Fig 1: The expression of APOBEC3 family genes negatively correlated with the LOXL2 status in cervical cancer. (A) Scatter plots of APOBEC3 family mRNA expression in the two groups clustered by LOXL2 expression in 176 core-set cervical cancer samples. Y axis shows the log2 of (RSEM (RNA-Seq by Expectation-Maximization)+1). Mean and SD were shown. P-values were calculated by Student's t-test. (B) qPCR of APOBEC3 family genes in SiHa from CON and sh-LOXL2 (n = 3). Mean and SEM were shown. P-values were calculated by Student's t-test. (* <0.05, ** <0.01) (C) Pearson correlation analysis of APOBEC3 family genes as APOBEC3A, APOBEC3B, APOBEC3D, and APOBEC3G with that of LOXL2 expression in TCGA dataset from GEPIA (21). P-value and Pearson correlation coefficient (r) were shown. (D) Representative images of IF assay in SiHa and HeLa cell lines. (E) Western blot assay of LOXL2, APOBEC3A, APOBEC3B, APOBEC3D, and APOBEC3G in SiHa and HeLa after transfected with sh-Con or sh-LOXL2. ß-tubulin served as the loading control.
Fig 2: FOLR1-mediated FA deficiency exhibited Inhibition of VSV replication by increasing the expression of APOBEC3B. A Bap (10 ?µmol/L) stimulated HeLa cells for 72 ?h, and APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H mRNAs were detected by qPCR analysis. B The expression of APOBEC3 mRNA in Bap-exposed mice lung tissues was detected by qPCR. C siNC or siAPOBEC3A, B, C, D, F, G, and H was transfected into Bap-exposed HeLa cells respectively with VSV infection for 24 ?h, and then VSV-G mRNA was detected by qPCR. D siAPOBEC3B was transfected into Bap-treated HeLa cells for 24 ?h, and VSV viral RNA was detected by qPCR assay. E–G HeLa cells were stimulated with Bap at different concentrations (1–10 ?µmol/L) for 72 ?h, transfected with 1 ?µg FOLR1 plasmid from 0 ?h to 72 ?h, or cultured with FA-free culture medium from 0 ?h to 72 ?h, respectively; APOBEC3B, GAPDH and FOLR1 proteins were measured by Western blot. Relative grey levels were analyzed using Image J. H HeLa cells were cultured with FA-free medium 72 ?h, and then they were supplied with different concentrations of FA, and APOBEC3B mRNA was detected by qPCR. I HeLa cells were stimulated with Bap at 10 ?µmol/L, overexpressed with FOLR1 (1 ?µg) plasmids, and cultured with FA-free culture medium for 72 ?h, respectively, and then they were supplied with FA (1 ?µmol/L) for 24 ?h. The expression of APOBEC3B, and GAPDH were measured by Western blot. J–K siAPOBEC3B knockdown or siNC HeLa cells were stimulated with Bap at 10 ?µmol/L for 72 ?h, overexpressed with FOLR1 plasmids, or cultured with FA-free culture medium, respectively, and then they were infected with VSV (MOI ?= ?1) for 24 ?h. The expression of VSV-G, APOBEC3B, and GAPDH protein were measured by Western blot, and the expression of VSV viral RNA was detected by qPCR. Data expressed as Means ?± ?SD (n ?= ?3–5, *P ?< ?0.05, **P ?< ?0.01, ***P ?< ?0.001).
Fig 3: MTX exhibits a broad antiviral activity via the upregulation of APOBEC3B. A, B HeLa cells were pretreated with MTX from 0.5 ?µmol/L to 10 ?µmol/L for 2 ?h, and then they were infected with VSV (MOI ?= ?1) for 24 ?h. The expression of VSV-G mRNA, VSV viral RNA and VSV-G protein were measured by qPCR and Western blot, respectively. C HeLa cells were pretreated with MTX 1 ?µmol/L for 2 ?h, and then they were infected with VSV (MOI ?= ?1) for 24 ?h. VSV viral RNA was detected by qPCR assay. D 10 female mice were divided equally into two groups, and then the infection of VSV (5 ?× ?104 ?PFU) following the model establishment of MTX (0.5 µg/mouse, n ?= ?5) or equal doses of DMSO (n ?= ?5) treatment in mice. E The expression of VSV-G mRNA and VSV viral RNA in the lung tissues of mice were detected 48 ?h post infection by qPCR analysis. F, G HeLa cells were pretreated with MTX 1 ?µmol/L, and then they were infected with VSV (MOI ?= ?1) for 24 ?h. Western blot and qPCR were used to detect APOBEC3B and GAPDH expression. H, I The expression of APOBEC3B mRNA and protein in HeLa cells were measured by qPCR and Western blot, respectively. J The expression of APOBEC3 mRNA in the lung tissues of mice were detected by qPCR analysis. K, L siAPOBEC3B knockdown or siNC HeLa cells were pretreated with MTX at 1 ?µmol/L for 2 ?h, and then they were infected with VSV (MOI ?= ?1) for 24 ?h. The expressions of VSV-G and GAPDH mRNA and protein were measured by qPCR and Western blot. Data expressed as means ?± ?SD (n ?= ?3–5, *P ?< ?0.05, **P ?< ?0.01, ***P ?< ?0.001, "ns" means no significant difference).
Fig 4: The schematic diagram showed that FOLR1 upregulation induced by Bap accelerates FA deficiency to affect VSV replication. Bap associated high level of FOLR1 accelerates the depletion of the limited extracellular FA (?, ?, ?, ?, ?), which resulting in FA deficiency intracellular (?). The status of intracellular FA deficiency increases the expression of APOBEC3B (?), which is associated with the inhibition of viral replication (?). Furthermore, MTX, as one of the anti-folates, exhibits antiviral effects by FA deficiency-related APOBEC3B upregulation.
Supplier Page from ABclonal Technology for APOBEC3B Rabbit pAb
Trial Size: 20 ul