Fig. 2. Metformin inhibits transcriptional expression of PD-L1 in an AMPK-downregulated AKT/β-catenin signaling-dependent manner. (A, B) The indicated cells were treated or not with metformin for 12 hrs. Then, mRNA and protein expression levels of PD-L1 were determined using quantitative real-time PCR (A) and immunoblotting (B) with the indicated primers and antibodies, respectively. (C) A431 cells were treated or not with metformin for 12 hrs and subjected to immunoblotting analysis using the indicated antibodies. (D) A431 cells were transfected with TOP-FLASH or FOP-FLASH, followed by metformin (10 mM) treatment for 12 hrs. Luciferase activity was measured. Relative luciferase activity levels were normalized to levels in untreated cells and to Renilla luciferase activity levels. (E–G) A431 cells stably expressing control vector or CA-β-catenin were transfected with TOP-FLASH or FOP-FLASH, followed by metformin (10 mM) treatment for 12 hrs. Luciferase activity was measured. Relative luciferase activity levels were normalized to levels in untreated cells and to Renilla luciferase activity levels (E). mRNA and protein expression levels of PD-L1 were determined using quantitative real-time PCR (F) and immunoblotting (G) with the indicated primers and antibodies, respectively. (H–J) A431 cells stably expressing the control vector or HA-myr-AKT1 were transfected with TOP-FLASH or FOP-FLASH, followed by metformin (10 mM) treatment for 12 hrs. Luciferase activity was measured. Relative levels of luciferase activity were normalized to levels in untreated cells and to Renilla luciferase activity levels (H). The mRNA and protein expression levels of PD-L1 were determined using quantitative real-time PCR (I) and immunoblotting (J) with the indicated primers and antibodies, respectively. (K, L) A431 cells were pretreated or not pretreated with compound C (5 μM) for 1 hr and then treated or not pretreated with metformin (10 mM) for 12 hrs. Protein and mRNA expression levels of PD-L1 were determined using immunoblotting (K and bottom panel of L) and quantitative real-time PCR (L, top panel) with the indicated antibodies and primers, respectively. Data are presented as mean±SD of three independent experiments (A, D, E, F, H, I, L). *P<0.05; **P<0.01; ***P<0.001, Student’s t-test or one-way ANOVA followed by Tukey post-hoc tests.
Abbreviations: PD-L1, programmed death ligand 1; AMPK, adenosine monophosphate-activated protein kinase; PCR, polymerase chain reaction; CA, constitutively active; HA, hemagglutinin.

Ann Lab Med 2024;44:426~436 https://doi.org/10.3343/alm.2023.0443