Fig 2: MET triggered an increase in PD-L1 protein expression through STAT4 pathway. (A–C) Western blot of the indicated proteins in N33 and LN229 cell lines on treatment with HGF (200 ng/mL), and HGF/PLB-1001 (100 µM) combined, for 24 hours. GAPDH, protein-loading controls. Quantitative results of western blot analysis and relative expression difference are shown on the right panel. Fisher’s exact test, *p<0.05, **p<0.01, ***p<0.001. (D) In the IHC analysis of the tissue microarray, the scatter plot shows the correlation between the expression of MET and PD-L1 proteins in GBM. (E) Photographs of IHC staining of two representative WHO 4 (IDH-wildtype) primary glioma. Positive cells are stained brown. Magnification, 400×. (F) The statistical result of chromatin immunoprecipitation assay confirming the binding of STAT4 to the PD-L1 DNA. Student’s t-test, ***p<0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GBM, glioblastoma; HGF, hepatocyte growth factor; IHC, immunohistochemistry; MET, mesenchymal-epidermal transition factor; PD-L1, programmed death-ligand 1; pMET, phosphorylated MET; pSTAT4, phosphorylated STAT, signal transducer and activator of transcription; PLB-1001, Bozitinib; NC, negative control.

Fig 3: Kaplan-Meier survival analysis of MET, STAT4, and PD-L1 in primary GBM of TCGA (A) and CGGA (B) data sets. Survival analysis was performed using Kaplan-Meier curve method in conjunction with two-sided log-rank test. CGGA, Chinese Glioma Genome Atlas; GBM, glioblastoma; MET, mesenchymal-epidermal transition factor; OS, overall survival; PD-L1, programmed death-ligand 1; TCGA, The Cancer Genome Atlas.

Fig 4: Vitamin D does not prevent Th1 differentiation. Relative TBX21 (A), IL-12Rβ2 (B), and IFNγ (C) expression in T cells activated for 24, 48, or 72 h in Th1-polarizing medium in the presence or absence of 100 nM 25(OH)D3. Data are normalized to unstimulated T cells (mean + SEM, n = 6). Representative Western blots (lower panel) and quantification (upper panel) of phosphorylated STAT1 and total STAT1 (D) and phosphorylated STAT4 and total STAT4 (E) with GAPDH as loading control from T cells activated for 0, 24, 48, and 72 h in Th1-polarizing medium in the presence or absence of 100 nM 25(OH)D3. Western blots including protein ladder are shown in the Figure S2 in Supplementary Material.

Fig 5: Pharmacological inhibition of both PTP1B and TC-PTP with specific inhibitor. (A) Specificity of 1B/TC inh (50 μM) to inhibit PTP1B and TC-PTP dephosphorylation (n = 3). (B) Titration of 1B/TC inh in mouse moDCs using IL-12 production as indicator of activation (n = 3). (C) IFNγ production by 1B/TC inh-treated mature moDCs (4 μM dose) (n = 4). (D) Expression levels (MFI) of CD40, CD80, CD86, MHC class I and II on 1B/TC inh-treated mature moDCs (4 μM dose) (n = 3). (E) Activation status STAT1 and STAT4 (n = 3). (F) Activation status of Src kinase and IkBα (n = 4). Therapeutic properties of 1B/TC inh-treated moDCs in a mouse model of E.G7 lymphoma: (G) Tumor volume before moDC treatment (10 d after implantation of 5 × 105 E.G7-OVA cells) and (H) after intraperitoneal (IP) injections of 5 × 106 1B/TC inh-treated or non-treated moDCs compared with control group (tumor-bearing mice without moDC treatments). The IP injections with moDCs were given 18 d after tumor implantation. (I) Images represent tumor-bearing mice 8 d after of moDC treatment. The results are representative of at least three independent experiments. The differences in more than two groups were determined using One-Way ANOVA (Holm–Sidak multiple comparison test) for parametric and Dunn's multiple test for non-parametric. The differences between two groups were determined with unpaired t test (two tails of distribution). Significant differences are represented by p values <0.05.