Fig. 7: TRIM2 accelerates proteasomal degradation of BNIP3.

A Caco-2 cells were transfected with either the HA-Vector or HA-TRIM2 plasmid using PEI transfection reagent. The protein expression of TRIM2 and BNIP3 was then detected by western blot. β-actin was used as a loading control (n = 3 biologically independent samples). B Caco-2 cells were transfected with HA-Vector or HA-TRIM2 plasmids using PEI transfection reagent. The relative mRNA level of BNIP3 in the two groups was then detected by qRT-PCR (n = 3 biologically independent samples). C HEK293T cells transfected with varying doses of HA-TRIM2 plasmids were harvested for western blot. β-actin was used as a loading control. D A correlation analysis was conducted to determine the relationship between the protein levels of TRIM2 and BNIP3 in cells (n = 32 biologically independent samples). (E) Caco-2 cells transfected with HA-TRIM2 plasmids were treated with dimethyl sulfoxide (DMSO), MG132 (a proteasome inhibitor), chloroquine (CQ, a lysosome inhibitor), and 3-methyladenine (3-MA, an autophagy inhibitor) for six hours. Western blot was employed to detect the protein expression levels of TRIM2 and BNIP3. The BNIP3 western blot bands were quantified in the different groups (n = 8 biologically independent samples). β-actin served as a loading control. F HEK293T cells transfected with HA-TRIM2 plasmids were treated with MG132 for 0 h, 1 h, 2 h and 3 h. Western blot was employed to evaluate the protein expression levels of TRIM2 and BNIP3 (n = 8 biologically independent samples). The quantification of the BNIP3 western blot bands at different time points is presented on the right. β-actin was used as a loading control. G The protein changes of BNIP3 in three groups of Caco-2 cells (transfected with HA-Vector plasmid and treated with DMSO, transfected with HA-TRIM2 plasmid and treated with DMSO, transfected with HA-TRIM2 plasmid and treated with MG132) after CHX treatment for indicated times. The western blot detected the half-life of BNIP3 in three groups, with β-actin serving as a loading control (n = 6 biologically independent samples). H The protein alterations of BNIP3 in three groups (treated with si-Control, treated with si-TRIM2, treated with si-TRIM2 and HA-TRIM2 plasmid) of Caco-2 cells following CHX treatment for the specified times. The half-life diagram of BNIP3 in the three groups is presented on the right. β-actin was used as a loading control (n = 9 biologically independent samples). I The protein changes of BNIP3 in three groups (co-transfected with Flag-BNIP3 and HA-Vector, co-transfected with Flag-BNIP3 and HA-TRIM2, and co-transfected with Flag-BNIP3-K130R and HA-TRIM2) of Caco-2 cells after CHX treatment for indicated times. Western blot detected the half-life of Flag-BNIP3 in the three groups (n = 8 biologically independent samples). β-actin was used as a loading control. J Caco-2 cells were treated with si-BNIP3, and immunofluorescence was performed to detect the co-localization and distribution of TRIM2 and Flag-BNIP3 in the different groups (si-BNIP3+Flag-BNIP3-WT + DMSO, the experimental groups were as follows: si-BNIP3+Flag-BNIP3-WT + MG132, si-BNIP3+Flag-BNIP3-K130R + DMSO, and si-BNIP3+Flag-BNIP3-K130R + MG132. Scale bars are 10 μm. All results are expressed as the mean ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s test, with the following levels of significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001.