Journal: Cell Research

Article Title: Pericytes augment glioblastoma cell resistance to temozolomide through CCL5-CCR5 paracrine signaling

doi: 10.1038/s41422-021-00528-3

Figure Lengend Snippet: a Apoptosis analysis of GBM-1 cells with indicated treatments. GBM cells were stimulated with CCL5 (10 ng/mL) or vehicle (PBS) followed by the administration of TMZ (500 μmol/L) or DMSO. ns, not significant. ** P < 0.01. b Cell survival analysis of GBM-1 cells with indicated treatments. CM from pericytes expressing shNT or shCCL5 (sh-1 or sh-2) was collected and added to GBM cells. GBM cells were treated with TMZ (500 μmol/L) after CM addition. ** P < 0.01. c Cell survival analysis of GBM-1 cells with indicated treatments. Pericyte CM was pretreated with neutralizing anti-CCL5 antibody or IgG before being added to GBM cells. GBM cells were treated with TMZ (500 μmol/L) after pericyte CM addition. ** P < 0.01. d Apoptosis analysis of GBM-1 cells with indicated treatments. GBM cells expressing shNT or shCCR5 (sh-1 or sh-2) were pretreated with pericyte CM or control GBM cell CM followed by TMZ (500 μmol/L) treatment. * P < 0.05; ** P < 0.01. e Schematic diagram of MVC-mediated inhibition of CCL5-CCR5 signaling. The binding of MVC to CCR5 leads to a conformational change of its extracellular domain to prevent ligand-stimulated CCR5 activation. f Apoptosis analysis of GBM-1 cells with indicated treatments. GBM-1 cells were pretreated with MVC (500 nmol/L) or DMSO for 1 h, followed by pericyte CM stimulation. Apoptosis and cell survival analyses were performed 48 h after TMZ treatment. ns, not significant. ** P < 0.01. g DNA damage after the indicated treatments was assessed by comet assay. Scale bars, 50 μm. h Quantification of percentage of cells with comet tails with indicated treatments. ns, not significant. * P < 0.05; ** P < 0.01. i , j Immunoblot analysis of phosphorylated AKT (Ser473) and AKT ( i ) or phosphorylated DNA-PKcs (Ser2056) and DNA-PKcs ( j ) in GBM-1 cells with indicated treatments. k Immunoblot analysis of phosphorylated AKT (Ser473), AKT, phosphorylated DNA-PKcs (Ser2056), DNA-PKcs, γ-H2AX in GBM-1 cells with indicated treatments. DNA-PKi (DNA-PKcs inhibitor) represents KU-57788; AKTi (AKT inhibitor) represents MK-2206. Experiments in a – k were independently performed three times.

Article Snippet: Human CCR5-specific shRNA vectors, CCL5-specific shRNA vectors and shNT vectors were purchased from Ribobio (China) with shRNA sequences listed in Supplementary information, Table .

Techniques: Expressing, Inhibition, Binding Assay, Activation Assay, Single Cell Gel Electrophoresis, Western Blot