Fig 2: miR-30d negatively regulated the expression of PSME3. (A) The matured sequence of miR-30d was aligned in chicken, pig, cow, human, monkey, mouse, and rat. (B) TargetScan analyzed the binding site of miR-30d to PSME3. (C) qPCR analysis of PSME3 expression in CPB2 toxin revulsive IPEC-J2 cells. (D) Comparison of the PSME3 3'UTR-WT and Mut vectors. (E) The relative luciferase activity of the IPEC-J2 cells after co-transfected with miR-30d mimic or mimic NC and PSME3 3'UTR-WT or Mut. (F,G) The miR-30d mimic and the inhibitor were transfected into IPEC-J2 for 36 h, (F) the mRNA level of PSME3 was evaluated through qPCR, (G) the protein level of PSME3 was evaluated via western blot. ns: not significant, *p < 0.05, **p < 0.01.

Fig 3: PSME3 repressed CPB2 toxin-induced inflammation in IPEC-J2 cells. The IPEC-J2 cells were transfected with pcDNA3.1, pc-PSME3 (1 μg), si-NC, and si-PSME3 (150 nM), and treated with CPB2 toxin. (A,B) qPCR analysis of PSME3 expression after transfection of pcDNA3.1, pc-PSME3 (1 μg), si-NC, si1-PSME3, and si2-PSME3 (150 nM). (C,D) Western blot analysis of PSME3 expression after transfection of pcDNA3.1, pc-PSME3 (1 μg), si-NC, and si2-PSME3 (si-PSME3, 150 nM). (E) ELISA analysis of IL-1β, IL-6, and IL-8 expression. (F) LDH activity was tested at 450 nm in a microplate reader. (G) The ROS level was tested by microplate reader at 488-nm excitation wavelength and 535-nm emission wavelength. ns: not significant, *p < 0.05, **p < 0.01.

Fig 4: PSME3 promoted cell proliferation and cycle progression in the CPB2 toxin revulsive IPEC-J2 cells. (A–F) The IPEC-J2 cells were transfected with pcDNA3.1, pc-PSME3 (1 μg), si-NC, and si-PSME3 (150 nM), and treated with CPB2 toxin for 36 h. (A) Cell viability was tested via CCK-8 on a microplate reader at 450 nm. (B) The EdU assay was performed. The IPEC-J2 cells in S-phase were stained red with EdU, whereas the nucleus was dyed blue with Hoechst. (C) Image J analysis of the proportion of proliferating cells. (D,E) Flow cytometry analysis of the IPEC-J2 cell cycle. (F) qPCR analysis of CDK4, PCNA, and p21 expression. *p < 0.05, **p < 0.01.