R cancer cells to doxorubicin-induced apoptosis. Exposure to 2 mM doxorubicin induced each apoptosis, as evidenced by detection of poly (ADP-ribose) polymerase (PARP)-cleaved fragments, and accumulation of mature 97 kDa NIS species in HepG2 cells (Figure 5a). Comparable final results have been obtained in CCSW1 cell (Supplementary Figure S7). Flow cytometry analysis of intact living cells exposed to doxorubicin showed a statistically substantial, even though moderate, increase of NIS expression both at the cell surface and intracellularly in HepG2 cells (Figure 5b). A small but consistent and considerable raise of NIS protein expression in the cell surface was also detected in CCSW1 CAA cells (Supplementary Figure S8), but not within the p53 null cell line Hep3b (Supplementary Figure S8). We also studied the response of HepG2 cells to doxorubicin after knockdown of endogenous NIS protein expression by particular siRNAs (Supplementary Figure S9). We located that NIS silencing triggered a reduction of PARP cleavage (Figure 5c) andDoxo two M+-+-+-+NIS mRNA Arbitrary Units30 20 10 – +H PH ep H*** *** – +2 G HDoxo 2 M- +7 uH H- +3B ep C C- +SWFigure 3 NIS gene promoter activity is induced by doxorubicin in liver cancer cell lines. (a) Luciferase activity normalized for transfection efficiency and expressed as fold induction with respect to manage. The indicated cell lines had been transfected together with the 2000/ 375 NIS luciferase construct and exposed for 24 h to doxorubicin (doxo 2 mM). Luciferase activity was assayed 30 h after transfection and expressed as in Figure 2a. Data are indicates .D. of at the least 3 experiments performed in duplicate. (b) NIS mRNA levels in untreated (NT) or doxorubicintreated (DOXO) cells have been analyzed and outcomes expressed as in Figure 2b. Information are indicates .D. of at the least 3 experiments performed in duplicate.Sincalide P-values had been determined employing the two-tails Student’s t-test.Magrolimab *Po0.05; **Po0.01; ***Po0.HepG2 Fold induction 1.00 0.75 0.50 0.25 – + – + – + 1.00 0.75 0.PMID:23376608 50 0.CCSW1 1.00 0.75 0.50 0.25 – + – + – + – +PHHPrimers ADoxo two M- +- +10 Fold induction 8 6 4 2 – + – + TP100 80 60 40 20 – + – + – +10 eight 6 four two – + TP73 – + – + Primers BDoxo 2 M – +TPFigure 4 p53 and p73 are actively recruited to NIS proximal promoter in response to doxorubicin in liver cancer cells. Cross-linked chromatin from untreated and doxorubicin-treated (two mM; 24 h) HepG2 cells, CCSW1 cells and PHHs was immune-precipitated using the relevant manage IgG or specific anti-p53 (aTP53), anti-p63 (aTP63) and anti-p73a (aTP73a) antibodies and analyzed by real-time PCR with the A and B pairs of NIS promoter selective primers. Manage reactions working with distant primers didn’t amplify anti-p53, anti-p63 or anti-p73a ChIPed solutions (data not shown). Histograms represent the signifies of 3 independent experiments. Bar, S.D.Cell Death and DiseaseNIS and p53-family members in liver cancer cells F Guerrieri et alRNAi NIS RNAi ctrl RNAi ctrl Intracellular surface Relative NIS expression 3 * 2 1 NT – + NT Cleaved Parp Relative expression 40 30 20 1 of Max ten + + NT three NIS of Max + DOXO * -cleaved PARP -ACTIND O XONT -Cleaved PARP -NIS -ACTINDOXO two M 60 Cleaved PAR Relative levels intracellular2 DOXO 2 M surfaceDoxoDoxo RNAi ctrl + RNAi NIS -++ + + – +NT Propidium IodideDOXODOXO iNIS-NIS -Cleaved PARP -ACTIN6 AnnexinV Cleaved PARP Relative expressionPCDNAHA-NIS -+Figure five NIS silencing reduces DNA damage-induced apoptosis in HepG2 cells. (a) Whole-cell extracts from untreated and doxorubicin-tr.