Fig. 3. Sempervirine induces nucleolar stress in p53-wt and p53-null cells.

A Light microscopy images of 2102EP(S) (EPS) and NCCIT nucleoli. Control, sempervine, or nutlin treatments were carried in EPS; control or sempervirine treatments were carried in NCCIT. Scale bar = 20 μM. Arrowheads pointing to nucleoli. B Immunofluorescence analysis on 2102EP(S) (EPS), NCCIT, and myometrial cells for nucleolin (left panel) at 0 h (left row), 6 h (middle row), 24 h (right row), and for RPA194 (right panel) at 0 h (left row) and 24 h (right row). Scale bar = 20 μM; n = 3. C Western blot analysis on extracts of 2102EP(S) (EPS) cell lines treated with 5 μM sempervirine at the indicated times, probed for RPA194, MDM2, p53, and nucleolin. Tubulin is the reference gene; n = 3. D Western blot analysis on cell extracts from 2102EP(S) (EPS) and NCCIT cell lines treated for 24 h with or without 5 μM sempervirine and probed for RNA Pol II, RPA194, UBF-1, and p53. Tubulin is the reference gene. Arrowhead points to the slower migrating band of RNA Pol II that corresponds to the phosphorylated form; n = 3. E Western blot analysis on cell extracts from 2102EP(S) (EPS) cell lines treated with or without 5 μM sempervirine in the absence or presence of 10 μM MG132. Extracts were probed for RPA194, p53, and MDM2. Tubulin is the reference gene; n = 3. F Western blot analysis on extracts from 2102EP(S) (EPS) cell lines treated for 24 h with or without 5 μM sempervirine or 5 μM nutlin in the presence or absence of 10 μM MG132 added for the last 6 h of culture, and probed for RPA194. Tubulin is the reference gene; n = 3. G Western blot analysis on cell extracts from sh-scrambled p53 2102EP(S) (p53-scr EPS) or sh-exon4 p53 2102EP(S) (p53ex4-sh EPS) cell lines that underwent MDM2 silencing with scrambled or MDM2 siRNAs (si-scr and si-MDM2, respectively) treated for 24 h with or without 5 μM sempervirine, and probed for RPA194, MDM2, and p53. GAPDH is the reference gene; n = 3.