Fig. 6: JQ1-TXNIP repressed histone H4 UFMylation.
From: BRD4 inhibition suppresses histone H4 UFMylation to increase ferroptosis sensitivity through TXNIP
A Histone H4 UFMylation in Control and TXNIP KO cells treated with DMSO and 2 μM JQ1. Histone H4 was immunoprecipitated and the precipitated proteins were immunoblotted with UFM1 antibody. B Overepression of TXNIP reduced H4 UFMylation. Empty vector (EV), Flag-TXNIP, and 6His-Flag-H4 were transfected into HEK293T cells. 6His-Flag-H4 proteins and their associated proteins were isolated using Ni-NTA beads and analyzed with indicated antibodies. C The interaction between histone H4 and UFBP1 in Control and TXNIP KO cells with DMSO and 2 μM JQ1 treatment. The protein complex was immunoprecipitated with UFBP1 antibody and probed with histone H4 antibody. D The interaction between histone H4 and cMYC in Control and TXNIP KO cells with and without JQ1 treatment. cMYC was immunoprecipitated and isolated proteins were subjected to Western Blotting analysis with a histone H4 antibody. E Protein UFMylation influenced the association between histone H4 and cMYC. cMYC was immunoprecipitated from Control cells treated with DMSO and JQ1 as well as TXNIP KO cells treated by JQ1 alone and JQ1 combined with DKM 2-93. The precipitates were analyzed using a histone H4 antibody. F Rescued the expression of cMYC target genes by TXNIP KO was depending on UFMylation. Control and TXNIP KO cells were treated by DMSO, 2 μM JQ1, 30 μM DKM 2-93 and a combination of JQ1 (2 μM) with DKM 2-93 (30 μM). The transcription levels of CYCLIN A2, MAD2, ORC2, and MCM6 were determined by RT-qPCR. G The interaction of cMYC with Flag-UFM1-H4 and Flag-H4. HepG2 cells were transfected with empty vector (EV), 6Hist-Flag-UFM1-H4, and 6Hist-Flag-H4 and their associated proteins were pulled down using Ni-NTA beads and probed for cMYC. H UFM1-H4 alleviated the repression of CYCLIN A2 by JQ1. HepG2 cells were transfected with Empty vector (EV), Flag-H4, and Flag-UFM1-H4 and treated with 2 μM JQ1. Total proteins were extracted and CYCLIN A2 expression was analyzed via Western Blotting. ****p value < 0.001, ***p value < 0.005, **p value < 0.01, *p value < 0.05 (one-way ANOVA test in F).
