Fig 2: Cells with genetic modification of locus 22 and silence of MAFG or NEF2L1 are failed to escape the T cell-mediated killing.A Representative images of SUM-159 cell before and after co-cultured with activated T cells. Representative images of activated T cells co-cultured with SUM-159 cells or genetic modified cells (sgVector, sg-22, sg-MAFG-1, sg-MAFG-2, sg-NFE2L1-1 and sg-NFE2L1-2; B Quantification of remaining SUM-159 cells and its genetic modified cells after co-cultured with activated T cells; C Real-time PCR was performed to examine the relative mRNA levels of Granzyme B in activated T cells co-cultured with control or genetic modified cells; D Real-time PCR was performed to examine the relative mRNA levels of IFNγ in activated T cells co-cultured with control or genetic modified cells; E The expression of MAFG, MAFF, MAFK, NFE2L1, NFE2L2, CD273 and CD274 in breast cancer cell lines. F The expression of MAFG is positively correlated with PD-L1 and PD-L2 in breast cancer cell lines. Data from (A) and (B) are representative of three independent experiments. Data from (C) and (D) are representative of two independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001.

Fig 3: LPS strongly induces PD-L1 expression via super-enhancer and its core DNA region.A SUM-159 cells and MDA-MB-231 cells were treated with indicated concentration of LPS for 24 h and western blot analysis was performed to examine the expression of PD-L1; B Relative mRNA level of PD-L1 and PD-L2 was examined by RT-PCR in LPS treated SUM-159 cells; C ML385 was used to pre-treat cells for 6 h, then LPS was added for treatment at indicated doses for 24 h. The expression of PD-L1 was examined by western blot; D JQ-1 was used to pre-treat cells for 6 h, then LPS was added for treatment at indicated doses for 24 h. The expression of PD-L1 was examined by western blot; E Western blotting was used to quantify the protein level of PD-L1 in LPS treated sgVector, sgPD-L1L2-SE and sgPD-L1L2-SE-C1 cells; F Western blotting was used to quantify the protein level of PD-L1 in LPS treated sgVector and sg-22 cells; G Relative mRNA level of PD-L1 was examined by RT-PCR in LPS treated sgV, sgSE, sgC1 and sg22 cells; H HCl was used to treat cell and the protein level of PD-L1 in sgV, sg-C1 and sg-22 was determined by WB. Data in (A–F) are representative of three independent experiments. Data in (G) and (H) are representative of two independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001.

Fig 4: The transcription factors MAFG and NFE2L1 are required for super-enhancer- and LPS-induced PD-L1 expression.A Western blot analysis of the expression of MAFG in MCF-7, SUM-159 and MDA-MB-231 cell lines; B The relative mRNA level of MAFG was qualified by real-time PCR in MCF-7, SUM-159 and MDA-MB-231 cell lines; C Western blot analysis of PD-L1 and MAFG in SUM-159 that stably transfected with control and sgRNAs targeting on MAFG; D The relative mRNA levels of PD-L1 and PD-L2 in SUM-159 that stably transfected with control and sgRNAs targeting on MAFG; E Immunofluorescence assays were used to determine the PD-L1 expression in MAFG-deficient cells; F The surface expression of PD-L1(PE) and PD-L2(APC) in control and MAFG-deficient cells was determined by FACS; G Western blot analysis of PD-L1 and NFE2L1 in SUM-159 that stably transfected with control and sgRNAs targeting on NFE2L1; H The relative mRNA levels of PD-L1 and PD-L2 in control and NFE2L1 deficient cells was determined by RT-PCR; I Immunofluorescence assays were used to identify PD-L1 expression in NFE2L1 deficient cells; J SUM-159 and MDA-MB-231 were treated with LPS in indicated doses and the level of MAFG and NFE2L1 was measured by Western blot. K Control and MAFG-deficient cells were treated by LPS for indicate doses. The level of PD-L1 in these cells was examined by Western blot; L Control and NFE2L1 deficient cells were treated by LPS for indicated doses and the level of PD-L1 was determined by Western blot. M The protein level of PD-L1 in sgV, sgNFE2L2, sgNFE2L1 and sgMAFG were analyzed by WB. N The protein level of PD-L1 in sgV and sgMAFF cells were determined by WB. O The protein level of PD-L1 in sgV and sgMAFK cells were determined by WB. Data in (A–D, G, H, J and K) are representative of three independent experiments. Data in (E, F, I, M–O) are representative of two independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001.

Fig 5: Precisely mapping the nucleotide acid in locus 22 and its resident transcriptional factors NFE2:MAF.A Cloning and direct sequencing of genetic modified locus 22 by T vectors. B DNA sequences of wild-type and two single cell colonies. One is deletion of TGT and another one is addition of T; C Three single cell colonies (1,2,5) were selected and expanded for experiments. Real-time PCR was performed to analyze the mRNA level of PD-L1 and PD-L2 in sgVector, pooled sg-22 and three single cell colonies separated from pooled sg-22; D Western blot was performed to analyze the protein level of PD-L1 in sgVector, sgC1, pooled sg-22 and three single cell colonies from pooled sg-22; E Immunofluorescence was used to examine the expression and distribution of PD-L1 in sgVector and three single cell colonies from pooled sg-22; F The potential transcription factors at 30 bp DNA region around locus 22 in wild-type and dTGT were analyzed by online programs AnimalTFDB (http://bioinfo.life.hust.edu.cn/AnimalTFDB). All potential transcription factors at each allele were listed. The binding of NFE2:MAF was disrupted after deletion of TGT. Data in (C) and (D) are representative of three independent experiments. Data in (E) are representative of two independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001.