Relationship between DOX, p53, and ORAI1 in CFs.

DOX increased the expression of p53 and induced apoptosis, cell cycle arrest, and ROS production. In addition, DOX increased the expression of ORAI1, not STIM1. Furthermore, the inhibition of ORAI1 negated the DOX-induced expression of p53, suggesting that the DOX-ORAI1-p53 pathway induces cardiotoxicity. (TIF)</p

Western blotting analysis for four groups to evaluate the effects of store-operated Ca²⁺ entry (SOCE) inhibition; CTRL group, DOX group, YM group, and YM+DOX group.

(A) YM-58483 significantly attenuated the DOX-induced upregulation of p53 protein. (B) YM-58483 significantly attenuated the DOX-induced upregulation of p21 protein. (one-way ANOVA followed by Tukey’s test, n = 6, *** p < 0.001, NS: no significant difference).</p

Protocol for western blotting.

(DOCX)</p

Western blotting analysis to evaluate the effects of ORAI1 gene knockdown.

(A) Expression of ORAI1 mRNA by qPCR. The expression level of ORAI1 mRNA was reduced in samples with siRNA against ORAI1. In samples with control siRNA, DOX significantly upregulated the expression level of ORAI1 mRNA (two-way ANOVA followed by Bonferroni’s post hoc test, n = 6, *** p p p p p p p p < 0.01, NS: no significant difference).</p

Original data (data shown in Fig 4).

(PDF)</p

Fig 1 -

1A and 1B. Western blotting analysis comparing doxorubicin (DOX) samples with control (CTRL) samples. (A) Human cardiac fibroblasts (HCFs) were exposed to 0.1 to 1.0 μM DOX for 24 h. DOX increased the expression of p53 protein in a dose-dependent manner (one-way ANOVA followed by Tukey’s test, n = 4, *** p p p 1C. Apoptosis assay comparing DOX samples with CTRL samples. Flow cytometry analysis showed that DOX significantly increased early apoptosis (Q3) (unpaired t-test, n = 4, ** p 1D and 1E. Cell cycle analysis comparing DOX samples with CTRL samples. (D) Western blotting analysis. HCFs were exposed to 0.5 μM DOX for 1 to 24 h. DOX increased the expression of p21 protein in a time-dependent manner (one-way ANOVA followed by Tukey’s test, n = 4, *** p p p 1F. ROS production measured by fluorescence 24 h after administration of DOX. DOX increased ROS production significantly in the samples with 5.0 μM DOX (one-way ANOVA followed by Tukey’s test, n = 6, * p < 0.05, NS: no significant difference).</p

Cell cycle assays for evaluating the effects of SOCE inhibition.

There were four groups: the CTRL group, DOX group, YM group, and YM+DOX group. DOX decreased the proportion of cells in the G1 and S phases. In contrast, DOX increased the proportion of cells in the G2 phase. This suggests that DOX induced cell cycle arrest in the G2/M phase. YM-58483 attenuated the changes induced by DOX. (TIF)</p

Apoptosis assay to evaluate the effects of SOCE inhibition.

(A) Apoptosis assay of four groups: CTRL group, DOX group, YM group, and YM+DOX group. Flow cytometry showed that YM-58483 significantly attenuated DOX-induced early apoptosis (one-way ANOVA followed by Tukey’s test, n = 6, *** p p p p < 0.01, NS: no significant difference).</p

Original data (data shown in Fig 1).

(PDF)</p

Original data (data shown in Fig 2).

(PDF)</p