Fig. 4: PHKG2 activates PP1, leading to reduced nuclear NRF2 expression.

STRING analysis predicted potential interactions between PHKG2 and the three catalytic subunits (γ, α, β) of PP1 (A). Western blot results showed that PHKG2 overexpression decreased nuclear NRF2 and GPX4 protein levels in 5–8F and FaDu cells, while PHKG2 knockdown exerted the opposite effects; total NRF2 remained unchanged (B, C). qRT-PCR analysis indicated that PHKG2 regulates GPX4 at the transcriptional level. PHKG2 overexpression decreased mRNA levels of GPX4, GCLC, GCLM, and GSS, while PHKG2 knockdown increased their expression (*p < 0.05, **p < 0.01, ***p < 0.001) (D, E).PP1 activity assays confirmed increased PP1 activation by PHKG2 overexpression, reversed by the PP1 inhibitor Calyculin A; PP1 activation was decreased following PHKG2 knockdown and restored by PP1 activator C2 Ceramide (**p < 0.01, ***p < 0.001) (F, G). qRT-PCR results showed that PHKG2 overexpression decreased GPX4 mRNA expression, which was reversed by Calyculin A. Conversely, PHKG2 knockdown increased GPX4 mRNA, and this effect was suppressed by C2 Ceramide (**p < 0.01, ***p < 0.001) (H, I). Western blotting further verified that changes in PP1 activity inversely correlated with nuclear NRF2 protein expression (J, K). Confocal microscopy demonstrated that PP1 inhibitor treatment reversed the nuclear export of NRF2 induced by PHKG2 overexpression (L).