Growth-inhibitory and molecular effects of sequential ITF2357/pemetrexed in LCSC in vitro and in vivo xenografts models. (A) Western blot analysis of TS, p62/SQTSM1, LC3B-I/II and PARP proteins in total cell lysates from LCSC143 cell line exposed to pemetrexed (PEM, 0.1 μM) or ITF2357 (1 μM) alone or in combination (24 h pemetrexed followed by 48 h ITF2357). HSP72/73 expression was used as loading and transferring control. (B) Analysis of cell viability by CellTiter-Glo assay and (C) drugs interaction evaluated on the basis of the Combination Index, which is plotted against fractional growth inhibition in LCSC143 line treated with ITF2357 and pemetrexed (drug ratio 1:1) alone or in combination. Treatment with pemetrexed for 24 h was followed by treatment with ITF2357 for 48 h. (●, ITF2357; ■ , pemetrexed; ▲, combination) The results are reported as "viability of ITF2357-treated cells/viability of untreated cells" × 100 and represent the mean ± SD of three independent experiments. (D) In vivo response of H1650 xenograft to pemetrexed (1000 mg/kg/weekly) or ITF2357 (100 mg/kg day for 4 days) alone or in combination (pemetrexed, 1000 mg/kg/weekly followed by ITF2357, 100 mg/kg/day for 4 days).