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. 2023 May 31;64:102769. doi: 10.1016/j.redox.2023.102769

Fig. 3.

Fig. 3

CH24H and 24HC inhibit VSV replication. A. N2a cells were transfected with the expression plasmid pCAGGS vector, pCAGGS-CH24H (named CH24H), or pCAGGS-CH25H (named CH25H), then the cells were infected with VSV at MOI 0.01 for 18 h, and viral titers were measured (n = 3). B. N2a cells were transfected as in (A), and then cells were lysed for 24HC concentration analysis by ELISA (n = 3). C. N2a cells were transfected with three different sets of CH24H-specific siRNAs (No.1, No.2, No.3) or negative control (NC), and CH24H mRNA levels were measured by qPCR at 36 h post-transfection (n = 3). D. N2a cells were transfected with three different sets of CH24H-specific siRNAs (No.1, No.2, No.3) or NC for 24 h, and then infected with VSV at MOI 0.01, CH24H protein levels were measured by WB at 18 h post-infection (h.p.i.) (n = 3). E. N2a cells were transfected with negative control siRNA (NC RNAi), CH24H-specific siRNA (CH24H RNAi), pCAGGS vector with CH24H RNAi, or CH24H with CH24H RNAi. Cells were infected with VSV at MOI 0.01 at 24 h post-transfection. After 12 h of VSV infection, viral titration (n = 3) and WB were used to measure viral load. F. N2a cells were transfected as in (C), and then cells were lysed for 24HC concentration analysis by ELISA (n = 3). G. N2a cells were treated with different concentrations of 24HC or ethanol (ETOH) for 36 h, then the supernatants were discarded and the cytotoxicity was measured (n = 5). H. N2a cells were pretreated with 5 μM 24HC for 12 h, and then infected with VSV at MOI 0.01 for the indicated periods, then supernatants were collected and viral titers were measured (n = 3). I. N2a cells were pretreated with 2.5 μM 24HC or 5 μM 24HC for 12 h, and then infected with VSV at MOI 0.01 for the indicated periods, WB was used to measure viral loads. J. N2a cells were pretreated with 5 μmol (μM) 24HC or 5 μM 25HC for 12 h, and then infected with VSV at MOI 0.01 for 12 h, viral titration (n = 3) (D) and WB (E) were used to measure viral loads. Statistical analysis of comparisons between groups was performed by Student's t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant). Bar graph shows mean ± SD. Western blot data are representative of at least two independent experiments.