Fig 2: Modulation of PD-L1 and MHC-I by GR is a common regulatory mechanism in PDAC cells.a Immunoblotting of GR, MHC-I, and GAPDH in human PDAC cell lines. b, c qPCR analysis of PD-L1, HLA-A, HLA-B, and HLA-C in HPAC (b) and BXPC-3 (c) cells with or without MIFE (20 µM, 72 h) treatment. n = 3 technical replicates. d, e Immunoblotting of PD-L1, MHC-I, and GAPDH in HPAC (d) and BXPC-3 (e) cells with or without MIFE treatment with the indicated doses for 72 h. f Left panel: qPCR analysis of Nr3c1, Pd-l1, H-2k, H-2d, and B2m in GR-knockdown HY24409 cells. Right panel: immunoblotting of GR and Gapdh in GR-knockdown HY24409 cells. g qPCR analysis of Pd-l1, H-2k, H-2d, and B2m in HY24409 cells with or without MIFE (20 µM, 48 h) treatment. h qPCR analysis of Pd-l1, H-2k, H-2d, and B2m in HY24409 cells treated with DEX (100 nM, 4 h) and MIFE (100 nM, 4 h), alone or in combination. n = 3 technical replicates in f–h. i–k Representative flow cytometry plots and quantification of cell-surface PD-L1 (i), MHC-I (j), and B2M (k) in HY24409 cells with or without MIFE (20 µM, 48 h) treatment. n = 3 biological replicates. l Left panel: qPCR analysis of Pd-l1, H-2k, H-2d, B2m, and Nr3c1 in GR-knockdown HY19636 cells. n = 3 technical replicates. Right panel: immunoblotting of GR and Gapdh in GR-knockdown HY19636 cells. m–o Representative flow cytometry plots and quantification of cell-surface PD-L1 (m), MHC-I (n), and B2M (o) in GR-knockdown HY19636 cells. n = 3 biological replicates. p qPCR analysis of Pd-l1, H-2k, H-2d, and B2m in HY19636 cells with or without MIFE (20 µM, 48 h) treatment. n = 3 technical replicates. q–s Representative flow cytometry plots and quantification of cell-surface PD-L1 (q), MHC-I (r), and B2M (s) in HY19636 cells with or without MIFE (20 µM, 48 h) treatment. n = 3 biological replicates. Statistical significance in b, c, and f–s was determined by a two-tailed unpaired t-test. Error bars are s.e.m. Source data are provided as a Source Data file.

Fig 3: A correlation between LC3B mRNA level and sensitivity of Sal in melanoma. (a) The mRNA levels of LC3B were analyzed using real-time PCR assay, and normalized with the amount of B2M mRNA. (b) Linear regression analysis was performed to identify the potential correlation between LC3B expression and inhibition rate of Sal treatment (1 μM, 7 days) on clinical melanoma samples. (c) Model of the action of Sal in SK-Mel-19 cells. Sal which disrupted the balance of Na+ and K+ triggered ER stress and a decrease of mitochondrial ΔΨ, thus leading to UPR and damaged mitochondria. To keep its stability balance and normal function, cells produced autophagosomes to degrade those damaged organelles. However, Sal not only induced autophagy, but also inhibited the fusion between autophagosomes and lysosomes, causing an aberrant autophagic flux. Finally, accumulations of autophagosomes lead to autophagic cell death.

Fig 4: Expression analysis of B2M and MHC class I molecules in B2M+/+ and B2M−/− cyiPSCs. (A) RT-PCR expression analysis of B2M mRNA. The RNA expression levels were normalized to the level of ACTB expression. Error bars denote means ± SD (n = 3 technical replicates). ***p < 0.001 by the two-tailed Student's t-tests compared with B2M+/+ cyiPSCs without IFNγ treatment. Data are representative of three independent experiments. (B) Western blot analysis of MHC class I molecules with or without IFNγ treatment. The data are representative of two independent experiments. (C) Flow cytometry analysis of cells without (blue line) or with (red line) IFNγ stimulation using anti-MHC class I antibodies. Black lines represent data from the isotype control. Data are representative of two independent experiments. IFNγ, interferon γ; MHC, major histocompatibility complex; reverse transcription PCR (RT-PCR); SD, standard deviation.

Fig 5: GR activates PD-L1 expression and represses MHC-I expression in human pancreatic cancer cells.a, b qPCR analysis of immune inhibitory and immune co-stimulatory genes (a) and genes involved in the MHC-I pathway (b) in SU86.86 cells with or without mifepristone (MIFE, 20 µM, 72 h) treatment. n = 3 technical replicates. c Immunoblotting of PD-L1, MHC-I, and B2M in SU86.86 cells with or without MIFE treatment with the indicated doses for 72 h. d–f Representative flow cytometry plots and quantification (by MFI: mean fluorescence intensity) of cell-surface PD-L1 (d), MHC-I (e), and B2M (f) in SU86.86 cells with or without MIFE (20 µM, 72 h) treatment. n = 3 biological replicates. g qPCR analysis of PD-L1, HLA-A, HLA-B, HLA-C, B2M, and NR3C1 in GR-knockdown SU86.86 cells. n = 3 technical replicates. h, Immunoblotting of PD-L1, MHC-I, B2M, and GR in GR-knockdown SU86.86 cells. i–k Representative flow cytometry plots and quantification of cell-surface PD-L1 (i), MHC-I (j), and B2M (k) in GR-knockdown SU86.86 cells. n = 3 biological replicates. l qPCR analysis of PD-L1, HLA-A, HLA-B, HLA-C, and B2M in GR-knockdown SU86.86 cells, with or without dexamethasone (DEX, 100 nM, 8 h) treatment. n = 3 technical replicates. m Immunoblotting of PD-L1, MHC-I, B2M, p-GR (Ser211), and GR in GR-knockdown SU86.86 cells, with or without dexamethasone (DEX, 100 nM, 8 h) treatment. n Normalized luciferase activity of the reporters containing the indicated human PD-L1 gene promoter regions in GR-knockdown SU86.86 cells, with or without IFN? (10 ng ml-1, 8 h) treatment. n = 4 wells. o Normalized luciferase activity of the reporters containing the indicated human HLA-B, HLA-C, and B2M gene promoter regions in GR-knockdown SU86.86 cells. n = 4 wells. p Normalized luciferase activity of the reporters containing the human GR-binding elements, PD-L1, HLA-B, HLA-C, and B2M promoter regions in SU86.86 cells with or without DEX (100 nM, 8 h) treatment. n = 4 wells. Statistical significance in a, b, d–g, i–l, and n–p was determined by a two-tailed unpaired t-test. Error bars are s.e.m. Source data are provided as a Source Data file.