Fig. 5: BNIP3 upregulation is related to ROS production under hypoxia in vitro.

HaCaT cells were subjected to hypoxia for 6 h. NAC was added 1 h before hypoxia exposure. a After hypoxia exposure, HaCaT cells were processed for immunostaining of DHE. Blue signals (DAPI) indicate nuclear staining, and representative images are shown. Scale bar = 100 μm. b Graphs indicate the relative fluorescence intensities in a as determined by ImageJ software. The results are represented by the mean ± SEM (n = 3). ^*P < 0.05 vs. Norm group, and ^#P < 0.05 vs. Hypo group. c Western blots were performed to analyze the expression of BNIP3 and LC3 as well as activities of p38/MAPK and JNK/MAPK in HaCaT cells subjected to NAC under hypoxia. HIF-1α was used as an indicator of hypoxia. GAPDH was used as the loading control. Immunostaining of BNIP3 (d) and LC3 (e) in indicated HaCaT cells. Nuclei were stained with DAPI. Scale bar = 25 μm. f–h Wound-healing assays and single-cell motility assays were performed using indicated cells. Representative images of wound healing, including keratinocyte trajectories. Scale bar = 100 μm. The results of the quantitative analysis are represented by the mean ± SEM (n = 3). ^*P < 0.05 vs. Norm group, and ^#P < 0.05 vs. the Hypo group