Fig 2: IL-6 from M2 macrophage elevates the expression of PD-L1 and miR-21 in ovarian cancer cells. (A) The markers of M2 macrophage, CD206 and CD11b, were measured by flow cytometry. (B) Protein level of PD-L1 after SKOV3 and M2 macrophage co-culture, in both cell types detected by western blotting. (C) The change of PD-L1 expression after SKOV3 co-culture with IL-6 depleted M2 macrophages was detected by western blotting. (D) The expression of PD-L1, IL-6 and PD-L1 were determined by Western blotting analysis. (E) The expression of miR-21 after SKOV3 co-cultured with IL-6 depleted M2 macrophages was determined by RT-qPCR. (F) Secretion of EVs was observed by TEM. (G) The markers for EVs isolated from SKOV3 were determined by Western blotting analysis. (H) The level of miR-21 in EVs isolated from SKOV3 co-cultured with IL-6 depleted M2 macrophages was assessed by RT- qPCR. * p < 0.05 vs. control or normal. # p < 0.05 vs. co-M2 or co-SKOV3. & p < 0.05 vs. SKOV3. Statistical comparisons were performed using unpaired t test when only two groups were compared or by Tukey’s test-corrected one-way ANOVA with when more than two groups were compared.

Fig 3: Experimental verification of the relationship between PLTP and prognosis and immune microenvironment in the DLBCL TMA cohort. A Kaplan–Meier curve for the PLTP high- and low- expression groups in our TMA cohort. The optimal cutoff point was obtained from X-tile 3.6.1 software. B Characterization of cell immunophenotypes with mIHC. A staining panel was developed to visualize DAPI, CD68, CD163 and PLTP simultaneously on the same tissue slide. C M2 macrophages content in the PLTP high- and low-expression groups. CD68+CD163+ indicated the content of M2 macrophages (p value by Wilcoxon rank-sum test). D Characterization of cell immunophenotypes with mIHC. A staining panel was developed to visualize DAPI, CD11B and PLTP simultaneously on the same tissue slide. E TAMs content in the PLTP high- and low- expression groups. CD11B+ indicated the content of TAMs (p value by Wilcoxon rank-sum test). F Characterization of cell immunophenotypes with mIHC. A staining panel was developed to visualize DAPI, PD-1, PD-L1, LAG3 and PLTP simultaneously on the same tissue slide. CD274 indicated the content of PD-L1 and PDCD1 indicated the content of PD-1. G–I Correlations between PLTP expression and immune checkpoints (including PD-1, PD-L1, and LAG3)

Fig 4: Representative images of the 10X magnified H and E sections and post-immunofluorescence showing the localization of C3 and GFAP in retinal tissues collected from (A) control and (B) diabetic retina, C3, and CD11b in (C) control and (D) diabetic retina, and CFH and CD11b in (E) control and (F) diabetic retina, magnification 20X, scale bar 200 μm. Co-localized expression of CFH and CD11b is highlighted in 40X magnification in panel (F).

Fig 5: G9a inhibitor, UNC0642, suppresses SLC7A2-mediated HCC immune escape, invasion, and metastasis.A, B MHCC97H and HCCLM3 HCC cells treated with 5-aza (2 μm, 3 days), EPZ-6438 (1 μm, 3 days), UNC0642 (5 μm, 2 days), ITF-2357 (1 μm, 3 days) then qRT-RCR and western blotting was used to test the expression of SLC7A2. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. C ChIP-quantitative real-time PCR analysis to detect H3K9me2 association with SLC7A2 gene after treatment of G9a inhibitor UNC0642. The data are shown as the mean ± SD from the at least three independent experiments; **P < 0.01. D Representative IHC staining images for SLC7A2 and G9a in human HCC tissues. Scale bars, 200 µm (upper), 50 µm (lower). E Bioinformatics analysis of G9a mRNA expression on the TCGA database in human HCC tissues. F Kaplan–Meier analysis of the associations between G9a expression and overall survival in the HCC cohort. G–I Treatment of UNC0642 promoted HCC cell proliferation and metastasis in vitro. G Usage of UNC0642 on HCC cell colony formation. H The effects of UNC0642 on HCC cell proliferation were measured by a CCK-8 assay. I Transwell assay shown the abilities of migration and invasion. The scale bar represents 100 μm. J Western blotting was used to detect the G9a, SLC7A2, and its downstream target genes expression in MHCC97H and HCCLM3 cells treated with UNC0642. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. K, L Tumor growth in mice subcutaneously injected with H22-control or H22-SLC7A2 cells, treated with UNC0642 daily by intraperitoneal injection at 5 mg/kg. The tumor volume was monitored every 3 days. n = 5. M Flow cytometric images of subcutaneous H22-control or H22-SLC7A2 cells treated with UNC0642 or its control at day 27. CD8+ T cells (upper) and MDSCs (lower). Flow cytometric analyses the ratio of CD8+ T cells and MDSCs between groups at day 27, respectively, n = 5. The data are shown as the mean ± SD from the at least three independent experiments; **P < 0.01. N Immunofluorescent staining of the CD11b, CD8, and granzyme B protein expression patterns in mice tumor cells. Scale bars, 20 µm. O–R In vivo assays shown that the treatment of UNC0642 can block knock-down SLC7A2-mediated HCC metastasis. O The representative Bioluminescence images were shown in the different groups, treated with UNC0642 or DMSO in the BALB/C injected with the indicated cells in the liver and Incidence of lung metastasis, n = 10. The Bioluminescence intensity in the tumors at the indicated time point was presented as the total photon flux. P The number of metastatic lung nodules in lung in the BALB/C mice. n = 10. Q Overall survival of the mice in each group. R Representative H&E-stained lung metastatic nodules. The scale bars represent 1 mm (upper panel) and 100 μm (lower panel). *P < 0.05, **P < 0.01. S A schematic diagram of the SLC7A2 signaling in HCC immune evasion. SLC7A2 deficiency upregulated CXCL1 expression through PI3K/Akt/NF-κB pathway. CXCL1 promoted HCC growth and metastasis through recruiting MDSCs to the tumors. Neutralizing or suppressing MDSC infiltration abolished deficient SLC7A2-mediated HCC growth and metastasis. Usage of G9a inhibitor (UNC0642) suppressed loss of SLC7A2-meidated HCC metastasis.