Figure 2. SLC28A3 expression affects the severity of DIC by regulating DOX uptake into cardiomyocytes.

A, Validation of CRISPR/Cas9–mediated SLC28A3 knockout (KO) in an isogenic hiPSC line detected by Sanger sequencing, showing 8 bp deletion downstream of the transcription start site (TSS). PAM, protospacer adjacent motif. B, Demonstration that 91% of the cell population acquire the introduced deletion. C, Validation of KO and AAVS1-based SLC28A3 overexpression (OE) by western blot and RT-PCR. D, Effect of DOX (72 h) on viability in ISO (n = 45), ISO–OE (n = 14), and ISO–KO (n = 6) hiPSC–CMs. E, Effect of doxorubicin (72 h) on apoptosis measured by activated caspase 3/7 in ISO (n = 8), ISO–OE (n = 10), and ISO–KO (n = 6) hiPSC–CMs. F, Assessment of DOX uptake via measurement of DOX intrinsic fluorescence using flow cytometry-based assay (n = 6–9). n = full independent experimental replicates, Error bars, s.e.m, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by unpaired two-tailed Student’s t-test (f). For (d and e) log-logistic non–linear regression model was used to estimate the value of the four parameters, and t-statistic was used to test for significant difference in LD₅₀ between different groups.