Phalloidin staining of F-actin.

<p>(A) Photographs depict EPCs pre-treated with or without MAPK inhibitors, and then treated with 0.25 µM doxorubicin (magnification 1000X). (B) Bar-graph showing cell size in various treatment groups. ct, control; Dox, 0.25 µM doxorubicin. SB, p38 inhibitor; SP, JNK inhibitor. *p<0.05 vs ct; § p<0.05 vs Dox.</p

Doxorubicin induces cell cycle alterations and impairs cell viability.

<p>(A) Cell cycle analysis: the blue area represents the control cells, the red line indicates treated cells. Cells were cultured for 24 h after treatment with doxorubicin 0,25 µM and then analyzed. Phases of the cell cycle are indicated: Sub G0 [M1], G0/G1 [M2], S [M3], G2/M [M4], >4N [M5]. (B) Cell proliferation evaluated by BrdU incorporation. Cells were cultured for 24 h after treatment with various doses of doxorubicin and then analyzed. BrdU data were normalized to untreated (media alone) cells (C), Number of viable cells evaluated by MTT assay. Cells were analyzed before treatment and 24 hours after treatment with various doses doxorubicin. ct, control; Dox, doxorubicin. *p<0.05 vs ct. § p<0.05 vs 0 h; # p<0.05 vs ct 24 hr.</p

EPC migration assay.

<p>Graph represents the percentage of migrating EPCs assessed by a modified Boyden chamber assay; EPCs were untreated or incubated with 0.25 µM doxorubicin and then exposed to: VEGF, medium, or conditioned medium from normal or doxorubin-induced senescent or apoptotic H9c2 cells. ct, control; Dox, doxorubicin; SP, JNK inhibitor; SB, p38 inhibitor; ct, control; *p<0.05 vs medium; # p<0.05 vs untreated cells exposed to VEGF; ¤ p<0.05 vs. all conditions.</p

Dose-effect curve.

<p>(A) Bar-graph showing the percentages of SA-b-gal and ssDNA positive cells after treatment with various doses of doxorubicin. (B) Photographs illustrating the effects of doxorubicin 0.25 and 1 µM. Cells were evaluated for (from top to bottom): SA-b-gal activity (magnification, ×200), ss-DNA positivity (magnification, ×200), and AV/PI staining (magnification, ×400). ct, control; Dox, doxorubicin. *p<0.05 vs control.</p

p38 and JNK control the cell response to sub-apototic doses of doxorubicin differently.

<p>(A):effects of exposure to 0,25 µM doxorubicin on MAPK activation by western blot using antibodies specific for phosphorilated (Ph) and total p38 and JNK (left panel). Bar graph showing values for ph-MAPK normalizzated to the amount of total enzyme and expressed as the relative increase above control value, which was set at 100 (right panel). (B) and (C): bar-graphs showing the effects of pre-treatment with the JNK inhibitor, SP600125, the p38 inhibitor, SB203580 and NAC on the percentage of SA-b-gal and ssDNA positive cells (panel B) and p16^INK4A protein levels (panel C). (D): merge of SA-b-gal staining (blue) and immunocytochemistry for p16^INK4A (brown) in cells exposed to doxorubicin and pre-treated with or without SP600125, SB203580, or NAC (magnification, ×400)<b>.</b> ct, control; Dox, 0.25 µM doxorubicin. SB, p38 inhibitor; SP, JNK inhibitor. *p<0.05 vs ct; § p<0.05 vs Dox.; ¤ p<0.05 vs. SP+Dox; # p<0.05 vs. SB+Dox.</p

Pulsed incubation of doxorubicin induces telomeric dysfunction and G2 cell cycle arrest.

<p>Cells were cultured for 24 h after treatment with doxorubicin 0,25 µM and then analyzed. (A)Western blot analysis of TRF2. (B) Metaphase spreads. Chromosomal abnormalities are indicated by arrows (magnification, ×1000).</p

Effect of exogenous IGF-1 on doxorubicin-induced apoptosis of H9c2 cells: TUNEL and caspase 3/7 activity.

<p>Frequency of apoptotic cells, as assessed by TUNEL (A; representative microphotographs are shown in B) and fluorescence (AUF) produced by the cleavage of a substrate of activated caspase 3/7 (C), 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with doxorubicin (Dox) ± IGF-1 at the indicated concentrations. ***, P <0.001 vs. Ctr. c, P <0.001 vs. Dox 0.1; d, P <0.001 vs. Dox 0.5; e, P <0.01 vs. Dox 0.1; f, P <0.05 vs. Dox 0.5. ¥, P <0.001 vs. Dox 0.1 + IGF-1 100; ¢, P <0.001 vs. Dox 0.1 + IGF-1 100 and Dox 0.5 + IGF-1 100.</p

Doxorubicin stimulates apoptosis and modulates IGF-1R/IGFBP-3 expression in H9c2 cells.

<p>Frequency of apoptotic cells (A) and IGF-1R (B) and IGFBP-3 (C) expression (densitometry of western blot bands) 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with 0.1, 0.5, or 1 μM doxorubicin (Dox). A representative western blot for IGF-1R and IGFBP-3 is shown in (D). *, P <0.05 vs. Ctr; **, P <0.01 vs. Ctr; ***, P <0.001 vs. Ctr. a, P <0.01 vs. Dox 0.5; b, P <0.05 vs. Dox 0.1; c, P <0.001 vs. Dox 0.1.</p

Involvement of p53 in the change in IGF-1R /IGFBP-3 levels caused by doxorubicin.

<p>(A) Representative western blot and band densitometry for p53 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with 0.1, 0.5, or 1 μM doxorubicin (Dox). (B and C) IGF-1R/IGFBP-3 expression (band densitometry and representative western blot, B) and annexin V/propidium iodide positivity (C) in H9c2 cardiomyocytes untreated or exposed to 1 μM Dox with or without pre-treatment with PFT-α. *, P <0.05 vs. Ctr; ***, P <0.001 vs. Ctr. ^, P <0.01 vs. Dox 1.</p

Effect of exogenous IGF-1 on doxorubicin-induced apoptosis of H9c2 cells: annexin V/propidium iodide.

<p>Frequency of apoptotic cells, as assessed by annexin V/propidium iodide staining, 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with doxorubicin (Dox) ± IGF-1 at the indicated concentrations. *, P <0.05 vs. Ctr; **, P <0.01 vs. Ctr; ***, P <0.001 vs. Ctr. e, P <0.01 vs. Dox 0.1; ¶, P <0.05 vs. IGF-1 0.01.</p