Fig 2: Construction of the PER2 stable knockdown cell line. (A) Western blot analysis of PER2 expression in RKO cells after shRNA transfection; (B) qPCR detection of PER2 mRNA expression in transfected RKO; (C) efficiency of control and shPER-2 lentiviral infection based on GFP fluorescence (approximately >90%) under an optical (left panel) or fluorescence (right panel) microscope, scale bar 50 µm. (D) The numbers of fluorescent cells were counted by ImageJ and are shown in the histogram. PER2, Period 2; RKO, human colorectal adenocarcinoma cells; qPCR: real-time quantitative polymerase chain reaction.

Fig 3: PER 2 reduces the sensitivity of colorectal cancer cells to doxorubicin. (A) Changes in PER2, cleaved-PARP, and p53 protein levels in RKO cells after .2 mM Dox treatment for 0, 6, 12, and 24 h. (B) Changes in PER2, cleaved-PARP, and p53 protein levels in RKO cells treated with different concentrations of Dox (0, .1 µg/µl, .2 µg/µl, and .4 µg/µl) for 24 h. (C) Representative images of flow cytometry of PER2-knockdown and control cells treated with .2 mM Dox at different times. (D) The percentage of apoptotic cells is shown (lower panel, **** P <.0001).PER2, Period 2; Dox, doxorubicin.

Fig 4: Immunoblot detection of circadian proteins in Huh-7 cells expressing the HCV core protein genotype 1b or 3a and GFP-expressing control cells.(A) 48 hours after transfection cells were lysed and equal amounts of proteins were loaded on a 10% polyacrylamide gel, separated by electrophoresis and immunoblotted with specific Rev-Erba, Rora, CLOCK, ARNTL, ARNTL2, PER1, PER2, CRY1 and CRY2 primary antibodies. ß-actin expression served as loading control. (B) Densitometric quantification of CRY2, PER2 and CLOCK proteins normalized to ß-actin expression of three different experiments.

Fig 5: Scheme illustrating the antagonism between HCV genotype 1b replication and PER2 in hepatocytes.HCV core protein genotype 1b, via a yet undefined mechanism, induces a downregulation of PER2 mRNA. PER2 protein produced in the cytoplasm accumulates in the nucleus, as observed in human liver biopsies infected with HCV genotype 1b, where could further inhibit the production of its own RNA [2], [3]. Alteration of this equilibrium by exogenous overexpression of PER2 protein hampers HCV genotype 1b replication.