), FL3 (100 nM), doxorubicin (1μM), and FL3 + doxorubicin. Doxorubicin elevated phosphorylated STAT3 levels, which were reduced by FL3 (n = 3; *p < 0.05, compared to vehicle; **p < 0.05, compared to doxorubicin treatment).
Fig 4.
FL3 rapidly induces phosphorylation of STAT3.
A. STAT3 coimmunoprecipitates (co-IP) with PHB1. An anti-FLAG antibody was incubated with extracts of the H9c2 cardiomyocytes. Immunoprecipitates were resolved by means of SDS-PAGE and probed for STAT3 and the FLAG tag to detect both PHB1 and PHB2. B. Representative western blots of protein lysates of H9c2 cells treated with FL3 (100 nM), by means of antibodies that recognize either phosphorylated (Tyr705) or total STAT3 protein. C. Quantitative analysis of the western blots (percentage of phosphorylated STAT3 in total STAT3, n = 4; *p < 0.05, compared to control; **p < 0.001, compared to control; ***p < 0.01, compared to control). D and E. Effects of the Janus kinase 2 (JAK2) inhibitor WP1066 on STAT3 phosphorylation: Representative western blots and quantitative analysis (percentage of phosphorylated STAT3 in total STAT3, n = 4; p < 0.05, compared to control; **p < 0.05, compared to FL3).
Fig 5.
FL3 protects H9c2 cardiomyocytes by acting on PHBs and their signaling target STAT3.
A. The histogram shows the percentage of apoptotic cells induced by doxorubicin (1 μM) among H9c2 control cells (transfected with nonspecific small interfering RNA [si-NT]) or the H9c2 cells where PHB1 or PHB2 were downregulated using specific small interfering RNA (siRNA). Knocking PHB1 or PHB2 down greatly diminished the cardioprotective effect of FL3 (100 nM; n = 4 to 5; *p < 0.05, compared to vehicle; **p < 0.05, compared to doxorubicin (doxo). B. The TUNEL assay shows the percentage of apoptotic cells in the 4’,6-diamidino-2-phenylindole (DAPI)-positive total cell population. C. Fluorescence-activated cell sorting (FACS) analysis shows the percentage of the maximum among annexin V-positive cells (n = 3; *p < 0.05, compared to vehicle; **p < 0.05, compared to doxorubicin treatment).
Fig 6.
Small-interfering-RNA (siRNA)-mediated downregulation of PHB1 proteins attenuates FL3-induced cardioprotection from doxorubicin toxicity.
A. Representative western blots show induction of STAT3 phosphorylation by the synthetic flavagline (FL3) in H9c2 cells transfected with nonspecific siRNA (left) or with anti-PHB1 siRNA (right). B. Quantification of phosphorylated STAT3, with normalization to actin (n = 3; *p < 0.05, compared to vehicle). FL3-mediated STAT3 activation by phosphorylation was abolished when the expression of PHBs were reduced. C. This histogram shows downregulation of PHB1 after transfection of H9c2 cells with anti-PHBs siRNA (n = 3; *p < 0.05, compared to vehicle). D. This histogram shows downregulation of PHB2 after transfection of H9c2 cells with anti-PHBs siRNA (n = 3; *p < 0.05, compared to vehicle).
Fig 7.
Proposed mechanism of FL3-induced cardioprotection from doxorubicin toxicity.
Doxorubicin induces the translocation of PHB1 and phosphorylated STAT3 in the nucleus of cardiomyocytes to induce apoptosis. On the opposite, FL3 induces the translocation of these signaling proteins into mitochondria to protect the cell against the adverse effects of doxorubicin.