Fig. 5: Metformin prevents PD-L1-CD-membrane association to induce PD-L1 degradation.

a Metformin reduces CD_260-290 interaction with DMPG/DH⁶PC bicelles. Superimposed 2D ¹H-¹⁵N TROSY-HSQC spectra of CD_260-290 in the bicelles (red) titrated with metformin at molar ratios of [metformin]:[DMPG] = 0.05 (orange), 0.1 (green), 0.2 (light blue), 0.4 (blue), 0.6 (dark blue), 1.0 (magenta), and 2.0 (purple). The addition of metformin shifts the correlations toward that of CD_260–290 in solution (black). The right panels 1–2 show the same spectral regions labeled on the full spectrum, highlighting the chemical shift changes for G273 and K270, respectively. b The comparison of chemical shift changes of CD_260–290 in DMPG/DH⁶PC bicelles between molar ratios of [metformin]:[DMPG] = 0 and 2.0. c Metformin decreases CD_260–290 insertion into DMPG/DH⁶PC bicelles. PRE of CD_260-290 spectral signals induced by the paramagnetic probe 16-DSA (2.5 mM) localized in the bicelle membranes were measured when the sample was titrated with metformin. The intensity recovery of broadened peaks for G273 (top) and K270 (bottom) at molar ratios of [metformin]:[DMPG] = 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 are shown. d FRET-detected effect of metformin on PD-L1-CD-membrane interaction. RKO cells expressing PD-L1-mTFP were treated with 5 mM metformin for 2 h. Fluorescent images of representative cells from the vehicle (DMSO) control (left) and the metformin treated (middle) samples are shown. The statistical results of FRET efficiency are represented as the mean ± SD for n = 24 or 26 cells from the control and treated samples, respectively (right). P values by unpaired two-sided Student’s t-test are indicated. Scale bars, 5 μm. e Cellular level of PD-L1^WT or PD-L1^3RE mutant. HEK293T cells expressing PD-L1^WT (top) or PD-L1^3RE mutant (bottom) were treated with the indicated concentrations of metformin for 24 h and analyzed by western blot. Similar results were obtained from two other independent experiments. f Surface level of PD-L1^WT or PD-L1^3RE mutant in HEK293T cells determined by flow-cytometric analysis. After metformin (5 mM) treatment for 24 h, the HEK293T cells expressing PD-L1^WT (left) or PD-L1^3RE mutant (right) were analyzed by flow-cytometry using anti-PD-L1 antibody with IgG, immunoglobulin G, as the control. Source data are provided as a Source Data file.