Figure 6.

Mechanistic insights in the regulation of CD271. (a) Immunoblot analysis of MeWo^Par cells treated with either cisplatin (Cis), etoposide (Eto) or temozolomide (TMZ) (all at a final concentration of 10 μM) for levels of CD271 (s/l=short/long exposure), p-p53^S15, p21^CIP, γH2AX, p-ERK1/2 and ERK1/2, p-AKT^S473 and AKT, p-IκBα, p-CHK1 and p-p38 kinase. (b) Immunoblot analysis of MeWo^Eto, MeWo^Cis or MeWo^Par cells following dose-dependent treatment with etoposide or cisplatin (3, 10 μM both) for 24 h, respectively. Levels of CD271, γH2AX and phosphorylated (activated) p53 (p-p53^S15) are shown indicating the different response of sensitive and resistant cells to drugs. (c) Regulation of p53-targets MDM2, p21^CIP (CDKN1A), CD95, NOXA, XIAP, as well as MSH6 and CD271 in MeWo^Par and MeWo^Eto cells treated with etoposide (3, 10 μM) for 24 h. Shown are relative expression levels of representative experiments (I, II). (d) Immunoblot analysis of T20/02 cells stably transfected with shCD271 or control, following dose-dependent treatment with etoposide for levels of CD271, p-p53^S15, p21^CIP and γH2AX. (e) Analysis of cells described in d but stably transfected with different CD271-targeting shRNAs (sh#2, sh#3, sh#4) or control (shCtl.) for levels of p53, p21^CIP, CD95, NOXA and BAX. Scale indicates relative expression levels±s.d. of independent triplicates, *P⩽0.05; **P⩽0.01; ***P⩽0.001. In (a, b, d), whole-cell lysates were analyzed, β-tubulin served as loading control. (f) Panel-based next-generation sequencing (NGS) revealed detection of a TP53 mutation (p.E258K, 5′-GAA->AAA-3' antisense (3′-CTT→TTT-5′)) in MeWo^Par cells, not present in wild-type T20/02 cells. Shown are sequences, as well as read depths. (g) Left panel: immunoblot analysis of cells described in d for levels of CD271 and p21^CIP following a dose-dependent treatment with MDM2 inhibitor nutlin-3a. Right panel: nutlin-3a treatment of MeWo^Par cells.