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. 2020 Jan 6;9:e51605. doi: 10.7554/eLife.51605

Figure 3. NEIL2 participates in A3B-mediated genomic DNA damage.

(A) Immunostaining of γH2AX foci in NEIL2-stable-knockdown Hs578T cell lines (shNEIL2#1 and shNEIL2#2) transfected with A3B-3HA. NEIL2 knockdown decreases the A3B-mediated γH2AX foci. EV, empty vector; shSCR, scramble shRNA. Scale bar, 50 μm. Right panel: Percentage of γH2AX foci, showing mean ± s.d., in at least 10 randomly selected microscopic fields in two replicate experiments for each condition. ***P < 0.001 by two-tailed unpaired Student’s t test. (B) In vitro deamination assay of nuclear extracts from NEIL2-stable-knockdown Hs578T cell lines with or without A3B-3HA expression. The substrate was a fluorescein-labeled single-stranded oligonucleotide (39 nt) containing -TCT- or -ACT- (negative control). Nuclear extract from HEK293T expressing A3B-3HA (A3B OE) was used as a positive control. NEIL2 knockdown does not affect A3B deaminase activity. Right panel: Quantifications of the cleaved products relative to total DNA loaded onto gel. S, substrate; P, product. (C) Quantification of the percentage of cells with γH2AX foci in NEIL2-stable-knockdown Hs578T cell line (shNEIL2#1) in the absence or presence of a NEIL2 expression vector pcDNA3.1(+)-NEIL2-3’HA. NEIL2 restoration increases A3B-triggered γH2AX foci. Data are represented as mean ± s.d. (n = 10 randomly selected microscopic fields in two replicate experiments). ***P < 0.001 by two-tailed unpaired Student’s t test. The corresponding images of γH2AX foci are shown in Figure 3—figure supplement 1C. (D) Immunostaining of γH2AX foci in MDA-MB-453 cells overexpressing A3B-3HA and NEIL2-HA. Percentage of cells with γH2AX foci is shown in the right panel. EV, empty vector. Scale bar, 50 μm. Data are represented as mean ± s.d. (n = 10 randomly selected microscopic fields in two replicate experiments). **P < 0.01; n.s., no significant difference by two-tailed unpaired Student’s t test.

Figure 3.

Figure 3—figure supplement 1. NEIL2 is required for A3B-mediated genomic DNA damage.

Figure 3—figure supplement 1.

(A) Western blot of A3B-3HA expression in NEIL2-stable-knockdown Hs578T cell lines shNEIL2#1 and shNEIL2#2. LaminB1 serves as a loading control. shSCR, scramble shRNA. EV, empty vector. (B) qRT-PCR shows NEIL2 rescue efficiency in NEIL2-stable-knockdown Hs578T cell line shNEIL2#1 (targets NEIL2 3’UTR) with NEIL2-HA overexpression. Error bars represent s.d., n = 3. ***P < 0.001 by two-tailed unpaired Student’s t test. (C) Immunostaining of γH2AX foci in NEIL2-stable-knockdown Hs578T cell line shNEIL2#1 with rescue by exogenous expression of NEIL2-HA. shSCR, scramble shRNA; shNEL2#1 targets NEIL2 3’UTR; EV, empty vector. Scale bar, 50 μm. The quantification data is shown in Figure 3C.