Figure 1.

Tb4 protects hiPSC-CM against hypoxic injury in vitro. To determine cyto-protective effect of Tb4, 2x10⁵ hiPSC-CMs/well were cultured in 12-well plate. After washing thrice with DPBS, hiPSC-CMs were cultured in 500 μL Hanks balanced salt solution (HBSS) supplemented with or without Tb4 protein and cultured in an incubator with hypoxia condition for 24 h: 5% CO₂, 94% N₂, and 1% O_2. (A) The concentrations of lactate dehydrogenase (LDH) in the cell culture medium were measured in the absence or presence of Tb4 and presented as a percentage of the measurements obtained in the absence of Tb4 (n = 6 for each concentration. One-way ANOVA analysis). (B) The concentration of DNA fragments in the cell culture medium were measured in the absence or presence of 600 ng/mL Tb4 (n = 6 for each sample. Independent T-test). (C) hiPSC-CMs cultured in HBSS medium supplemented without or with 600 ng/mL Tb4 were TUNEL-stained and counter-stained with DAPI after 24 hours' hypoxia. (α-SA: α-sarcomere actin. Bar = 100 μm). (D) hiPSC-CM apoptosis was quantified as the number of TUNEL⁺hiPSC-CMs over the total hiPSC-CMs per field (n = 3 for each sample. Independent T-test). (E) Representative Western Blot images of hiPSC-CMs cultured in HBSS medium supplemented without or with 600 ng/mL Tb4 for protein expressions of pAKT, AKT, and Bcl-XL. Protein expression level of GAPDH was used as an internal control. Cells were cultured under hypoxic condition and were harvested at 0, 1, 3, and 6 h after treatment. (F) Quantification of pAKT protein expressions, which were expressed as percentages of AKT protein levels after normalized with GAPDH protein. (n = 5 for each sample. Independent T-test). (G) Quantification of Bcl-XL protein expressions, which were presented as percentages of GAPDH protein levels. (n = 5 for each sample. Independent T-test). (*: p < 0.05; **: p < 0.01; ***: p < 0.001). Values are presented as the means ± SD.