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. 2021 Jul 4;69:103456. doi: 10.1016/j.ebiom.2021.103456

Fig. 6.

Fig 6

TRIM21 negatively regulates p62-Keap1 aggregation and anti-ferroptotic effect in cardiomyocytes. (a) H9c2 immortalized rat cardiomyocytes were infected with lentiviral none-target control (shNTC) or two independent shRNAs for TRIM21. Relative mRNA level of TRIM21 was measured by quantitative RT-PCR. Data shown are mean ± standard deviation. Student's t-test was used to compare the results. ****p<0.0001. (b) Cells were treated with doxorubicin (1 μM) for 48 hours and observed under phase-contrast microscope. (c) Cell death was determined by trypan blue staining. Data shown are the mean plus SD of three countings (500 cells each). Student's t-test was used to compare the results. n.s., non-significant; ****p<0.0001. (d) H9c2 cells infected with lentiviral shNTC or shTRIM21 were treated with or without doxorubicin (Doxo, 1μM) alone or together with ferrostatin-1 (Fer-1,10 μM), DXZ (100 μM), zVAD (10 μM), or necrostatin-1 (Nec, 0.5 μM). Cell death was determined by trypan blue staining after 36 h. Shown is the average plus SD of triplicate experiments. Student's t-test was used to compare the results. n.s., non-significant; ****p<0.0001. (e) H9c2 cells infected with lentiviral shNTC or shTRIM21 were treated with doxorubicin (1 μM) for 4 h. Cells were lysed in IP lysis buffer containing 1% Triton X-100. The insoluble fraction was dissolved in RIPA buffer containing 1% SDS. Both the Triton X-100 soluble and insoluble fractions were subjected to IB. The ratio of insoluble to soluble bands was determined by ImageJ. (f) HA-TRIM21WT, HA-TRIM21C16A or HA-TRIM21W381/383A mutants was overexpressed in H9c2 cells. (g) H9c2 cells reconstituted with vector, HA-TRIM21WT, HA-TRIM21C16A or HA-TRIM21W381/383A mutants were treated with doxorubicin (1 μM) for indicated times. Cells were lysed in IP lysis buffer containing 1% Triton X-100. The insoluble fraction was dissolved in RIPA buffer containing 1% SDS. Both Triton X-100 soluble and insoluble fractions were subjected to IB. The ratio of insoluble to soluble bands was determined by ImageJ.