CCR5 Antibody - #AF6339

Fig. 5 CCL5 promotes prostate cancer invasion and PCSCs self-renewal via activating the CCR5/β-catenin/STAT3 pathway. a–c Heatmaps of 94 metastasis-related DEGs (a), 42 stemness-related DEGs (b), as well as 30 STAT3 pathway-related DEGs (c). RNA-Seq analysis was conducted to characterize the cellular responses of PC3 cells to 40 ng/ml CCL5 treatment. Differential gene expression analysis was conducted to identify the DEGs (n = 3). d Venn diagram of the DEGs in the indicated groups. CTNNB1, also known as β-catenin, was the most significant one of them. e Western blotting assay indicated that the CD133+ PCSCs sorted from PC3 cells by MACS method exhibited increased expression of CCR5, β-catenin, and STAT3 when compared with the CD133- subpopulation (n = 3). f CCL5 treatment significantly elevated the promoter activity of STAT3 in PC3 cells while XAV-939, the specific inhibitor of β-catenin, partly abrogated that (n = 6). g Immunofluorescence assay indicated that CCL5 treatment (40 ng/ ml) could significantly induce the expression and nuclear translocation of β-catenin in prostate cancer cells. Scale bar, 10 μm. h CHIP assay suggested that β-catenin could bind to the promoter region of STAT3, while the specific inhibitor of β-catenin (XAV-939) partly decreased their binding activity. i CCR5 specific siRNAs could significantly abrogate the activation effect of CCL5 on β-catenin/STAT3 pathway (n = 3). All values are presented as the mean ± SD, *p < 0.05, **p < 0.01.

Fig. 4 Inhibiting the CCL5/CCR5 signaling pathway attenuates hyperalgesia in DRG PP2Cm-deficient mice a, Quantification for release of CCL5 in the L4-L5 DRGs of PP2Cm-ctrl and PP2Cm-cKO mice, as detected by ELISA. n = 4 mice/group. b, Illustrative cartoon depicting the intrathecal administration of an anti-CCL5 neutralizing antibody. c, Quantification for release of CCL5 in the L4-L5 DRGs of PP2Cm-cKO + IgG, PP2Cm-cKO + anti-CCL5 1d and PP2Cm-cKO + anti-CCL5 3d mice. n = 4 mice/group. d-f, Mechanical withdrawal thresholds (d), withdrawal latencies to heat (e) and cold stimulus (f) on day 0 (baseline, BL), 1, and 3 post-injection in PP2Cm-cKO mice treated with IgG or anti-CCL5 neutralizing antibody. n = 5–6 mice/group. g, The L4-L5 DRGs from PP2Cm-ctrl and PP2Cm-cKO mice were double stained with PP2Cm (green) and CCR5 (red). The corner image in white square is the zoomed-in image of the area in the smaller white square. Scale bar: 20 μm. h, i, Fluorescent quantifications for PP2Cm (h) and CCR5 (i) in DRG sections from PP2Cm-ctrl and PP2Cm-cKO mice. n = 3 sections from 3 mice/group. j, A representative cartoon illustrating the intrathecal injection of CCR5 antagonist maraviroc or Ccr5 small interfering RNA (siCcr5). k-m, Mechanical withdrawal thresholds (k), withdrawal latencies to heat (l) and cold stimulus (m) at BL, 1 h, and 24 h post-injection in PP2Cm-cKO mice treated with Vehicle or maraviroc. n = 6 mice/group. n, The mRNA levels of Ccr5 in the L4-L5 DRGs from PP2Cm-cKO + siCtrl, PP2Cm-cKO + siCcr5 2d, and PP2Cm-cKO + siCcr5 8d mice (to β-actin). n = 3 experimental repeats (6 mice)/group. o-q, Mechanical withdrawal thresholds (o), withdrawal latencies to heat (p) and cold stimulus (q) at BL, 2d, and 8d post-injection in PP2Cm-cKO mice treated with siCtrl or siCcr5. n = 5–6 mice/group. Data are shown as the mean ± SEM. Statistical tests used were unpaired two-tailed Student’s t-test (a, h, i), one-way ANOVA with Tukey’s multiple comparisons test (c, n), and two-way repeated-measures ANOVA with Bonferroni’s post hoc test (d-f, k-m, o-q). *p 

Figure 2. The chemokine (C-C motif) ligand 5 (CCL5)/chemokine (C-C motif) receptor 5 (CCR5) pathway regulates the migration of 3T3-L1 preadipocytes to C2C12 myotubes. A and B: representative bright-field micrographs and quantification results of crystal violet-stained transwell membranes for 3T3-L1 preadipocyte migration by adding recombinant CCL5 (rCCL5) at concentrations of 0, 5, 10, and 20 ng/mL. Scale bar, 200 μm. n = 3. C: the relative mRNA expression of Ccl5 in C2C12 myotubes transfected with negative control siRNA (siNC) or with siRNA silencing CCL5 (siCCL5), normalized to siNC. Gapdh was used as the internal reference gene. n = 3. D: Western blot analysis and quantification of CCL5 in C2C12 myotubes transfected with siNC or with siCCL5. n = 3. E and F: representative bright-field micrographs of crystal violet-stained trans well membranes and corresponding quantification for each treatment group. The treatment involved the migration of 3T3-L1 preadipocytes in a coculture system with C2C12 myotubes that were transfected with siNC, siCCL5, siNC + rCCL5 (20 ng/mL), and siCCL5 + rCCL5 (20 ng/mL). Scale bar, 200 μm. n = 3. G and H: the relative mRNA expression of Ccr5 and Western blot analysis of CCR5 in 3T3-L1 preadipocytes treated with DMEM (NC) or conditioned media from differentiated C2C12 cells for 0 and 6 days. n = 3. I: the relative mRNA expression of Ccr5 in 3T3-L1 cells transfected with pcDNA3.1 vectors or overexpression vectors containing the coding sequence (CDS) region of CCR5 (pc-CCR5). n = 3. J: representative bright-field micrographs and quantification results of crystal violet-stained transwell filters for 3T3-L1 preadipocyte migration. The treatments involved the 3T3-L1 preadipocyte migration in a coculture system with C2C12 myotubes. The 3T3-L1 preadipocytes were transduced with pcDNA3.1 or pc-CCR5 vectors. Scale bar, 200 μm. n = 3. K: representative bright-field micrographs and quantification results of crystal violet-stained trans well filters for 3T3-L1 preadipocyte migration. The coculture system involved C2C12 myotubes that were treated with MARAVIROC (MVC) (0.1 μm) or not. Scale bar, 200 μm. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. Validation of CCR1, CCR5 and CCR7 in Guangxi cohort. (A) Expression of CCR1, CCR5 and CCR7 in HCC and para-carcinoma live tissues detected with IHC assay; (B) expression of CCR1, CCR5 and CCR7 in HCC and para-carcinoma live tissues detected with qPCR assay; (C) Matrix graphs of Pearson correlations for CCR1, CCR5 and CCR7; (D–F) ROC curves for CCR1, CCR5 and CCR7; (G–I) survival analysis for OS in terms of CCR1, CCR5 and CCR7; ** P