Fig 1: SERINC5 promotes apoptosis by inhibiting autophagy in CSFV infected cells. (a-d) The protein expression of Cleaved-PARP, Cleaved-Caspase3, Cleaved-Caspase9, Npro and Tubulin were assayed. PK-15 (a and c) and 3D4/2 (b and d) cells were transfected 3 × Flag-SERINC5 or siSERINC5, and then were infected or uninfected with CSFV (MOI = 0.1) for 24 h. The level of proteins was carried out using Image-Pro Plus 6.0 software.
Fig 2: glycoGag rescues retroviral infectivity in the presence of IFITM3. (A) 293T were cotransfected with env-deficient MLV encoding or not encoding glycoGag (2.5 µg), pBabeLuc (0.6 µg), xenotropic Env (0.5 µg), and empty pCMV6, pCMV6-IFITM3 (0.83 µg), or pBJ5-SERINC5 (0.093 µg). (B) 293T were cotransfected with env-deficient MLV (2.5 µg), pBabeLuc (0.6 µg), xenotropic Env (0.5 µg), and empty pCMV6, pCMV6-IFITM3 (0.27 µg). Under the indicated conditions, pCMV-glycoGag-myc of pCMV-glycoGag Y36A-myc (0.01 or 0.03 µg) were included. The specific infectivities of viruses produced by transfected cells were measured as described in Materials and Methods. (C) Transfected cells were lysed at 72 h posttransfection, and virus-containing supernatants from 48 and 72 h posttransfection were filtered, ultracentrifuged through 20% sucrose. Then, SDS-PAGE and Western blotting were performed. The data represent the averages of three independent experiments; for each experiment, infectivities were measured in triplicate and averaged. Statistical analysis in panel A was performed with the Student t test. Statistical analysis in panel B was performed using one-way ANOVA. *, P < 0.05; **, P < 0.0005.
Fig 3: The RNA interference of SERINC5 enhanced CSFV replication in PK-15 and 3D4/2 cells. siRNA knockdown of SERINC5 in PK-15 (A,C) and 3D4/2 (B,D) cells transfected with siNC or SERINC5 siRNA-1/-2/-3. The expression of SERINC5 was assessed by western blotting (A,B) and qRT-PCR (C,D) at 24 hpi. PK-15 (E,G) and 3D4/2 (F,H) cells were infected with CSFV (MOI = 0.1) for 24 and 48 h after transfection with SERINC5 siRNA. Statistical analysis of the influence of SERINC5 siRNA on viral titers (E,F) and viral genome replication (G,H). The effect of overexpression and siRNA on cell viability of PK-15 (I) and 3D4/2 (J) cells. Cells were transfected with p3 × Flag-CMV, p3 × Flag-SERINC5, siNC and SERINC5 siRNA were evaluated by CCK-8 assay. The relative levels of the targeted proteins were analyzed with ImageJ software, and the ratios were calculated relative to the GAPDH control. Error bars represent the mean ± SD; n = 3; *P < 0.05; **P < 0.01; ***P < 0.001; NSP > 0.05.
Fig 4: Melanoma differentiation-associated protein 5 (MDA5) activation of the type I IFN signaling pathway is enhanced by SERINC5. HEK-293T cells were co-transfected with p3 × Fag-SERINC5 (0, 50, 100, and 200 ng), HA- RIG-I, MDA5, MAVS, IRF3, and IRF7. Luciferase activities of IFNß (A) and ISRE (B) were detected by a dual-luciferase reporter assay after transfection with the IFNß or ISRE luciferase reporter and pTK-Rluc for 24 h. pTK-Rluc was used as an internal control. PK-15 cells were co-transfected with p3 × Flag-SERINC5 (0, 50, 100, and 200 ng) together with HA-MDA5 (200 ng), HA-RIG-I (200 ng), or HA-CMV (200 ng) for 24 h. The gene expression of IFNa (C), IFNß (D), Mx1 (E), and OAS1 (F) were analyzed by qRT-PCR. Error bars represent the mean ± SD; n = 3; *P < 0.05; **P < 0.01; ***P < 0.001; NSP > 0.05.
Fig 5: Serine incorporator 5 (SERINC5) positively regulates the type I IFN signaling pathway. (A) PK-15 cells were transfected with p3 × Flag-SERINC5 followed by infection with CSFV (MOI = 0.1) for 24 and 48 h. The expression of IFNa, IFNß, TNFa, IL-18, and IL-6 mRNA was determined by qRT-PCR. (B) HEK-293T cells were transfected with p3 × Flag-SERINC5 together with the IFNß, ISRE or NF-?B luciferase reporter and pTK-Rluc for 24 h. The cells were then infected with SeV for another 24 h. Luciferase activities were detected via a dual-luciferase reporter assay. pTK-Rluc was used as an internal control. (C) HEK-293T cells were transfected with p3 × Flag-SERINC5 along with the IFNß or ISRE luciferase reporter and pTK-Rluc. Luciferase activities were detected after incubation with poly (I:C) (10 mg/mL) or 5'-ppp-RNA (2 mg/mL) for 24 h. pTK-Rluc was used as an internal control. The protein expression of p3 × Flag-SERINC5 in HEK-293T cells was assayed by western blotting. GAPDH was used as a loading control. Error bars represent the mean ± SD; n = 3; *P < 0.05; **P < 0.01; ***P < 0.001; NSP > 0.05.
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