Niltubacin

Fifty microliters of cell culture medium were added per well to 96-well electronic microtiter plate (E-Plate) for impedance background measurement. CCHE-45 cells were then plated at 12000 cells/well in 24 hours prior to treatment. The E-Plate was incubated at 37 °C with 5% CO₂ and monitored on the real time cell analysis xCELLigence (ACEA Biosciences) at 5-minute time intervals. The next day, cells were treated with different concentrations of tubacin or niltubacin (Enzo Life Science). Cells were monitored for up to 72 hours post treatment. Cell index (CI) was plotted against different concentrations of tubacin or niltubacin. Experiment was performed three times and three wells/drug concentration were used.

Neuroblastoma SH-SY5Y cell line is a kind gift from Dr. Juma Mora at Sant Joan De Deu, Barcelona, Spain. SH-SY5Y cells were authenticated using AmpFlSTR® SGM Plus® PCR Amplification Kit (Applied BioSystems). SH-SY5Y and HEK293-T cells were cultured in RPMI and DMEM (Lonza) respectively supplemented with 10% FBS (Lonza). For induction of autophagy, cells were serum starved in Hank’s balanced salt solution (HBSS) (Lonza) for 2 and 6 hours. For HDAC6 inhibition, cells were treated with 20 μM tubacin or niltubacin (Enzo Life Sciences). For 5-aza-2′-deoxycytidine (5-AZA-dC) treatment (Sigma Aldrich), cells were treated with either DMSO or 10 μM 5-AZA-dC for four successive days.

Mouse inner medullary collecting duct (IMCD) cells transfected with somatostatin receptor 3 fused to GFP were a generous gift of Bradley K. Yoder of University of Alabama at Birmingham. Cells were cultured on fibronectin-coated coverslips and slides to 70% confluence in growth medium (DMEM F-12 with 10% FBS, 1% pen/strep and 200 µg/ml geneticin) and serum-starved for 72 h to promote cilia formation. For the tubacin experiment, cells were cultured as described above and treated with 0.5 mM of tubacin or niltubacin (Enzo Life Sciences) for 4 h prior to exposure to flow. For the HDAC6 knockdown experiment, cells were cultured to 60% confluence in growth media and transfected with scrambled control or HDAC6 siRNA (sc35545; Santa Cruz Biotechnology) using Lipofectamine 2000 (Life Technologies). Cells were serum-starved the following day for 72 h and then used in flow experiments. The average cilium length measured during the flow experiments was 3.9±0.2 µm (n=33).