Figure 8
Exosomes from NSCLC plasma patients modulate osteoclastogenesis in human primary osteoclasts. (a) Representative western blotting of ALIX and TSG 101 in 30 µg of exosomes isolated from plasma (1, 5 ml) of four NSCLC patients (Pz 1:stage I, Pz 2:stage IB, Pz 3:stage IIIA, Pz 4:stage IIIA). Original uncropped WBs were reported in Figure S5F. (b) Representative western blotting of AREG in 30 µg of exosomes isolated from plasma of ten NSCLC patients at different disease stages (Pz 1:stage I, Pz 2:stage IB, Pz 3:stage III, Pz 4:stage IIIA, Pz 5:stage IV, Pz 6:stage IV, Pz 7:stage IV, Pz 8:stage IV, Pz 9:stage IV, Pz 10:stage IV). Original uncropped WBs were reported in Figure S5G. (c) TRAP staining of human pOCs cultured in differentiation medium and incubated with: REC-AREG, CRL-2868 exosomes, NSCLC-patient exosomes, NSCLC-patient exosomes plus AREG neutralizing antibodies. Scale bar 10 µm. Evaluation by Real Time PCR of TRAP (d) and MMP9 (e) mRNA expression, in primary human pre-osteoclasts, treated for 4 days, with: CRL-2868 exosomes, AREG neutralizing antibodies, NSCLC patient exosomes, CRL-2868 exosomes plus AREG neutralizing antibodies, NSCLC patients exosomes plus AREG neutralizing antibodies. (f) MMP9 protein levels assessed by ELISA, in primary human pre-osteoclasts treated for 4 days with: CRL-2868 exosomes, AREG neutralizing antibodies, NSCLC patient exosomes, CRL-2868 exosomes plus AREG neutralizing antibodies, NSCLC patients exosomes plus AREG neutralizing antibodies. (g) Evaluation by real time PCR analysis of mRNA expression of TRAP and MMP9 in primary human pre-osteoclasts treated for 4 days with: exosomes released by Control, Mock, Scramble, and AREG-knockdown CRL-2868 cells. Values are the mean ± SD of three independent experiments *p ≤ 0.05, **p ≤ 0.01.
