Figure 4.

ZBTB7A regulation of ROS-controlling factors contributes to cell tolerance of HCoV-229E

(A) Huh7 cell viability after H₂O₂ treatment. Control-Huh7 cells and ZBTB7A-Huh7 cells were incubated with 1.5 mM H₂O₂. After 24 h, live cells were stained.

(B) The area of live cells from (A) was calculated via Image J analysis. n = 4 images from two independent experiments.

(C) ROS activity during infection. ZBTB7A-Huh7 cells and control-Huh7 cells were infected with HCoV-229E at 0.5 MOI. ROS were detected via CellROX and imaged at 2 DPI.

(D) Fluorescence intensity of the ROS sensor was calculated from (C) by Image J. n = 4 biological replicates.

(E) ROS assay after superoxide scavenger treatment. Huh7 cells were infected with HCoV-229E at 0.5 MOI. 10 mM tiron or 200 μM trolox were added at 2 hpi and ROS sensor fluorescence was imaged at 2 DPI.

(F) Fluorescence intensity of the ROS sensor calculated from (E) by Image J. n = 4 biological replicates.

(G) Viral RNA detection after superoxide scavenger treatment. Huh7 cells were infected and treated as described in (E). Cells were collected at 2 DPI for qRT-PCR. n = 5 biological replicates. ND, not detected.

(H) Viral titer after superoxide scavenger treatment. Huh7 cells was treated as described in (E). Cells were collected at 2 DPI. n = 4 biological replicates.

(I) Cell viability after superoxide scavenger treatment. Huh7 cells were infected and treated as described in (E) and MTT assays were conducted at 3 DPI. n = 4 biological replicates.

(J and K) Survival of Huh7 cells after superoxide scavenger treatment. Huh7 cells were infected and treated as described in (E). The surviving cells were stained at 3 DPI (J) and 5 DPI (K).

(L) HCoV-229E N staining in surviving cells. Huh7 cells were infected and treated as described in (E). HCoV-229E N was stained in surviving cells at 5 DPI.

(M and N) ZBTB7A knockdown efficiency. Huh7 cells were transfected with ZBTB7A siRNA or negative control siRNA. After 48 h, samples were collected for qRT-PCR (n = 4 biological replicates) (M) and western blot (N).

(O) Viral RNA detection via qRT-PCR. Huh7 cells were transfected with ZBTB7A siRNA or control siRNA and infected with HCoV-229E at 0.5 MOI after 24 h. Viral RNA was measured via qRT-PCR at the indicated time points. n = 4 biological replicates.

(P) Viral titer assessed via plaque assay. Huh7 cells were treated as described in (O). n = 4 biological replicates.

(Q) ROS sensor fluorescence after ZBTB7A knockdown. Huh7 cells treated as described in (O). Samples were stained at 2 DPI and analyzed by flow cytometry. n = 4 biological replicates.

(R) Proportion of live cells after ZBTB7A knockdown. Huh7 cells treated as described in (O). Dead cells were stained at the indicated time points and analyzed by flow cytometry. n = 4 biological replicates. p values were calculated by two-way ANOVA.

Unless otherwise indicated, all panels are representative of two independent experiments. For all panels, p values were calculated by unpaired two-tailed Student’s t tests. ^∗p < 0.05; ^∗∗p < 0.001; ns, not significant. Scale bar, 100 μm. Data shown as mean ± SD.