Journal: Signal Transduction and Targeted Therapy

Article Title: Cardiomyocyte-specific knockout of ADAM17 alleviates doxorubicin-induced cardiomyopathy via inhibiting TNFα–TRAF3–TAK1–MAPK axis

doi: 10.1038/s41392-024-01977-z

Figure Lengend Snippet: TNF-α enhanced TRAF3 expression in NRCMs treated with DOX, and interaction of TRAF3 and TAK1 leads to activation of MAPKs pathway. a , b Representative western blot images and comparison of protein expression of TRAF3 among three groups of NRCMs treated with NS, TNF-α and TNF-α + infliximab, respectively ( n = 6 in each group). c Comparison of mRNA expression of TRAF3 among three groups of NRCMs treated with NS, TNF-α and TNF-α + infliximab, respectively ( n = 6 in each group). d , e Representative western blot images and comparison of protein expression of TRAF3 among three groups of NRCMs treated with DMSO, DOX and DOX + infliximab, respectively ( n = 6 in each group). f Comparison of mRNA expression of TRAF3 among three groups of NRCMs treated with DMSO, DOX, DOX + infliximab, respectively ( n = 6 in each group). g Representative western blot images of cleaved PARP, PARP, cleaved caspase 3 and caspase 3 expression among three groups of NRCMs treated with DMSO, DOX and DOX + infliximab, respectively. h Comparison of cleaved PARP/PARP expression among three groups of NRCMs treated with DMSO, DOX and DOX + infliximab, respectively ( n = 6 in each group). i Comparison of cleaved caspase3/caspase3 expression among three groups of NRCMs treated with DMSO, DOX, DOX + infliximab, respectively ( n = 6 in each group). j , k Co-IP assay in NRCMs showing endogenous TRAF3 binding to TAK1. l , m Co-IP assay in HEK293T cells co-transfected with Myc-TRAF3 and Flag-TAK1 showing exogenous TRAF3 binding to TAK1. n Representative western blot images of phosphorylated TAK1, TAK1, phosphorylated JNK, JNK, phosphorylated P38 MAPK, P38 MAPK, phosphorylated ERK and ERK among four groups of NRCMs treated with siNC + DMSO, siNC + DOX, siTRAF3 + DMSO and siTRAF3 + DOX, respectively. o Comparison of protein expression of phosphorylated TAK1/TAK1 among four groups of NRCMs treated with siNC + DMSO, siNC + DOX, siTRAF3 + DMSO and siTRAF3 + DOX, respectively ( n = 6 in each group). p Comparison of protein expression of phosphorylated JNK/JNK among four groups of NRCMs treated with siNC + DMSO, siNC + DOX, siTRAF3 + DMSO and siTRAF3 + DOX, respectively ( n = 6 in each group). q Comparison of protein expression of phosphorylated P38 MAPK/P38 MAPK among four groups of NRCMs treated with siNC + DMSO, siNC + DOX, siTRAF3 + DMSO and siTRAF3 + DOX, respectively ( n = 6 in each group). r Comparison of protein expression of phosphorylated ERK/ERK among four groups of NRCMs treated with siNC + DMSO, siNC + DOX, siTRAF3 + DMSO and siTRAF3 + DOX, respectively ( n = 6 in each group). s Representative western blot images of phosphorylated TAK1, TAK1, phosphorylated JNK, JNK, phosphorylated P38 MAPK, P38 MAPK, phosphorylated ERK and ERK among three groups of NRCMs treated with DMSO, DOX and DOX + 5Z-7-ox, respectively. t Comparison of protein expression of phosphorylated TAK1/TAK1 among three groups of NRCMs treated with DMSO, DOX and DOX + 5Z-7-ox, respectively ( n = 6 in each group). u Comparison of protein expression of phosphorylated JNK/JNK among three groups of NRCMs treated with DMSO, DOX and DOX + 5Z-7-ox, respectively ( n = 6 in each group). v Comparison of protein expression of phosphorylated P38 MAPK/P38 MAPK among three groups of NRCMs treated with DMSO, DOX and DOX + 5Z-7-ox, respectively ( n = 6 in each group). w Comparison of protein expression of phosphorylated ERK/ERK among three groups of NRCMs treated with DMSO, DOX and DOX + 5Z-7-ox, respectively ( n = 6 in each group). x Representative western blot images of cleaved PARP, PARP, cleaved caspase 3 and caspase 3 expression among three groups of NRCMs treated with DMSO, DOX and DOX + 5Z-7-ox, respectively. y Comparison of cleaved PARP/PARP expression among three groups of NRCMs treated with DMSO, DOX and DOX + 5Z-7-ox, respectively ( n = 6 in each group). z Comparison of cleaved caspase3/caspase3 expression among three groups of NRCMs treated with DMSO, DOX and DOX + 5Z-7-ox, respectively ( n = 6 in each group). Values shown were mean and SEM. One-way ANOVA were applied in ( b , c , e , f , h , i , o – r , t – w , y and z ). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: In the eighth proportion of the in vitro experiment (Supplementary Fig. ), in order to verify that the downstream MAPKs pathway was activated by TAK1, NRCMs were treated with DMSO, 1 μM DOX or 1 μM DOX + TAK1 inhibitor 5Z-7-ox (MCE, USA), respectively, for 24 h before cell collection.

Techniques: Expressing, Activation Assay, Western Blot, Comparison, Co-Immunoprecipitation Assay, Binding Assay, Transfection