Jennifer S. Yu

Table S4. Echocardiograph data analysis in chronic heart failure mouse model after tamoxifen was given 150 days.

Figure S4. Proposed mechanism of anthracycline-induced cardiotoxicity and prevention by TOP2B ASO therapy.

Doxorubicin induces TOP2B overexpression in human AIC specimens. A, Western blot analysis showing the expression levels of TOP2B and GAPDH in heart tissue samples from ND patients and patients with AIC. B, Hematoxylin and eosin stains of heart tissue sections from ND patients and patients with AIC, illustrating tissue morphology and cellular integrity. C, IHC staining showing the localization and expression of TOP2B in heart tissue sections from ND patients and patients with AIC. D, Masson’s Trichrome staining of heart tissue sections from ND patients and patients with AIC, highlighting fibrosis in purple color in the tissue.

Doxorubicin induces TOP2B overexpression in the AIC mouse model. A, Western blot analysis showing increased expression of TOP2B protein in the AIC mouse model treated with doxorubicin (5 mg/kg/week, i.v., for four consecutive weeks) in mice with AIC. B, IHC staining confirming TOP2B overexpression in tissue samples from the same model. C, Immunofluorescence staining of cardiac sections demonstrating increased nuclear TOP2B (red) in cardiomyocytes following doxorubicin treatment. cTnI (green) marks cardiomyocytes, and DAPI (blue) labels nuclei. Merged images show enhanced nuclear localization of TOP2B in cardiomyocytes of doxorubicin-treated hearts relative to controls. Scale bars as indicated.

TOP2B binds to the S domain of SMYD1. A, Schematic diagram of SMYD1 truncation mutants fused to an N-terminal eGFP tag. Seven constructs were generated: a, full-length SMYD1 (residues 1–490); b, residues 1–290 lacking the C-terminal domain (CTD); c, residues 1–258 lacking post-SET and CTD; d, residues 1–180 lacking SET, post-SET, and CTD; e, residues 1–100 containing only the MYND domain; f, residues 1–50 containing only the S domain; and g, residues 100–490 lacking the S domain. B, Coimmunoprecipitation (co-IP) analysis of interactions between FLAG-tagged TOP2B and each eGFP-tagged SMYD1 truncation mutant in HEK293 cells. Anti-FLAG antibody was used for IP, and bound proteins were detected by immunoblotting with anti-FLAG and anti-GFP antibodies. Strong interaction was observed for constructs containing the S domain (constructs a–f), whereas the mutant lacking the S domain (g) showed loss of interaction. C, Representative confocal images of AC16 cells coexpressing FLAG-TOP2B and each eGFP-SMYD1 truncation mutant. Immunofluorescence staining of TOP2B (red), eGFP-SMYD1 (green), and nuclei (blue, DAPI). Colocalization of TOP2B and SMYD1 was evident for constructs containing the S domain (a–f), whereas the mutant lacking the S domain (g) exhibited no colocalization with TOP2B. **, P < 0.01; Scale bar, 10 μm.

Figure S1. Doxyrubicin modestly increases TOP2B mRNA expression in mouse heart tissue.

Figure S2. Doxorubicin significantly increases TOP2B protein expression in mouse heart tissue.

Table S6. Analyses of TOP2B pull down proteins by LS/MS. (LTS= low tissue specificity)

TOP2B binds SMYD1. A, Western blot analysis after immunoprecipitation (IP) of TOP2B in transgenic mice, probed for SMYD1, TITIN, and ENO3. B, IHC analysis showing the localization and expression levels of SMYD1, TITIN, and ENO3 in tissue sections from TOP2B transgenic mice. C, Western blot analysis after IP of TOP2B in patient samples with AIC, probed for SMYD1, TITIN, and ENO3. D, IHC analysis showing the localization and expression levels of SMYD1, TITIN, and ENO3 in tissue sections from AIC patient samples.

Table S5. Echocardiograph data analysis in chronic heart failure mouse model after tamoxifen with or without doxorubicin treatment.