Fig 2: Correlation between differentially expressed CBXs and infiltration of immune cells using TIMER. The expression of (A) CBX1 was positively related to tumor purity and the infiltration of CD8+ T cells, macrophages, neutrophils and dendritic cells; (B) CBX2 expression was positively related to tumor purity and the infiltration of macrophages; (C) CBX3 expression was positively related to the infiltration of CD8+ T cells, macrophages, neutrophils and dendritic cells; (D) CBX4 expression was positively related to the infiltration of B cells, macrophages and neutrophils; (E) CBX5 expression was positively related to tumor purity and the infiltration of CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells; (F) CBX6 expression was positively related to the infiltration of B cells, CD8+ T cells and macrophages; (G) CBX7 expression was positively related to the infiltration of B cells, CD4+ T cells, macrophages, and negatively associated with the infiltration of CD8+ T cells and dendritic cells; (H) CBX8 expression was positively associated with tumor purity, the infiltration of macrophages and negatively associated with the infiltration of neutrophils and dendritic cells. TPM, transcripts per million; CBX, chromobox; TIMER, Tumor Immune Estimation Resource.

Fig 3: Protein levels of CBXs in normal (from healthy controls) bladder mucosal and BLCA tissues from the Human Protein Atlas. The protein levels of (A and B) CBX1, (C and D) CBX2, (E and F) CBX3, and (G and H) CBX4 were increased in BLCA tissues compared with in normal bladder tissues, whereas (I and J) CBX5 levels were unaltered. The protein levels of (K and L) CBX6 and (M and N) CBX7 were reduced in BLCA tissues compared with in normal bladder tissues, whereas those of (O and P) CBX8 were increased. Original magnification of the upper panels was ×50. Original magnification of the lower panels was ×400. The difference between groups was evaluated by unpaired Student's t-test. *P<0.05, **P<0.01. BLCA, bladder urothelial carcinoma; CBX, chromobox.

Fig 4: Protein expression levels of CBXs with prognostic value in different cell types. (A) Protein expression levels of CBX1, CBX2 and CBX7 in different cell types, and the bottom GAPDH blot is for both CBX2 and CX7. Semi-quantification of (B) CBX1, (C) CBX2 and (D) CBX7 protein expression levels determined by densitometry normalized to GAPDH. The difference between groups was evaluated by one-way ANOVA followed by the LSD post hoc multiple comparisons test. Data are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. CBX, chromobox.

Fig 5: Promoter methylation status of CBXs in BLCA using UALCAN. The promoters of (A) CBX1 and (B) CBX2 were hypomethylated in BLCA tissues. The promoter methylation level of (C) CBX3 and (D) CBX4 did not change. The promoters of (E) CBX5, (F) CBX6 and (G) CBX7 were hypermethylated in BLCA tissues, whereas (H) CBX8 promoter methylation did not change. The beta value (percentage of methylation signal strength) is the most commonly used quantitative method of methylation levels. The differences in promoter methylation levels were compared by Welch's t-test. ***P<0.001. BLCA, bladder urothelial carcinoma; CBX, chromobox.