Sirna oligos

The HUVECs or HCASMCs cells were transfected with Lipofectamine 2000 (Invitrogen) and AMPK-α1/α2-targeted or a control small interfering RNA (siRNA) oligos (Dharmacon, Lafayette, CO, USA) according to the manufacturer’s instructions (Takara Biotechnology (Dalian) CO., LTD). The siRNA sequence was as follows: 50-ACC GAG CUA UGA AGC AGC UGG GUU U-30. The efficiency of gene silencing was confirmed by Western blotting analysis.

Silencing was performed as previously described (Paul et al., 2011 (link)) using siRNA oligos purchased from Dharmacon. For protocol and sequence details refer to the Supplemental Experimental Procedures.

Electroporation of siRNA into CLL cells was performed using Amaxa Human B-cell Nucleofection Kit (Amaxa, Cologne, Germany). 1x10⁷ PBMCs were mixed with 100 μL of Amaxa B-cell nucleofector solution, and 2 μg of siRNA was nucleofected using program X-005 [29 (link)]. Transfection efficiency, assessed by transfection with 2 μg pMaxGFP plasmid, was 30–60% with cell viability of 50–80% at 24 hours. siRNA oligos were synthesized by Dharmacon (Lafayette, CO). Sense strand against human ASK1: GCUCGUAAUUUAUACACUGUU.

The siRNA oligos (Dharmacon, Lafayette, CO, USA) targeting PGC-1β, ERRα, PCK2 or non-targeting controls were used for targeted depletion of the colorectal cancer cells. For pooled transfections, two validated, individual ON-TARGET PLUS siRNAs were used at a final RNAi concentration of 40 nM and were added to 5 µL of RNAiMAX (ThermoFisher, Waltham, MA, USA, 13778150) and 500 µL Hank Buffer Salt Solution without sodium bicarb. The mixture was added to 300,000 cells in 1.5–2 mL of media without antibiotics in a 6-well plate. All transfections were conducted for 72-h before analysis.
RNA sequences for transfections are listed in Supplemental File S4.

To induce autophagy in MSC, cells were treated with rapamycin (200 nM, Enzo Life Sciences, Farmingdale, NY, USA, BML-A275), EBSS (Sigma, E2888) for 4 h and human TNFα (hTNFα) (10 µg/mL) for 2 and 4 h. For autophagy flux experiments, MSC cells were transfected with vectors encoding human Pacer-Flag or empty vector (OriGene) using TransIT-x2 dynamic delivery system (Mirus Bio, Madison, WI, USA, MR.MIR6000) according to manufacturer´s instructions or siRNA oligos targeting mouse Pacer or scrambled siRNAs as a control (Dharmacon) transfected with Dharmafect Transfection Reagents (Dharmacon, T-2001-01) using the manufacturer´s protocol. To induce autophagy, cells were treated with hTNFα (10 µg/mL) (Sigma, SRP3177) for 30 min, 2 h or 4 h. To inhibit autophagosome-lysosome fusion, cells were pretreated with Bafilomycin A1 for 30 min (0.5 µM, Sigma, B1793-10UG) and then stimulated with hTNFα at the same times described above. Autophagy flux was calculated as described in Ref. [37 (link)]: LC3II flux per sample equals LC3 II densitometric values (after normalization to β-Actin) of lysosomal inhibitor-treated samples minus lysosomal inhibitors untreated controls. This was performed for each independent experiment, and the 3 N were graphically represented. The same was performed for p62 flux.