Chloroquinone

DMEM, propidium iodide (PI), acridine orange, monodansyl cadaverine, 3-(4,5-dimethylthiazole-2-yl)-2,5 diphenyltetrazolium bromide (MTT), bafilomycin, chloroquinone (CQ), sodium fluoride, sodium orthovanadate, fetal bovine serum were purchased from Sigma-Aldrich, Missouri, USA. Akt inhibitor IV and VIII, antibodies of caspase-3, cyclin A, cyclin B, cdc2, PARP-1, β-Actin and Akt siRNA were from Santa Cruz Biotechnology, Texas, USA. Antibodies to cdc25, CDK-1, cyclin E were purchased from Piercenet, Illinois, USA. All other antibodies and chemicals were purchased from Cell signaling technology, Massachusetts, USA. Electrophoresis reagents, reagents for protein estimation and protein molecular weight markers were from Bio-Rad Laboratories, California, USA. Polyvinyldifluoride (PVDF) membrane was purchased from Millipore, Massachusetts, USA. K-LISA mTOR activity kit were purchased from Calbiochem, San Diego, USA.

Autophagy in C2C12 or 7304.1 cultures was induced by amino-acid starvation by incubating cultures in Hanks Balanced Salt Solution (HBSS) for 3 hours [17 (link)], in F10 supplemented with 0.5% horse serum (Invitrogen) incubation for 48 hours. Cell fusion, as marked by MF20 expression, was not found in this condition. Chloroquinone (20µM) (Sigma-Aldrich) treatment was carried out for 4 hours in growth medium. In the IM2 cell model, autophagy was induced by incubation in DMEM supplemented with 2% HS for two days.

Vero (ATCC No. CCL-81) cell line was maintained using MEM (Himedia, Mumbai, India), supplemented with 10% FBS (Gibco, USA), and antibiotic–antimycotic solution (Sigma-Aldrich, Saint Louis, MO, USA) at 37°C and 5% CO₂. Dengue virus (DENV) serotype-2 (Strain No. 803347) and chikungunya (CHIKV, Strain No. 061573, P-2, African genotype) were used for this study. DENV-2 stock was prepared in C6/36 (mosquito cell line) and CHIKV was propagated in Vero cells and stored at −80°C.
A total of 25 different parts from eight plants, i.e., Vitex negundo (RMRC-1355), Plumeria alba (RMRC-1357), Ancistrocladus heyneanus (RMRC-1359), Bacopa monnieri (RMRC-1361), Anacardium occidentale (RMRC-1356), Cucurbita maxima (RMRC-1362), Simarouba glauca (RMRC-1358), and Embelia ribes (RMRC-1360) (Table 1), selected based on their ethno botanical use in the treatment of infectious diseases, were collected from various sites in Belagavi, identified, and authenticated at ICMR-National Institute of Traditional Medicine, Belagavi, where voucher herbarium specimens were deposited. Four purified compounds, i.e., anacardic acid (Sigma-Aldrich, Saint Louis, MO, USA), chloroquinone (Sigma-Aldrich, Saint Louis, MO, USA), methyl gallate (Sigma-Aldrich, Saint Louis, MO, USA), and glaucarubinone (Dr. John Beutler’s Lab, NIH USA) were also included in this study.