Figure 5.

IGFBP2 co-localizes with p21 in the cytoplasm of senescent psoriatic keratinocytes and its expression is positively regulated by p16. (A) Immunofluorescence analysis was conducted on pso KC cultures (n = 3) at passage 4 (P4) to evaluate IGFBP2, p16, p21 and p-p21 subcellular localization. Cells were immunostained with anti-IGFBP2 Ab, followed by Cy3-conjugated secondary Ab (orange, panel i) or by Alexa Fluor 488-conjugated secondary Ab (green, panel v, ix), or, alternatively with p16 followed by Alexa Fluor 488 secondary Ab (green, panel ii), and p21 or p-p21 primary antibodies followed by Alexa Fluor 555 secondary Ab (orange, panels vi, x). Nuclei were counterstained with DAPI (blue) and the merging of three patterns was shown within the same field (merge, panels iv, viii, xii). Bars, 100 μM (B) IGFBP2, p16, p21 and p-p21 expression was analysed by WB on nuclear and cytosolic protein fractions obtained from two different pso KC strains (pso KC s1; pso KC s2), left untreated or treated with M4 for 18 hours. The quality of nuclear and cytosolic fractions was assessed by detection of lamin A/C and α-tubulin, respectively. (C) WB analysis was performed on lysates from primary human KC transduced with empty vector (mock) or p16 sense (p16S) vector and analysed for p16 and IGFBP2 expression. (D) Similarly, p16 and IGFBP2 expression was evaluated by WB on protein lysates obtained from primary KC cultures transduced with empty (mock) or antisense (p16AS). In (C, D), graphs show the means of the densitometric intensity (D.I.) of the bands ± SD, obtained from three independent experiments. *p ≤ 0.05, as calculated by Mann–Whitney U test.