Tcs sp5 instrument

MDA-MB-231 cells were plated on coverslips and co-transfected with UGDH or negative control siRNA plus two µg of EGFP-LC3 (# 11546, Addgene). 24 h after transfection, tumor cells were treated with 1 µM EPI as described above. After the treatment, the medium was removed, and cells were mounted on glass slides after being washed twice with PBS and fixed in 4% formaldehyde for 30 min. LC3 subcellular localization was analyzed by confocal microscopy using a Leica TCS SP5 instrument (Leica, Milan, Italy). The experiment was also performed treating each well with 20 µM chloroquine (an inhibitor of autophagy). Chloroquine was added to the cells concomitantly with EPI.

AoSMCs were nucleofected with the plasmid pcDNA3-GFP-RELA (Addgene) coding for the GFP–p65 protein fusion, plated on poly-l-lysine (100 ng/liter) pre-coated glass coverslips (35 mm in diameter), and treated with 0.1 μm TNFα alone or in combination with 1 μm SRT1720, 100 μm RESV, or 10 μm PDTC (Sigma). After 48 h, cell culture medium was removed, and AoSMCs were washed three times with PBS and fixed with a 4% paraformaldehyde/PBS solution for 10 min at room temperature. Coverslips were mounted on glass slides, and p65 subcellular localization was analyzed by confocal microscopy using a Leica TCS SP5 instrument.

Coding sequences for the KEA3 cTPs were amplified from the oeKEA3 constructs (Armbruster et al., 2016 (link)) and assembled into pEarleyGate100 (Earley et al., 2006 (link)) linearized with Xbal and Xhol restriction enzymes downstream of the 35S promoter by using Gibson Assembly (Gibson et al., 2009 (link)). Colonies of Agrobacterium tumefaciens strain GV3101 transformed with the cTP_KEA3−YFP constructs were resuspended in induction medium (10 mM MgCl₂, 10 mM MES-KOH pH 5.6, 150 μM acetosyringone) to an OD₆₀₀ of 0.5. After 2 h at 28°C, suspensions were inoculated onto sections of well-watered N. benthamiana leaves by injecting into the bottom side of a punctured leaf (Blatt and Grefen, 2014 (link)). Transfected plants were grown for 2 d in room light before detached leaves were analyzed for the localization of the YFP fluorescence signal by confocal microscopy. For microscopy, the Leica TCS SP5 instrument was used with 63×/1.4 objective and water immersion. Fluorophores were excited by using an argon laser at 514 nm with 30% intensity, YFP fluorescence was collected between 524 and 582 nm (930 V gain), and Chl fluorescence between 618 and 800 nm (650 V gain).