Fig 1: Inhibited expression of Col-III and a-SMA following loss of function of Adamts4. IF with anti-Col-III (shown in green) and anti-a-SMA (shown in red) antibodies show inhibited expression of these markers under ATSsi + H2O2 and ATSsi + Hyp conditions when compared with H2O2 and Hyp treated cells (a) Col-III showed a reduction from 4 and 4.25 fold observed for the H2O2 and Hyp treatment groups in comparison with the control group to 1.8 and 2.2 fold observed in ATSsi + H2O2 and ATSsi + Hyp groups (a and b). Similarly, for a-SMA, H2O2 and Hyp treated cells show a significant 4.5 and fourfold increased expression when compared to control, the ATSsi + H2O2 and ATSsi + Hyp treated cells show a fold change of 2.5 and 2.4 increments when compared to the control group (a and c). DAPI (shown in blue) was used as nuclear stain. n = 3, p < 0.05 is considered as significant for differences among groups. Data analysed and expressed as mean ± SD.
Fig 2: Reduced expression of Periostin following Adamts4 loss of function. IF with anti-Periostin antibody (shown in green colour) shows a fold change of 4 and 3.7 increase in the H2O2 and Hyp treated group in comparison to the control set, this elevation is significantly reduced 2.4 and 2.2 in ATSsi + H2O2 and ATSsi + Hyp groups following siRNA mediated knockdown of Adamts4 (a). DAPI (shown in blue) was used as nuclear stain. Its quantification is depicted in the graph (b). n = 3, data analysed and expressed as mean ± SD. Differences were considered statistically significant for p < 0.05.
Fig 3: Periostin expression is reduced after ALKI pre-treatment and successful knockdown of Adamt4 mRNA in cultured H9c2 cells. IF with anti-Periostin antibody (shown in green) shows inhibition of Periostin expression under ALKI + H2O2, and ALKI + Hyp conditions as compared to only H2O2 and Hyp treated conditions. A reduced expression of 2.3 and 2 folds for ALKI + H2O2, and ALKI + Hyp was observed which was a decrease from the 4 and 3.7 fold change found for only H2O2 and Hyp treatments (a and b) in comparison to control. Successful knockdown of Adamts4 via Adamts4 siRNA transfection is shown by Adamts4 WB (shown in c and d), a 0.46 fold expression in the knockdown group against the control group was found. Further, Adamts4 IF (shown in green) also validated the successful knockdown of Adamts4. A decrease in the mean fluorescence intensity from 1 as observed for Scsi treatment set to 0.33 for ATSsi tr group was found. (e and f). DAPI (shown in blue) was used as nuclear stain. Finally, from qPCR Adamts4 mRNA levels were downregulated from onefold observed for Scsi tr set to 0.25-fold for ATSsi tr group (g). n = 3, data analyzed and expressed as mean ± SD. Differences were considered statistically significant for p < 0.05.
Fig 4: Dynamic expression of Adamts4 protein in embryonic and adult heart. Adamts4 expression is shown in developing E10.5 (a), E12.5 (b), E14.5 (c), E18.5 (d) adult (e) murine hearts. IHC with anti-Adamts4 antibody (green colour) and Topro3 (blue colour) used as nuclear stain, the expression pattern of Adamts4 is observed in (a)–(e). While the expression pattern of Adamts4 is more widespread in the embryonic stages throughout the RA, LA, RV, LV and IVS (a–d), the expression drastically reduces and is mainly only confined at the IVS in the adult stage (e) displaying a sharp contrast between the expression of Adamts4 in developing and adult stages. (f) Highlights the colocalization of Adamts4 (green) in the chamber myocardium with E13.5 chamber cardiomyocytes shows colocalization of Adamts4 with MF20 (shown in yellow colour) across the LV of chamber myocardium. The arrowheads point towards cells that have colocalized with both the markers ie, Adamts4 and MF20. This confirms the expression of Adamts4 in embryonic cardiomyocytes. n = 4.
Fig 5: Injury induced overexpression of Adamts4 and fibrosis related markers in H9c2 cells. Relative mRNA expression assessed by quantitative real-time PCRs of Catalase (a) shows an upregulation by 3.8 fold, Hif-1a (b) was found to be elevated by fourfold, Adamts4 (c) was found to be upregulated by 5 and 4.5 fold for H2O2 and Hyp treatment sets respectively, Col-III (d) was found to be elevated by 4 and 3.5 fold H2O2 and Hyp treatments, Tgf-ß1 (e) levels were upregulated by 8 and 7.4 folds for H2O2 and Hyp treatments respectively. Also, elevated expression of proteins analyzed by WB with Adamts4 (f and g) showed an enhanced expression of 3.7 and fourfold for Hyp and H2O2 treatments, a-SMA (f and h) showed an upregulation of 3 and 3.5 folds Hyp and H2O2 treatment groups, Vimentin (f and i) under stress induced conditions of hypoxia and H2O2 showed upregulated expression by 4.8 fold for hypoxia and 4.7 fold for H2O2 treatments. Elevated expression of markers-Catalase and Hif-1a signified successful injury inductions while upregulated expressions of Adamts4, Col-III, Tgf-ß1, a-SMA and Vimentin indicated development of injury related fibrosis. ß-actin was used to normalize gene expression for qPCR assay and total protein was used as loading control for WB. n = 3. Data analyzed and expressed as mean ± SD. Differences were considered statistically significant for p < 0.05. (j) H9c2 morphology after injury induction as compared to control taken with the help of a DIC microscope.
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