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Journal: bioRxiv
Article Title: Glioma-associated fibroblasts promote glioblastoma resistance to temozolomide through CCL2-CCR2 paracrine signaling
doi: 10.1101/2024.03.05.581575
Figure Lengend Snippet: (A) Gene Set Enrichment analysis of differentially expressed genes on the MAPK pathway in glioma cells (U87 and A172) cultured in control medium or GAF conditioned medium. Adjusted p value, the estimated probability that a set with a given Normalized Enrichment Score represents a false positive finding (n = 6 per group). (B) WB analysis of three key molecules, such as phosphorylated ERK1/2(Thr202/Tyr204) and total ERK1/2, phosphorylated p38MAPK and total p38MAPK, and phosphorylated JNK1/2/3 and total JNK1/2/3, of MAPK signaling pathway of U87 cells cultured with Ctr-CM or GAF-CM for 48 hours following TMZ treatment respectively (C) Quantification of protein levels (4 different GAFs were used for this experiment). (D) Representative flow cytometry plots of apoptotic analyses of U87 cells following TMZ treatment for 48 hours. U87 cells were pretreated with Trametinib (100nmol/L) or DMSO for 6 hours, followed by U87 cells or GAFs coculture for 24 hours. (E) Quantification of apoptotic GBM cells (n = 4 per group). (F) Representative flow cytometry plots of apoptotic analyses of U87 cells following TMZ treatment for 48 hours. U87 cells were pretreated with Trametinib (100nmol/L) or DMSO for six hours, then cultured with Ctr-CM or GAF-CM for 48 hours. (G) Quantification of apoptotic GBM cells (n = 4 per group). (H) Representative flow cytometry plots of apoptotic analyses of U87 cells. U87 cells were pretreated with Ravoxertinib (20µmol/L) or DMSO for six hours, then cultured with Ctr-CM or GAF-CM for 48 hours. (I) Quantification of apoptotic GBM cells (n = 4 per group). (J) WB analysis of cleaved-caspase9, cleaved-caspase3, and phosphorylated ERK1/2 (Thr202/Tyr204) and total ERK1/2 in U87 cells with indicated treatment. (K-M) Quantification of protein levels (4 different GAFs were used for this experiment). (N) WB analysis of cleaved-caspase3 and phosphorylated ERK1/2 (Thr202/Tyr204), and total ERK1/2 in U87 cells with indicated treatment. (O-P) Quantification of protein levels (3 different GAFs were used for this experiment). Data in C, E, G, I, K-M, and N-P were mean±SEM. p values from empirical phenotype-based permutation tests and adjusted using the False Discovery Rate (FDR) procedure (A), two-tailed student-t-test (C, E, G, I, K-M, and N-P). * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, ns = no significant.
Article Snippet: Information of primary antibodies was as follows: Fibronectin (BD Biosciences #610077), α-SMA (Proteintech #55135-1-AP), Cleaved-caspase3 (Cell Signaling Technology #9661), Cleaved-caspase9 (Cell Signaling Technology #9509), Bax (Millipore #ABC11), β-Tublin (Millipore #MAB3408), β-Actin (Cell Signaling Technology #3700), GAPDH (Proteintech #CL594-60004), Phospho-ERK1/2(Thr202/Tyr204) (Cell Signaling Technology #4370S), total-ERK1/2 (Cell Signaling Technology #4696), Phospho-p38 MAPK (T180/Y182) (Abclonal #AP1165), total-p38 MAPK (Abcam #ab308333), Phospho-JNK1-T183/Y185+JNK2-T183/Y185+JNK3-T221/Y223 (Abclonal #AP0276),
Techniques: Cell Culture, Flow Cytometry, Two Tailed Test