Fig 2: Experimental verification reveals FOXP3 involved in TMZ resistance (a). RT-qPCR analysis displayed the expression level of 4 genes in GBM-resistant strains and normal groups. (b) FOXP3 stable overexpression by LN229 and U87MG cells was verified by western blot. (c). The cell viability assay revealed that FOXP3-overexpressed GBM cell lines had a higher IC50 than the control group. ∗p < 0.05; ∗∗p < 0.01;∗∗∗p < 0.001; ns p > 0.05.

Fig 3: p65 promoted Tregs recruitment via directly modulating miR‐23a. A, A p65‐binding site could be found in miR‐23a promoter region. B, Overexpression of p65 significantly reduced the luciferase activity driven by miR‐23a promoter. Mutagenesis in the p65‐binding site released such repression. C, ChIP assay confirmed that p65 could bind to the promoter region of miR‐23a. D, p65 inhibition upregulated miR‐23a level. E, p65 inhibitor significantly reduced the p‐p65, p65, and CCL22 protein levels in HepG2 and HepG2.2.15 cells. F, The gray scale analysis of p‐p65, p65, CCL22 after p65 inhibitor treatment. G, p65 inhibition dampened CCL22 mRNA level. H, CD4+CD25+ Foxp3+ human Tregs were identified. I, HBV+ HepG2.2.15 cells caused more Tregs to migrate through transwell membrane than HBV− HpeG2 cells. Pretreatment with p65 inhibitor attenuated Tregs transmigration in both cell lines. Error bars represented mean ± SD. *P < .05, **P < .01, and ***P < .001

Fig 4: High expression of FoxP3 could promote migration, invasion, and metastasis of ccRCC in vitro, and it might be associated with epithelial–mesenchymal transition (EMT). (A) Gene Set Enrichment Analysis (GSEA) images showing the difference in the expression of tumor-associated pathways between FoxP3-silenced 786-O cells and control cells (shFoxP3: FoxP3-silenced 786-O cells; shNC: normal control 786-O cells). Each colored line segment in the figure corresponds to the pathway of the corresponding color on the right side, respectively. The vertical lines in the lower panel represent each gene in the specific pathway, respectively. The horizontal lines of the upper graph represent the genes at the corresponding positions in the lower graph, and the vertical lines represent the enrichment score (ES). The peaks of its vertical line coordinate the ES value of the gene set. The peak appearing at the front end of the sequenced gene set (ES value > 0) indicates that the pathway was up-regulated, and the peak appearing at the back end (ES value < 0) indicates that the pathway was down-regulated. (B) The Gene Set Enrichment Analysis (GSEA) image showing the difference in expression of the “Hallmark_epithelia_mensenchymal_transition_targets” pathway between the FoxP3 high-expression group and the FoxP3 low-expression group. The top part of the graph was described previously. The red part in the middle of the graph indicates that the gene was highly expressed in the FoxP3 high-expression group. The blue part indicates that the gene was highly expressed in the FoxP3 low-expression group. The lower part of the graph shows the rank in the ordered dataset, where a value greater than 0 means that the gene was more highly expressed in the FoxP3 high-expression group. The lower the vertical line is above 0, the higher the expression of the corresponding gene in the FoxP3 low-expression group. (C) The images of the wound healing assay illustrate the difference in cell migration ability between the FoxP3-silenced and control groups of 786-O cells and ACHN cells. The images were recorded at 0, 24, 48, and 72 h after scratching, where the black line indicates the front end of the migrating cells. (D) Representative images were taken from the Transwell assay, and box plots were counted for migrating and invading cells in each group with the magnification of 100X under the microscope. The 786-O cells and ACHN cells were selected as the FoxP3-silenced group and the normal control group, respectively. For the Transwell assay, the 4 groups of images on the left represent 2 types of cells grouped for migration and invasion, respectively. The dark-colored cells of the images are tumor cells. The right panel compares the number of migrating or invading cells in the two types of experimental groups with the control group, respectively.

Fig 5: In ccRCC tissue microarray, FoxP3 was highly expressed in the tumor and might correlate with ccRCC stages. (A) Representative images of adjacent normal kidney tissues (upper panel) and kidney tissues (lower panel) after detection by IHC. (B) Histogram showing the FoxP3 average IHC score in grade T1 (53), T2 (23), and T3 (6) of ccRCC in the tissue microarray, respectively. IHC staining was graded as follows: 0 for 0%, 1 for ≤25%, 2 for 25–50%, 3 for 50–75%, and 4 for ≥75%. The IHC intensity was scored as follows: 0 for no staining, 1 for weakly positive staining, 2 for moderately positive staining, and 3 for strongly positive staining (ns: no statistical significance). (C) Histogram showing the FoxP3 average IHC score in stage Ⅰ (55), Ⅱ (23), and Ⅲ (8) of ccRCC in the tissue microarray, respectively. (D) Histogram representing the FoxP3 average IHC score in metastatic and non-metastatic ccRCC in tissue microarray, respectively (meta: metastasis occurred; no-meta: no metastasis occurred).