Fig. 8.

The senescence phenotype promoted by YBX1 depletion in epidermal progenitors is dependent on the secretion of senescence-associated CXC cytokines. a, b YBX1-mediated induction of paracrine senescence. a Conditioned medium from human primary keratinocytes transfected with control and YBX1 siRNA (collected 4 days after transfection) was added to normal proliferating epidermal progenitors. Four days later, the cultures were stained with SA-β-Gal. Total and SA-β-Gal-positive cells were counted under light microscopy. Error bars represent mean SD, n = 7, *p < 0.05, unpaired t-test. b A portion from the same conditioned medium from control and YBX KD cells (as in a) was added to epidermal progenitors, seeded at clonal density (100 cells per well) on the top of feeder layer from mitomycin C-treated Swiss3T3 fibroblasts. Ten days later, the samples were fixed, the colonies were stained with SRB, and counted using ImageJ, **p < 0.01, unpaired t-test. c CXCR2 small-molecule inhibitor reverts keratinocytes senescence caused by YBX1 depletion. Primary cultures of human keratinocytes transfected either with control or YBX1 siRNA were treated with vehicle or the small-molecule antagonist of CXCR2 and stained for SA-β-Gal. SA-β-Gal-positive cells were counted under light microscopy (×40) and shown as percentage of total cells (*p = 0.012, unpaired t-test). d Blockade of the CXCL1/IL-8 signaling at the level of receptor activation rescues the effect of YBX1 knockdown on epidermal progenitor renewal: primary human keratinocytes transfected with control and YBX1 siRNA were seeded at clonal density (100 cells per well) over Swiss3T3 feeder layer and treated with vehicle control or a mix of blocking antibodies (3 μg each) for the two receptors interacting with CXCL1 and IL-8, CXCR1 and CXCR2. Ten days later, the cultures were fixed, the colonies were stained with SRB, counted using ImageJ, and fold changes in the numbers relative to the control are presented as average values, *p < 0.05, **p < 0.01, unpaired t-test