Fig 2: Differentially expressed genes between the ApoM-OE and NC groups. (A) Scatter plot. The X-axis represents the NC group, and the Y-axis represents the ApoM-OE group. (B) Volcano plot. The X-axis represents the logarithm conversion of the fold difference to base 2, and the Y-axis represents the logarithm conversion of the corrected significant levels to base 10. Red indicates all the probes with |Fold Change| > 2 and P < 0.05. (C) Heat map of hierarchical clustering. Each column represents a sample, and each row represents a differentially expressed gene. The upper dendritic structure indicates the aggregation or classification of all samples; the left dendritic structure indicates the aggregation of expression patterns of differentially expressed genes. (D) The relative RPS27A mRNA level in Caco-2 cells was determined by qPCR. (E) The RPS27A protein level in Caco-2 cells was determined by Western blotting. Both the ApoM-OE and NC groups include four replicates. GAPDH was the loading control. (F) The relative RPS27A protein level was significantly higher in the ApoM-OE group than in the NC group. The data are presented as the means ± SD. Student's t test was used to analyze significant differences; *P < 0.05 versus NC. ApoM, apolipoprotein M; qPCR, quantitative PCR; RPS27A, ribosomal protein S27a; OE, overexpression; NC, negative control.

Fig 3: Rad6 co-sediments with elongating ribosomes(A) Immunoblot of co-immunoprecipitation (coIP) of Rad6-FLAG with ribosomal proteins uS3 and uL5.(B) Rad6 presence on the ribosome pellet is independent of cellular lysate incubation with 10 mM DTT, 1 M NaCl, 1 mM H2O2, and DNAse (0.1 U/µg protein) and RNAse I (100 U/µg protein). 1% SDS was used to disrupt molecular interactions. Anti-uS3 and -uL5 were used as loading control for proteins from the 40S and 60S ribosome subunits, respectively.(C) Sucrose density sedimentation analysis and anti-HA immunoblot of lysates from cells expressing Rad6-HA, GFP-HA, or GFP-Rad6-HA constructs. Anti-uS3 and -uL5 were used as markers for the 40S and 60S ribosome subunits, respectively.(D) Purified Rad6, but not GFP, relocalizes to the polysomal fraction after incubation with ribosomes for 10 min at room temperature followed by sucrose density-sedimentation separation. Ribosomes were isolated from rad6?bre1? strain. Anti-uL5 was used as a marker for the 60S ribosome subunit. *Anti-Rad6 antibody unspecific band.(E) 3D classification of ribosomes incubated with purified Rad6 (RiboRad6mix) and ribosomes co-immunoprecipitated with FLAG-tagged Rad6 (RiboRad6IP). Ribosomes were observed in the pre-translocation (PRE), post-translocation (POST), and transition (PRE-to-POST) states.(F) Structural detail of RiboRad6IP at the pre-translocation stage with tRNAs in hybrid A/P and P/E positions. Rigid fit of the tRNAs was performed using coordinates from the 80S ribosome (PDB: 6GZ5) (Flis et al., 2018).(G) Focused classification of RiboRad6IP shows two classes with distinct conformations for the 40S beak and ribosomal proteins eS12 and eS31. The extended beak rRNA model was built using COOT (Emsley and Cowtan, 2004).

Fig 4: RPS27A expression in CRC tissues. (A) RPS27A expression in tumors and corresponding tumor-adjacent tissue specimens from 46 CRC cases was quantified via qPCR and normalized to GAPDH expression. Fold-change was calculated using 2-?Ct. The data are compared using a Student's t test and are shown as the means ± SD; ***P < 0.001. (B) The correlation between RPS27A expression and ApoM expression in CRC tissues was analyzed using the Pearson correlation method, and r is defined as the Pearson correlation coefficient. CRC, colorectal cancer; RPS27A, ribosomal protein S27a; qPCR, quantitative PCR; NC, negative control. ApoM affects the biological roles of Caco-2 cells through RPS27A

Fig 5: BoHV-1 infection promoted Nrf2 depletion through a proteasome pathway. (a, c, and i) MDBK cells in 60 mm dishes were infected with BoHV-1 at an MOI of 0.1 in the presence of MG132 (1 µM) or DMSO control. After infection for 24 h, the cell lysates were prepared for Western blotting to detect the expression of Nrf2 (a) or were prepared for IP using antibodies against Nrf2 (c), DJ-1 (d), and unrelated IgG (k). The IP samples were subjected to immunoblots using antibodies against ubiquitin (Cell Signaling Technology, cat# 3936, 1 : 1000) and Nrf2 (Abcam, cat# ab137550, 1 : 500), respectively. (d and e) MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 (MOI = 0.1) for an indicated time length. The cell lysates were then prepared for Western blots to detect the expression of KEAP1 and DJ-1 using antibodies against KEAP1 mAb (Cell Signaling Technology, cat# 8047, 1 : 1000) and DJ-1 (ABclonal, cat# A0201, 1 : 1000), respectively. Data shown are representative of three independent experiments. (f, h, and j) The band intensity was analyzed with software ImageJ. Each analysis was compared with that of control, which was arbitrarily set as 100%. These images are representative of those from three independent experiments. The significance was assessed with Student's t-test (*P < 0.05). (g) MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 (MOI = 0.1) for 24 hpi. Then, the nuclear proteins were prepared using a commercial kit, and the levels of protein DJ-1 were detected by immunoblots. Data shown are representative of three independent experiments. WCE: whole cell extract.