Fig 1: CBX6 expression is downregulated in multiple cancer types(A) Statistical analysis of The Cancer Genome Atlas (TCGA) Pan-Cancer RNA sequencing (RNA-seq) database shows CBX6 expression is downregulated in various cancer types. (B) CBX6 expression is significantly suppressed in brain cancer, breast cancer, lung adenocarcinoma (abbreviated as LUAD), and prostate cancer, based on analysis of TCGA tumor microarray data using the BRowse All Variants Online (BRAVO) database method. (C and D) Comparison of CBX6 expression in patient-derived GBM tissues and primary tumor cells relative to respective normal controls using qRT-PCR. GAPDH was used as a loading control. (D) Reduced CBX6 expression is significantly associated with poor prognosis in patients with glioma, as shown in Kaplan-Meier survival curves derived from a public patient-derived microarray database,34p = 1.94∗ e−7.PBT, primary brain tumor; RBT, recurrent brain tumor.
Fig 2: Effects of CBX6 modulation on invasion and migration of U-251 MG cells(A and B) Representative images and cell quantification from transwell migration assays of U-251 MG cells with modulated CBX6 expression compared to corresponding controls. (C and D) Representative images and cell quantification from transwell invasion assays of U-251 MG cells with modulated CBX6 expression compared to respective controls. VC, vector control; OE, CBX6 overexpression; KD, CBX6 knockdown. “∗” Indicates p < 0.05.
Fig 3: CBX6 modulation affects tumor cell morphology and proliferation(A) CBX6 modulation affects the morphology of U-251 MG cells in two-dimensional culture. (B) Downregulation of CBX6 increases growth and invasion of U-251 MG cells in 3D culture. (C) Overexpression of CBX6 significantly suppresses U-251 MG proliferation on days 3 and 5 post-plating, as measured by MTT assay. (D) MTT assay of KLuc cells stably overexpressing human CBX6 or transfected with control plasmid on days 3 and 5 post-plating. (E) Silencing CBX6 increases U-251 MG proliferation compared to shRNA controls. Images were captured randomly from different fields under 20× magnification. VC, vector control; OE, CBX6 overexpression; KD, CBX6 knockdown. “∗” indicated p < 0.05.
Fig 4: Effect of human CBX6 overexpression on glioma tumor growth(A and B) Bioluminescent imaging of NSG mice (A) and quantification of bioluminescence signal intensity as fold change (B) on days 4 and 8 (n = 5). (C) Bioluminescent imaging at week 2 showing reduced Kluc tumor size in mice with human CBX6 overexpression compared to controls (n = 10). (D and E) Kaplan-Meier survival analysis demonstrating that overexpression of human CBX6 in U-251 MG cells (D) (n = 5) and Kluc cells (E) (n = 10) improved mouse survival. (F) Histological analysis of tumor invasion and microsatellite metastasis in CBX6-overexpressing tumors. “∗” Indicates p < 0.05.
Fig 5: CBX6 binds to the CA9 promoter(A) Genes related to tumor cell invasion, proliferation, or migration were selected based on RNA sequencing data from U-251 MG cells with CBX6 knockdown compared to negative controls. (B) qRT-PCR results show that CA9 expression is affected by CBX6 dysregulation in U-251 MG cells, with overexpression or shRNA-mediated knockdown of CBX6, using GAPDH as a reference gene. (C) Gene expression correlation analysis from CGGA reveals an inverse relationship between CBX6 and CA9 expression patterns (http://www.cgga.org.cn). (D) Western blot and qRT-PCR data demonstrate changes in CBX6 and CA9 expression in U-251 MG and PBT030 cells under normoxic (N) and hypoxic (H) conditions for 24 and 48 h, using 28S as a reference gene. (E) ChIP assay results from CBX6-overexpressing U-251 MG cells show detection of the CA9 promoter sequence using two primer sets in CBX6/Flag pull-down products compared to a negative IgG control via qRT-PCR. VC, vector control; OE, CBX6 overexpression; KD, CBX6 knockdown. “∗” Indicates p < 0.05.
Supplier Page from OriGene Technologies for CBX6 Human shRNA Plasmid Kit (Locus ID 23466)