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. 2016 Feb 22;7(11):12254–12266. doi: 10.18632/oncotarget.7601

Figure 6. Impairment in autophagy does not account for enhanced Mtx-induced death observed in SESTRIN 2 knockdown cells.

Figure 6

A. HCC1806 cells were treated with 20 μM Mtx for the indicated time after which lysates were immunoblotted for phospho-MTOR, total MTOR and ACTIN. B. Autophagic flux was evaluated in HCC1806 cells treated with 20 μM CQ alone, 20 μM Mtx alone, or a combination of 20 μM CQ and 20 μM Mtx for 48 h and cell lysates immunoblotted for SESTRIN 2, LC3-I/II and ACTIN. C. Ctrl and SESTRIN 2 siRNAs transfected HCC1806 cells were treated with 20 μM Mtx ± 20 μM CQ for 48 h. Lysates were immunoblotted for SESTRIN 2, LC3-I/II and ACTIN. D. HCC1806 cells were treated with 20 μM Mtx alone, 10 μM Spautin-1 (Spa-1) alone or a combination of Mtx and Spa-1 for up to 72 h after which cell viability was assessed by PI uptake. E. HCC1806 cells were treated with 20 μM CQ alone, 10 μM Spa-1 alone or a combination of CQ and Spa-1 for 24 h and lysates immunoblotted for LC3-I/II and ACTIN. F.-G. Ctrl and BECLIN1 siRNA transfected HCC1806 cells were treated with 20 μM Mtx for the indicated time. (F) Lysates were immunoblotted for SESTRIN 2 and ACTIN and (G) Cell viability assessed at each time-point by PI uptake.