Fig 2: Changes in gene expression in total leukocytes in patients treated with MTL-CEBPA. A, Expression of indicated genes in leukocytes from 12 patients. Gene expression was evaluated by qRT-PCR. Individual results, mean, and standard deviation are shown. P values are calculated using two-sided Student t test. B, the presence of M-MDSCs (CD66b- CD14+ HLA-DR-/loCD15- CD11b+CD38+) and PMN-MDSCs (CD66b+CD14-CD15+CD11b+ LOX1+) among mononuclear cells was analyzed by flow cytometry and represented as frequency of gated cell population at 60K event per 1 × 106 cells (n = 3). C, Total circulating population of monocytes and neutrophils in the same patients was measured as a percentage of total PBMC.

Fig 3: Pro-phagocytic stimulation does not influence protein diversity but alters abundance of specific proteins. (A) For 3 biological replicates, donor-derived macrophages are stimulated with IF-g (+Stim) or PBS (-Stim) prior to lysis, digestion, and MS acquisition. (B) Summed spectral counts across 3 donors show comparable spectral counts between -Stim and +Stim. Differentially detected protein IDs follow a similar trend. (C). Spectral counts for CD11b, CD74 and IDO1 are consistent across donors and are altered in response to pro-phagocytic stimulation. (D). Ratios observed by MS are recapitulated via western blot.

Fig 4: The M1-to-M2 ratio is increased in aged skin. (a) Representative CD86 (green) and CD68 (red) immunofluorescence-stained samples of young (mean age = 34.2 years) and old (mean age = 74.5 years) human skin. The arrows indicate CD86+ CD68+ cells in the dermis. (b) Representative CD206 (green) and CD68 (red) immunofluorescence-stained samples of young and old human skin. The arrows indicate CD206+ CD68+ cells in the dermis. (c) Representative CD11b (green) and CD68 (red) immunofluorescence-stained samples of young and old human skin. The arrows indicate CD11b+ CD68+ cells in the dermis. (d) Quantitation of CD86+ CD68+ cells/mm2 in the dermis. (e) Quantitation of CD206+ CD68+ cells/mm2 in the dermis. (f) Quantitation of CD11b+ CD68+ cells/mm2 in the dermis. (g) Quantitation of the M1/M2 ratio (number of CD86+ CD68+ cells/number of CD206+ CD68+ cells) in the dermis. Nuclei were stained with Hoechst (blue). All data are expressed as mean ± SD values. n = 10 in both the young and old groups. *P < 0.05 and **P < 0.01. To test for significance, the Mann?Whitney U test was utilized. Bar = 100 µm. n.s., not significant.

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-?B 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.