Fig 2: Alpha4 protein knockdown alters expression of BCL-2 family of pro- and anti-apoptotic proteins. H9c2 cardiomyocytes were transfected with either 50 nM non-targeting control (NTC) siRNA or 50 nM siRNA specific to rat α4 protein (siα4) for 1–4 days. The expression of Bax and Bcl-2 protein was determined by Western analysis using antibodies raised against human Bax and Bcl-2 protein (a). Quantification of Bax (b) and Bcl-2 (c) protein expression was determined by densitometry. The Bax:Bcl-2 ratio was calculated by dividing the Bax signal by the Bcl-2 signal (d). The expression of Bcl-xL was determined by Western analysis using an antibody raised against human Bcl-xL, in H9c2 cardiomyocytes transfected with either 50 nM non-targeting control (NTC) siRNA or 50 nM siRNA specific to rat α4 protein (siα4) for 4 days (e). Where appropriate, statistical significance was determined either by a one-tailed Student’s T-test or by ANOVA followed by a multiple comparison Tukey’s modified test. All data represents mean values ± SEM of 5 individual experiments. Expression of β-actin was used to confirm equal protein loading between samples.

Fig 3: Alpha4 protein regulates doxorubicin-induced DNA damage and cytotoxicity in cardiomyocytes. H9c2 cardiomyocytes were transfected with either 50 nM non-targeting control (NTC) siRNA or 50 nM siRNA specific to rat α4 protein (siα4) for 4 days. H9c2 cardiomyocytes were exposed to either DMEM vehicle control (CTR) or 1 µM doxorubicin (DOX) for 24 h on day 3. DNA fragmentation in H9c2 cardiomyocytes imaged at × 100 magnification was determined by TUNEL assay. Nuclei were stained with DAPI and samples were excited at 405 nm, whereas DNA fragmentation in samples was determined by fluorescein-conjugated TUNEL and excited at 488 nm. White arrows in merged images indicate positive TUNEL stained nuclei, n = 3, scale bar = 50 µm (a). DNA damage was quantified by counting DAPI stained nuclei followed by the number of TUNEL positive nuclei in 5 randomly selected regions of interest within the field of view, n = 3 (b). H9c2 cardiomyocytes were transfected with either 50 nM non-targeting control (NTC) siRNA or 50 nM siRNA specific to rat α4 protein (siα4) for 4 days. H9c2 cardiomyocytes were exposed to either DMEM vehicle control (CTR) or 100 nM doxorubicin (DOX) for 24 h on day 3. Phosphorylation of H2AX at Ser139 was then determined by Western analysis using a phosphospecific antibody raised against residues surrounding Ser139 of human H2AX, n = 4 (c) and cell viability was determined by MTT assay, n = 3 (d). All data represents mean values ± SEM and statistical significance was determined by ANOVA followed by a multiple comparison Tukey’s modified test. Expression of β-actin was used to confirm equal protein loading between samples.