Fig 2: YY1 transcriptionally up‐regulates IL32 expression in pericytes. A) Promoter prediction study of IL32 reveals the presence of transcriptional factor YY1 putative binding sites. Schematic diagram showing the position of the two predicted YY1 putative binding sites. B,C) Western blot or RT‐PCR analysis of YY1 and IL32 expression in PCs transfected with siNSC or siYY1‐1/‐2. The quantification of the Western blot results is provided below the blots. D) Chromatin immunoprecipitation (ChIP) assay showed an increased binding of YY1 to both of its putative binding sites in siNSC transfected PCs as compared to siYY1 transfected PCs. E) The bar chart illustrates the relative luciferase activity in PCs following co‐transfection with a luciferase vector containing the full‐length IL32 promoter sequence and varying levels of YY1 overexpression plasmid. F) The bar chart displays the relative luciferase activity in PCs following co‐transfection with a luciferase vector containing either the full‐length IL32 promoter sequence (WT) or a promoter sequence with mutated proximal or distal YY1 putative binding sites, along with or without a YY1 overexpression plasmid. G) The bar chart shows the percentage of pericyte‐YY1 positive blood vessels in each patient. H) Representative triple immunostaining images of YY1, α‐SMA, and CD34 on tumor sections derived from each group are given. I) Correlation study between pericyte‐YY1 and pericyte‐IL32 expression in our NSCLC patient cohort (n = 20 patients). J) Almonertinib/Osimertinib IC50 experiments of HCC827 cells treated with CM from PCs transfected with either siNSC or YY1 targeting siRNA‐1/‐2. K) Representative fluorescent images of GFP fluorescently labeled HCC827 cells co‐cultured with RFP fluorescently labeled siNSC or siYY1‐1/‐2 transfected PCs in the presence of Almonertinib/Osimertinib. The bar chart shows the relative number of GFP (left) or RFP (right) positive cells in each group. L,M) Colony formation assays of HCC827 cells after being treated with CM derived from siNSC or siYY1‐1/‐2 transfected PCs in the presence/absence of Almonertinib/Osimertinib. The bar chart shows the relative colony number in each group. N,O) Transwell invasion assays of HCC827 cells after being treated with CM derived from siNSC or siYY1‐1/‐2 transfected PCs in the presence/absence of Almonertinib/Osimertinib. The bar chart shows the relative number of invaded cells in each group. NS: non‐significant difference. *p < 0.05, ** p < 0.01, ***p < 0.001. C–F,K–O) One‐way ANOVA. I) Pearson correlation coefficient. Scale bars in H,N,O) represent 100 µm. K) 200 µm.

Fig 3: Effects of ectopic USP15 and UbV expression on YY1 mRNA level. 293T were transfected with pNL4-3, pNL4-3 plus pUSP15-Myc (1:1 ratio) or pNL4-3 plus pFlag-UbV15.1 (1:1 ratio). pcDNA3 was included to equalize the total DNA amounts among the transfections. The cells were harvested for total RNA isolation and qRT-PCR. YY1 mRNA levels were calculated based on the RQ Min and Max of the replicates, normalized to the internal control GAPDH, and compared to the transfection with pNL4-3 only, which was set at 1.0. The data were Mean ± SD of RQ Min and Max of multiple replicates of three independent experiments.

Fig 4: The suppressed BM of NSCLC and attenuated SPHK2/S1P/STAT3 pathway in xenograft and zebrafish model by RPTOR blockade. A-J PC-9 cells were injected intravenously to construct the BM mouse model. Fluorescence microscopy showed that RPTOR blockade significantly suppressed the BM formation, the fluorescence of tumor cells in the ROI of BM, the expression of the proliferation marker Ki67 and MVD in the peritumoral area, prolonging overall survival (200 × magnification, scale bar = 50 μm). RPTOR overexpression produced the opposite effects. K Effects of RPTOR expression on the expression of proteins in BM tissue samples in nude mice. RPTOR knockdown reduced the expressions of RPTOR, YY1, SPHK2, and S1P1, with RPTOR overexpression yielding the opposite effects (200 × magnification, scale bar = 50 μm). L-O H1299 and PC9 cells bearing vectors for RPTOR knockdown or overexpression were injected into the perivitelline space of zebrafish embryos. The number of cancer cells at days 1 and 3 after the injection was significantly higher in the RPTOR-overexpressed group than in their respective control groups, whereas the number of cancer cells at days 1 and 3 after injection was significantly lower in RPTOR-knockdown cells than in control cells

Fig 5: The lactylation of YY1 in microglia plays an important role in regulating angiogenesis. a Heatmap shows the top 20 DELPs with increased and decreased lactylation under hypoxia for 24 h. b The enrichment analysis of DELPs. c Using the STRING database, DELPs specifically target the binding process. The node size corresponds to the relative fold-change of lactylation. d Collision-induced dissociation (CID) analysis was used to determine the modification sites. The MS/MS spectrum of modified “(Kla)SYLSGGAGAAGGGGADPGNK” is shown. e Hypoxia increases YY1 lactylation. Lactylation of YY1 in HMC3 cells under normoxia or hypoxia for 24 h was detected by YY1-Kla antibody (n = 3 per group). f–i HMC3 cells overexpressing WT or K183R YY1 were pretreated with 1% O2 for 24 h. Then HRMECs were cocultured with the pretreated HMC3 cells. The tube formation, spheroid sprouting, migration, and proliferation assays were performed as shown in the “Methods” (n = 3 independent experiments, 3 images for each group); scale bars, 50 μm (f, g, and i) or 100 μm (h). *p < 0.05; **p < 0.01; ***p < 0.001