Figure 3
CRL-2868 exosomes induce the activation of EGFR pathway and osteoclast markers gene expression. (a) Western blotting analysis of pEGFR and EGFR in whole lysate of RAW 264.7 cells treated, for 6 days, with 20 μg/ml CRL-2868 exosomes (Exo) and RANKL (positive control) compared to untreated cells (Ctrl). Original uncropped WBs were reported in Figure S5C. GAPDH was used as loading control. (b) Evaluation by quantitative Real Time PCR of mRNA RANKL expression in RAW 264.7 cells treated, for 6 days, with 20 and 50 μg/ml CRL-2868 exosomes. (c) msRANKL protein levels assessed by ELISA, in RAW 264.7 cells treated, for 6 days, with 20 and 50 μg/ml CRL-2868 exosomes. (d) Evaluation by quantitative Real Time PCR of mRNA expression of TRAP and MMP9 in RAW 264.7 cells treated, for 6 days, with 20–50 μg/ml CRL-2868 exosomes and RANKL (positive control). (e) MMP9 protein level assessed by ELISA, in RAW 264.7 cells treated, for 6 days, with 20–50 μg/ml CRL-2868 exosomes and RANKL. Values are the mean ± SD of 3 three independent experiments *p ≤ 0.05, **p ≤ 0.01. (f) TRAP staining of RAW 264.7 cells incubated with CRL-2868 exosomes and 25 ng/ml of RANKL (positive control), for 6 days, compared with untreated cells. Scale bar 10 µm. (g) Evaluation by quantitative Real Time PCR of mRNA expression of TRAP and MMP9 in human primary pre-osteoclasts treated, for 4 days, with 20–50 μg/ml of CRL-2868 exosomes. (h) MMP9 protein levels assessed by ELISA, in human primary pre-osteoclasts treated, for 4 days, with 20–50 μg/ml CRL-2868 exosomes. Values are the mean ± SD in three independent experiments *p ≤ 0.05, **p ≤ 0.01.
