Fig. 6.

CM-SASP induces senescence in a paracrine manner. a Schematic representation of the experiment. Biliary cells are cultured with SASP-conditioned media (CM-SASP) and Control-conditioned media (CM-Ctrol). After 1 week, different markers of senescence were evaluated. b Number of total Ctrol-treated biliary cells increase (suggesting that they continue proliferating). Total number of CM-SASP-treated biliary cells plateau, indicative of an impaired proliferative response. **** denotes p < 0.0001 (Mean ± SEM). Mann−Whitney test. N = 7–8 biological replicates. c qRT-PCR shows a significant increased expression of cdkn1a (p21), cdkn1b (p27) and cdkn2a (p16) in the CM-SASP-treated biliary cells at day 7. * denotes p < 0.05 (Mean ± SEM). Mann−Whitney test. N = 4 biological replicates. d Morphological changes observed in the cell cultures. CM-SASP-treated biliary cells become flatter, larger and their content is vacuolized. e Increased SA-βGal (red) expression in CM-SASP-treated biliary cells with decreased BrdU incorporation (green). Far right, total percentage of SA-βGal or BrdU per DAPI-positive nuclei per field. f Increased expression of different senescence markers in CM-SASP-treated biliary cells compared with the CM-Ctrol-treated biliary cells. Below, total percentage of those markers per DAPI-positive nuclei. * denotes p < 0.05 (Mean ± SEM). Mann−Whitney test. N = 4 biological replicates. Scale bars = 50 µm