Lc3 2

Scutellarin (purity 98%) was purchased from Honghe Qianshan Bioengineering Co., Ltd. (Honghe, China). Streptozocin (STZ) and rosiglitazone were obtained from Multi Sciences (Lianke) Biotech, Co., Ltd. (Hangzhou, China). Antibodies to detect β-actin, procaspase-3, procaspase-8, procaspase-9, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax, Cyt-C, Bcl-2, Beclin-1, and LC3-II were ordered from Santa Cruz Biotechnology. The procaspase-12 polyclonal antibody was ordered from Abcam Company. Adult male Sprague-Dawley (SD) rats (180 to 200 g) were obtained from the Experimental Animal Center of Kunming Medical University, China. Two weeks after the adaptive feeding of the rats, they were randomly divided into control group, model group, SCU low-dose (100 mg/kg/d), high-dose (200 mg/kg/d) treatment group, and rosiglitazone (5 mg/kg/d) positive control group (each group of ten). The model group was given a high-sugar and high-fat diet for 8 weeks and injected with STZ by 35 mg/kg intraperitoneally, while the control group was given a normal diet. The SCU groups were administered orally for 8 weeks [12 (link)].

Propranolol was obtained from the Chinese materials research center (Beijing, China). Fetal bovine serum (FBS) was got from Gibco, Gaithersburg, MD, USA). N-Acetyl-L-cysteine (NAC), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Hoechst 33258 staining, and Annexin V-PE Apoptosis Detection Kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The enhanced chemiluminescence (ECL) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). JNK inhibitor (SP600125) and 3-MA (3-Methyladenine) were obteined from sigma (St. Louis, MI, USA). Antibodies against Cyclin B1, phospho-cdc2, cdc2, JNK, phosphorylated JNK, and p21 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Cdk1, Bcl-2, cleavage caspase-3, cleavage caspase -8, cleavage caspase -9, BAX, LC3-I, LC3- II, Becline-1, p62, GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other common chemicals and buffers were from Boster (Wuhan, China).

Macrophages were seeded on coverslips and treated with 100 μM simvastatin overnight as above. Cells were then infected with green fluorescent protein (GFP) expressing- L. major (IL81) GFP-IL81 (MHOM/IL/81/FEBNI) at MOI of 10. After 24 hours, cells were washed and fixed in 4% paraformaldehyde followed by labelling of phagosome markers with fluorescent antibodies against Lysosome Associated Membrane Protein-3 (LAMP-3, Santa Cruz) and a lysosomal protease (Cathepsin D, Santa Cruz). Macrophages were also labelled for Light Chain 3-II (LC3-II, Santa Cruz), a marker for autophagy. These markers were then visualized by counter-stained with Alexa 546 antibodies (Molecular probes, Invitrogen) followed by nuclear stain (DAPI). Coverslips were then mounted using mowiol-containing anti-fade on glass slides. Images were captured under Carl Zeiss 510 confocal microscope and analysed using Zen Blue software as previously described14 (link).