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. 2023 Jul 18;120(30):e2217128120. doi: 10.1073/pnas.2217128120

Fig. 1.

Fig. 1.

SSH1 inhibits Nrf2/ARE target gene expression independent of SSH1 phosphatase activity. (A1) Schematic of the Nrf2 reporter construct pREP-8xARE-GFP-SV40-BFP. (A2) Schematic of SSH1 and SSH1-CS proteins showing the catalytic domain (CAT) and binding sites for cofilin and p62. (B) Representative images of HT22 cells coexpressing the Nrf2 reporter (green and blue), with vector control, myc-Nrf2, and/or Flag-SSH1 (red). (C) Quantification of Nrf2 reporter [one-way ANOVA, F (2, 218) = 18.62, P < 0.0001, post hoc Dunnett, ****P < 0.0001, **P = 0.0076, n = 15 to 20 images/condition/experiment from three experiments]. (D) Representative images of HT22 cells coexpressing the Nrf2 reporter (green and blue) and vector or Flag-SSH1 (red), ± 200 μM H2O2 (2 h). (E) Quantification of Nrf2 reporter [one-way ANOVA, F (2, 519) = 124.7, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 15 to 20 images/condition/experiment from four experiments]. (F) Representative images of HT22 cells coexpressing the Nrf2 reporter (green and blue), vector, Flag-SSH1, or Flag-SSH1CS (red), ± 250 μM H2O2 (3 h). (G) Quantification of Nrf2 reporter [one-way ANOVA, F (3, 308) = 61.03, P < 0.0001, post hoc Dunnett, ****P < 0.0001, ns = not significant, n = 8 to 12 images/condition/experiment from four experiments]. (H) Representative images of HT22 cells cotransfected with the Nrf2 reporter (green and blue) and control siRNA or SSH1 siRNA, stained for SSH1 (red), ± 250 μM H2O2 (3 h). (I) Quantification of Nrf2 reporter [one-way ANOVA, F (3, 855) = 41.31, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 15 to 20 images/condition/experiment from four experiments]. (J) Representative immunoblots from lysates of HEK293T cells expressing vector or Flag-SSH1 ± 15 µM NaAsO2 (18 h). (K) Quantification of HMOX1 and NQO1 proteins [one-way ANOVA; HMOX1: F (2, 21) = 310.2, P < 0.0001; NQO1: F (2, 21) = 34.82, P < 0.0001; post hoc Dunnett, ****P < 0.0001, ***P < 0.001, *P < 0.05. n = 8 samples/condition]. (L) Representative immunoblots from lysates of HEK293T cells coexpressing vector or myc-Nrf2 plus vector, Flag-SSH1, or Flag-SSH1-CS. (M) Quantification of HMOX1 protein [one-way ANOVA, F (2, 6) = 32.61, P = 0.0006, post hoc Dunnett, ***P = 0.006, **P = 0.0022, ns = not significant, n = 3 samples/condition]. (N) Representative immunoblots from lysates of HEK293T cells transfected with control or SSH1 siRNA ± 15 µM NaAsO2 (18 h). (O) Quantification of HMOX1 protein [one-way ANOVA, F (2, 15) = 185.4, P < 0.0001, post hoc Dunnett, ****P < 0.0001, n = 6 samples/condition]. (P) Quantification of NQO1 protein [one-way ANOVA, F (2, 14) = 34.49, P < 0.0001, post hoc Dunnett, ****P < 0.0001, ns = not significant, n = 6 samples/condition].