β actin or gapdh

Spheroids with and without treatments (24 h) were lysed in RIPA buffer containing Na₃VO₄ (1 mM) and protease inhibitors (1 μg/ml). For subcellular fractionation, spheroids (in 24-well plates) were treated with GW-exo-miR for 24 h, harvested, and the pellet re-suspended in the digitonin cell permeabilization buffer (Trevigen, Gaithersburg, MD). The supernatant containing the cytosolic fraction was collected. The remaining pellet (organelle fraction) was lysed in the RIPA buffer containing Na₃VO₄ (1 mM) and protease inhibitors (1 μg/ml). Protein concentration was assessed by the Bradford assay. The lysates (10 μg of protein) were separated using 4–12% SDS-PAGE (Life Technologies) and transferred onto a nitrocellulose membrane (Protran). After blocking with 5% non-fat milk in PBS-Tween (0.1%), the membranes were incubated overnight at 4  °C with primary antibodies against LC3B, phospho-mTOR (p-mTOR^ser-2448), mTOR, phospho-p70S6K^ser-235/236, p70S6K, PARP-1, BID, BAX, Caspase-8, Caspase-9, Caspase-3, cyt-c, AIF, pAMPK^thr-172, AMPK, pULK1^ser-555 and ULK1 (all Cell signaling). β-Actin or GAPDH (Cell Signalling) was used as a loading control. After incubation with HRP-conjugated secondary IgG (Cell Signalling), the blots were developed using ECL (Pierce). The band intensities were visualized and quantified with ChemiDoc using Quantity One software (Bio-Rad Laboratories).