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  • br Cell proliferation was also evaluated by using EdU

    2020-08-12


    Cell proliferation was also evaluated by using EdU Kit (Millipore, USA). The proportion of EdU positive cells was determined by fluor-escence microscopy and FACS. Total cells were counted following nu-clear DAPI counterstaining.
    2.5. Cell viability assay
    2.6. Cell cycle analysis
    Cells were seeded and treated with Mar-C or DMSO for 5 d as de-scribed. The cells were collected, then fixed overnight and incubated with RNaseA (Solarbio, China) for 30 min. Cells were stained with PI for 15 min at 4 °C. Cell suspensions were subjected to flow cytometer (BD, USA). Modfit LT 5.0 software was used for analysis. Untreated cells served as controls.
    2.7. Quantitative PCR analysis
    Total RNA was extracted from A549 cells and cDNA was prepared as described previously [16]. Quantitative PCR was performed with SYBR Green qPCR master mix (Takara, China).
    2.8. Western blot analysis
    Whole cells were lysed in RIPA lysis buffer containing phosphatase and protease inhibitors at 4 °C. Protein concentration of the supernatant was determined using a BCA assay (Beyotime Biotechnology). Samples were resolved on suitable gradient SDS-PAGE gels and blotted to ni-trocellulose membranes (Millipore, USA). The membranes were wrapped in plastic bags with primary CHIR-265 overnight at 4 °C, subsequently washed and incubated with secondary antibody (KPL, USA) for 1 h at room temperature. Target proteins were detected by chemiluminescence detection system with Kodak films.
    2.9. Immunofluorescence
    Cells were fixed, permeabilized and blocked with 3% bovine serum albumin containing 0.1% Triton X-100 for 20 min. After washed with PBST (PBS buffer containing 0.02% Triton X-100) for 5 min, the cells were incubated with primary antibodies at an optimal concentration at 4 °C. Next, the cells were washed with PBST for 3 times and incubated with fluorophore-conjugated secondary antibodies at room temperature for 2 h. Then followed by 3 times wash with PBST, the nuclei were counterstained with DAPI for 15 min. The LSM800 (Carl Zeiss, Germany) was used for observation.
    A549 cells were exposed to Mar-C, doxorubicin or DMSO for 5 d. Conditioned medium were collected, centrifuged and stored in liquid nitrogen. Cytokines in conditioned medium were quantified using Quantibody Human Inflammation Array 1 (RayBiotech Inc.). The glass chips were scanned by Gene microarray scanner and the signals were
    (caption on next page)
    Fig. 1. Mar-C induces cellular senescence of lung cancer cells at a low concentration with less effect on normal cells. (A) Chemical structure of Mar-C. (B and C) MTT assay showed slight inhibition on A549 or H1299 cells by 2 μM Mar-C compared with obvious inhibition of 10 μM. (D) A549 or H1299 cells under different concentrations of Mar-C. The cells displayed senescence-like phenotype with enlarged and flattened morphologic changes at low concentrations (2 μM) instead of apoptosis. (E) A549 or H1299 cells exhibited positive β-gal staining after treatment with Mar-C (2 μM) for 5 days. (F) Representative images and histogram of diminished clone formation of the Mar-C (2 μM)-treated cells. (G) MTT assay revealed that Mar-C (2 μM) showed no difference on the inhibition of NHF cells, compared with the control. (H) EdU incorporation assays by FACS showed that 2 μM of Mar-C had no effects on the proliferation of NHF cells after 5 days treatment.
    (I) the representative images and histogram of fluorescence in EdU incorporation assay under the treatment of Mar-C (2 μM) to NHF cells. (J) Morphological changes of NHF cells exposed to Mar-C (2 μM) were the same as the control, with negative β-gal staining, whereas doxorubicin-induced senescence showed positive β-gal staining. (K) Compared with the control, Mar-C (2 μM) exhibited little inhibition on IMR90 and HBE cells. Data information: In B, C, F, G, I, and K, data represent mean ± SEM (n = 3); *P < .05 or **P < .01.
    (caption on next page)
    Fig. 2. Mar-C reduces tumor growth potential and prolongs the survival of tumor bearing mice. (A) Primary tumors were quantified using bioluminescence IVIS spectrum live imaging at the day 17 after initial implantation. The bioluminescence intensities (Photon flux; photons·s−1·cm−2·sr−1) were shown in the histogram.
    (B) Image of primary A549 tumors after treatment with vehicle, DOX and Mar-C for 17 days. Tumor volumes were measured over 3 weeks of time period by caliper. Mar-C suppresses A549-luc xenograft tumor growth, compared with the vehicle. (C) Changes in body weight of A549 tumor-bearing mice treated with vehicle, DOX and Mar-C for 17 days. (D) Changes of liver enzymes in the serum of mice after 17 days. Aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT) and alanine aminotransferase (ALT). (E) The survival time were compared between the mice continuously treated with Mar-C and those that received the vehicle or DOX. The cumulative survival plots showed that Mar-C significantly prolonged the survival time of the A549 tumor-bearing mice. (F) SA-β-gal staining of tissue sections which were obtained from A549 tumors under Mar-C treatment, compared with the vehicle group.