Fig 2: LPS induced HK-2 cell damage. (a) CCK-8 assays were used to detect cell viability; (b) flow cytometry was used for assessment of cell apoptosis; (c) ELISA was performed for estimation of TNF-a, IL-1ß, IL-6, and IL-8 expression and secretion. (d) Western blot was performed to determine the protein levels of caspase-3, caspase-9, Bax, Bcl-2, CHOP, GRP78, E-cadherin, a-SMA, Snail, and Vimentin, where GAPDH was used as an internal control; (e) RT-qPCR assay was used for evaluation of the mRNA levels of caspase-3, caspase-9, Bax, Bcl-2, CHOP, GRP78, E-cadherin, a-SMA, Snail, and Vimentin, in which GAPDH was used as an internal control gene to calculate the relative expression of the target genes. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control group.

Fig 3: Liver and Lung tumor cells express higher levels of GRP78 and HSP27 than non-transformed cells from the same tissue from the same patientA liver tissue micro-array was purchased (http://www.novusbio.com/Liver-Tissue-Micro-Array_NBP2–30221.html) and antigen retrieval performed on the tissue sections. Immuno-histochemical staining was performed to determine and compare the expression of PDE5, HSP70, Phosopho-ERBB1, HSP90, GRP78 and HSP27 in normal liver alongside paired tumor tissue from the same patient.

Fig 4: [Sorafenib + Sildenafil + Afatinib] treatment inactivates mTOR, STAT3 and NF?B; over-expression of [GRP78 + HSP27] or knock down of Beclin1 / ATG5 / eIF2a prevents these effects(A) ADOR cells were treated with vehicle control or [sorafenib (2.0 µM) + sildenafil (2 µM)] and/or afatinib (1 µM) or the three drugs in combination, for 6 h after which the phosphorylation of eIF2a, mTOR, ATG13, ERK1/2, AKT (T308), STAT3 (Y705) and p65 NF?B was determined at 10× by immuno-fluorescence in the Hermes WiScan system. (B) ADOR cells were transfected with an empty vector plasmid, or plasmids to express HSP27 and GRP78 together. Twenty four h after transfection cells were treated with vehicle control or with [sorafenib (2.0 µM) + sildenafil (2 µM)] +/- afatinib (1 µM) for 6 h after which cells were fixed in place and permeabilized using 0.5% Triton X100. Immuno-fluorescence was performed to detect the phosphorylation levels of mTOR, STAT3 and p65 NF?B. (C) ADOR cells were transfected with an empty vector plasmid, or plasmids to express: GRP78 and HSP27 together; an activated form of STAT3; an activated form of mTOR; or the dominant negative super-repressor I?B S32A S36A. Twenty four h after transfection cells were treated with vehicle control or with [sorafenib (2.0 µM) + sildenafil (2 µM) + afatinib (1 µM)] for 12 h after which cell viability was determined (n = 3 +/- SEM). (D) ADOR cells were transfected with a scrambled siRNA or siRNA molecules to knock down Beclin1 or ATG5 or eIF2a. Twenty four h after transfection cells were treated with vehicle control or with [sorafenib (2.0 µM) + sildenafil (2 µM) + afatinib (1 µM)] for 12 h after which cell viability wasdetermined (n = 3 +/-SEM).

Fig 5: Killing by [pemetrexed + sorafenib + afatinib] requires ER stress signaling as well as inactivation of AKT and PERK dephosphorylationA. BT474 and SUM149 cells were transfected with a scrambled siRNA or with an siRNA molecule to knock down the expression of PERK. Twenty four h after transfection cells were treated with vehicle control, [pemetrexed (0.5 µM) and sorafenib (2 µM)], Afatinib (0.5 µM), Neratinib (0.5 µM) or the drugs in combination as indicated. Twelve h after treatment cells were treated with live / dead reagent and cells examined using a Hermes WiScan microscope where red/yellow cells = dead; green cells = alive. B. BT474 and SUM149 cells were transfected with an empty vector plasmid (CMV) or with a plasmid to express GRP78. Twenty four h after transfection cells were treated with vehicle control, [pemetrexed (0.5 µM) and sorafenib (2 µM)], Afatinib (0.5 µM), Neratinib (0.5 µM) or the drugs in combination as indicated. Twelve h after treatment cells were treated with live / dead reagent and cells examined using a Hermes WiScan microscope where red/yellow cells = dead; green cells = alive. C. SUM149 cells were treated for 6h with vehicle control, [pemetrexed (0.5 µM) and sorafenib (2 µM)], Afatinib (0.5 µM) or the drugs in combination as indicated. Cells were fixed and permeabilized in situ and immuno-fluorescence was performed to determine the total phosphorylation level at 10X magnification of PERK T981; PERK T799; and AKT T308. D. SUM149 cells were transfected with a scrambled siRNA or with siRNA molecules to knock down the expression of AMPKa1/a2, PERK, ATF4 or CHOP. Twenty four h after transfection cells were treated with vehicle control, [pemetrexed (0.5 µM) and sorafenib (2 µM)], Afatinib (0.5 µM) or the drugs in combination as indicated. Twelve h after treatment cells were treated with live / dead reagent and cells examined using a Hermes WiScan microscope where red/yellow cells = dead; green cells = alive (+/- SEM) * p < 0.05 less than siSCR control value.