Figure 4. Expression of GPx8 normalizes ER calcium dysregulation, downstream of Nrf2 signaling.

HeLa cells were transfected with GPx8 (+) or pcDNA3 (−) for 48 h, and then with Nrf2 siRNA (+) or control siRNA (−) for 24 h. All data are presented as mean ± SEM. (A) Representative Western blot of GPx8 overexpression and β-actin as loading control. (B) Resting ER calcium measured by erGCaMP3. n = 317, 186, 253, 337 cells from three sets of experiments. P values between indicated groups by Kruskal–Wallis test with Dunn’s correction are shown. (C) Resting cytosolic calcium levels measured with GCaMP6f. n = 325, 331, 505, 402 cells from three sets of experiments. P values between indicated groups by Kruskal–Wallis test with Dunn’s correction are shown. (D) ER calcium released upon 100 μM histamine stimulation, measured by drop in erGCaMP3 fluorescence. n = 36, 36, 52, 77 cells from three sets of experiments. P values between indicated groups by paired one-way ANOVA test with Sidak’s correction are shown. (E) Cytosolic calcium transient peak induced with 100 μM histamine, measured by GCaMP6f. n = 41, 49, 61, 73 cells from three sets of experiments. P values between indicated groups by Kruskal–Wallis test with Dunn’s correction are shown. (F) Proposed model linking ER protein folding and ER calcium through H₂O₂ signaling, Nrf2 nuclear translocation, and GPx8 expression.