Persaud L , Mighty J , Zhong X , Francis A , Mendez M , Muharam H , Redenti SM , Das D , Aktas BH , Sauane M .

SC1 CA2005 National Institutes of Health, SC1 CA200518 NCI NIH HHS , SC1 CA2005 NIH HHS

	Figure 1. IL-24 activates ATF4 in a dosage dependent manner. MCF-7 cells were treated for 72 h with Ad.IL-24 (25, 50, and 100 plaque-forming units (pfu) per cell) or Ad.vector (100 pfu per cell). Cells were collected, protein purified, and subjected to Western blot analysis to detect phospho-ATF4, total ATF4, BiP, and β-actin.
	Figure 2. IL-24 activates protein kinase A (PKA) in a concentration-dependent manner. (A) MCF-7 cells were treated for 72 h with Ad.IL-24 (25, 50, and 100 pfu per cell) or Ad.vector (100 pfu per cell). Cells were collected, protein purified, and subjected to Western blot analysis to detect phospho-PKA substrates, PKA-Cα subunits, and β-actin proteins. (B) MCF-7 cells were treated with Ad.vector (100 pfu/cell) or Ad.IL-24 (25, 50, and 100 pfu per cell) and then assayed for the production of cAMP after 20 h of treatment. Numbers represent mean cyclic adenosine monophosphate (cAMP) (nM) concentration after normalization to control. An average of three independent experiments is shown ± SE (n = 9). (C,D) MCF-7 cells were treated for 72 h with Ad.vector (control) or Ad.IL-24 at 100 pfu/cell, and either untreated or treated with 10 μM H-89 for 72 h. Cells were collected, protein purified, and subjected to Western blot analysis to detect phospho-PKA substrates, PKA-Cα subunit, phospho-ATF4, total ATF4, and β-actin.
	Figure 3. The IL-24 killing effect is decreased in the presence of PKA inhibitors. (A) Human breast MCF-7, MDA-MB-231, MDA-MB-157, and T47D cancer cells were incubated with 10 μM of PKA inhibitor, H-89, or PKI, with or without Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell), and cell viability was determined by the MTT proliferation assay five days after treatment. Numbers represent the ratio of specific treatments to values in control cells (Ad.vector). An average of three independent experiments is shown ± SD as errors bars. *, p < 0.001 comparted to Ad.vector. (B) Cells were treated as described in A, and then assayed for cell death using annexin V staining, a measure of apoptosis, was determined 48 h later by fluorescence-activated cell sorting (FACS) analysis using the CellQuest software (Becton Dickinson). An average of three independent experiments is shown ± SD as errors bars, *, p < 0.001 comparted to Ad.vector. (C) MCF-7 cells were treated for 24 h with Ad.IL-24 (25, 50, and 100 pfu per cell), 10 μM H-89 or Ad.vector (100 pfu per cell). Cells were collected, protein purified, and subjected to Western blot analysis to detect phospho-p38 MAPK, total p38 MAPK, and β-actin. (D) MCF-7 cells were infected with either Ad.vector (control) or Ad.IL-24 at 100 pfu/cell, and either untreated or treated with 10 μM H-89 for 72 h. Western blot analysis was performed with antibodies for phospho-p38 MAPK, total p38 MAPK, and β-actin.
	Figure 4. IL-24 induces TP53 expression, and promotes nuclear translocation in a PKA-dependent manner. (A,B) MCF-7 cells were treated for 72 h with Ad.IL-24 (25, 50, and 100 pfu per cell), 10 μM H-89, or Ad.vector (100 pfu per cell). Cells were collected, protein purified, and subjected to Western blot analysis to detect to detect phospho-TP53, total TP53, and β-actin proteins. (C) Semi-quantitative measurements of the mean fluorescein isothiocyanate (FITC) intensity were taken using Nikon NIS Elements whereby, the average intensity of five cell nuclei were taken under each experimental condition. Error bars are expressed as the standard deviation of FITC intensity values. (D) Cells were fixed and phospho-Ser15 TP53 was detected by immunofluorescence using anti-phospho-TP53 antibodies.
	Figure 5. The inhibition of PKA blocks IL-24 activation of extrinsic apoptosis. (A) MCF-7 cells were infected with either Ad.vector (control) or increasing concentrations of Ad.IL-24 (25, 50, 100 pfu per cell) for 72 h. Western blot analysis was performed with antibodies for FasL, Fas, FADD, DR4, and β-actin. (B) MCF-7 cells were infected with either the Ad.vector (control) or Ad.IL-24 at 100 pfu/cell, and either untreated or treated with 10 μM H-89 for 72 h. Western blot analysis was performed with antibodies for FasL, Fas, FADD, DR4, and β-actin.
	Figure 6. IL-24 activates PKA to induce apoptosis in breast cancer cells. A schematic of the molecular mechanisms underlying Ad.IL-24 induced apoptosis of cancer cells, involving PKA activation, ATF-4 phosphorylation, p38MAPK signaling, p53 phosphorylation, Fas apoptotic signaling, and the inhibition of translation initiation (red arrow indicates downregulation; green arrow indicates upregulation; black arrows indicate pathway activation, black dotted arrows indicate potential mechanism of action, black bar headed arrow indicates inhibition).

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